Title [125I] iodomelatonin binding sites in the avian brain and retina

Author(s) Yuan, He.; 袁和

Citation

Issued Date 1993

URL http://hdl.handle.net/10722/34852

Rights The author retains all proprietary rights, (such as patent rights)and the right to use in future works.

THESIS ENTITLED: "[! 251]IODOMELATONIN BINDING SITES IN THE AVIAN BRAIN AND RETINA"

SUBMITTED BY: HE YUAN

FOR THE DEGREE OF: Ph. D

A T THE UNIVERSITY OF HONG KONG IN SEPTEMBER 1993

ACKNOWLEDGMENTS

This thesis is dedicated to my loving wife, L i Ning and my son, Xiao Hu.

I would like to express my deepest and lasting appreciation to my supervisor. Dr. S. F.

Pang for his supervision, encouragements and help in making this achievement possible. I would

like to thank all my colleagues and friends, Mr. Peter Lee. Mrs. Elizabeth Ayre, Miss Kimmy

Tsang, Mr. T.K. Yung, Ms L u Yan and Mrs Celia Pang for their enthusiastic support and

dependable friendship. I am indebted to Dr. A. E. Allen, Miss Cathi Brown and Miss Catherine

Tenn for their thoughtful suggestions for my thesis. Thanks is also extended to Dr. G. M . Brown

for providing me the opportunity to work in the Clarke Institute of Psychiatry during the last year

of my Ph. D program.

ABSTRACT

The radioreceptor binding assay set up in the present studies is a sensitive, stable and

reliable assay. The successful melatonin iodination, using iodogen and the effective separation

of iodomelatonin by HPLC, provides a ligand with high specific activity and purity. These

characteristics are supported by the binding results of high affinity, high specificity and low

non-specific binding from avian brain membrane preparations.

Our results confirmed and extended the findings of [125I]iodomelatomn binding sites in

the brain tissue of birds. The binding characteristics satisfied all the criteria for a hormone

binding site. Regional distribution studies showed that the high affinity [125I]iodomelatonin

binding was found in all brain regions examined. The order of binding density in those regions

was hypothalamus > midbrain > pons-medulla > telencephalon > cerebellum. Subcellular

distribution studies demonstrated that the binding sites localize in the crude membrane

fractions. The [125I]iodomelatonin binding characteristics that were demonstrated in the present

studies are similar in the brains of different avian species including; chickens, pigeons，quails and ducks. Compared to the binding sites in the brain tissue of mammals, the high affinity [125I]iodomelatonin binding sites are widely distributed throughout the whole brain suggesting differences in the activity and/or functions of pineal melatonin between birds and mammals.

The development of [125I]iodomelatonin binding sites in chicken embryo brain tissues begins earlier than in mammals. The developmental pattern and the effect of Guanosine S'-ty-thio]triphosphate (GTPyS) on the binding density suggest that functional melatonin receptors

may be expressed in the early development of the chicken embryo brain. The diumal variation of [125I]iodomelatonin binding sites in the chicken brain was

inversely related to the rhythm of serum melatonin. Melatonin administration decreased and pinealectomy increased the binding capacity suggesting that the circulating melatonin concentration can regulate the density of [125I]iodomelatomn binding sites in chicken brains.

The physiological significance of the high affinity [ 125I]icxiomelatonin binding sites in the avian brain was demonstrated in the present studies by: 1) The affinity of the binding sites in the brain was of the same order of magnitude as the melatonin concentration in the circulation; 2) The uneven regional distribution of the binding sites was parallel with that of endogenous melatonin; 3) Melatonin and related indoles had the same order of potency in the inhibition of [125I]iodomelatomn binding in the chicken brain membrane preparations and in the inhibition of p H ] dopamine release from the chicken retina; 4) GTP analogues decreased

the binding capacity, both in embryo and chicken brains indicating that the chicken brain

[125I]iodomelatonin binding sites belong to the family of G-protein-linked receptors.

The [125I]iodomelatonin binding sites demonstrated in chicken retina have similar

binding characteristics, diumal variation and developmental pattern to the binding sites in the

chicken brain. It is speculated that the binding sites in the chicken brain and retina belong to a

similar group of high-affinity binding sites which reside in different tissues.

CONTENT

I. INTRODUCTION 1

A . PHYLOGENY OF THE PINEALOCYTE 1

1. Pineal photoreceptor cells in lower vertebrates

2. Pineal photoreceptor cells in birds

3. Pinealocytes in mammals

B . MELATONIN RECEPTORS IN BRAIN AND RETINA 8

1. The speculation of melatonin receptors in brain and retina

1). Pharmacological study - the structure-activity relationships of melatonin

2). Physiological study - localization of melatonin receptors in the brain and retina

3). Biochemical study - The uneven distribution of exogenous and endogenous

melatonin in the brain

2. Identification of melatonin receptors in the brain and retina by receptor binding studies

1). The radioligands: Tritiated and iodinated melatonin in receptor identification

2). Affinity and density of pH]-melatonin binding sites in the brain and retina

3). Affinity and density of [ ^I]Icxlomelatomn binding sites in the brain and retina 3. Multiple [•'•^IJIodomelatomn binding sites in the brain and retina 4. Receptor agonists and antagonists 5. Receptor regulation 6. The mechanism of melatonin receptor actions

1) Guanine nucleotide-binding proteins (G-protein) related signal transduction systems 2) Melatonin receptors and G protein related signal transduction systems

IL MATERIALS AND METHODS A. MATERIALS:

2 5 2 5

1 • Chemicals 2 . Animals

B. METHODS 27

1 • Sample collection 2 . Tissue preparation 3 . Binding assay

1) Iodination of melatonin 2) Saturation study 3) Kinetic study 4) Specificity 5) Cytosol binding assay 6) Data analysis of binding study

4 . Protein determination 5 . Radioimmunoassay of melatonin 6 . Pinealectomy 7 • Melatonin administration 8 . Statistics

III. RESULTS 4 1

A . METHODOLOGY 4 1

1 • Iodination of melatonin 1) • Separation of the radioligand 1)• [125j;]iodomelatoniii purification and stability

2 . Conditions for the binding assay 1). Effects of p H and ions on [^^Ijiodomelatonin binding to chicken brain

membrane preparations 2). The effect of temperature on the [^^I]iodomelatomn binding to chicken brain

membrane preparations 3 • Tissue concentrations 4 . Sample stability

B. [1 2 5I]IODOMELATONIN BINDING SITES IN BRAIN TISSUE OF BIRDS: G E N E R A L BINDING CHARACTERIZATION AND REGIONAL DISTRIBUTION 47

1 • Kinetic Studies 2 . Saturation Studies 3 . Specificity 4 . Regional Distribution 5 . Subcellular Distribution 6 . Effects of GTP analogues on chicken brain [^^Ijiodomelatomn binding sites

C. D I U R N A L V A R I A T I O N A N D E F F E C T S O F M E L A T O N I N MANIPULATIONS O N [ 1 2 5 I ]IODOMELATONIN BINDING SITES IN THE CHICKEN BRAIN 5 1

1. Diumal rhythms of serum melatonin levels and binding capacities of [^^IJiodomelatonin binding sites

2 . Effects of melatonin administration on serum melatonin levels and [^^I]iodomelatonin binding capacities

3 . Effects of pinealectomy on serum melatonin levels and [^^I]iodomelatonin binding in the brain

D . DEVELOPMENT OF [ 1 2 5 I]IODOMELATONIN BINDING SITES IN THE CHICKEN BRAIN 5 4

1. Comparison of [^^I]iodomelatomn binding characteristics between the 17-day-old embryo and young chicken brain membrane preparations 1). Kinetic study 2). Saturation Study 3). Specificity

2 . Development of chicken brain [ i o d o m e l a t o n i n binding sites 1). Development of chicken brain weight and the protein content 2). Development of chicken brain [^^jiodomelatomn binding sites

3 • Development of the diumal rhythm of [ 1 2 5 I ] iodomelatonin binding sites in the chicken brain

4 . The effect of GTPyS on the [^^1]iodomelatonin binding sites in embryonic chicken brain

E . BINDING CHARACTERISTICS AND DIURNAL VARIATION OF [ 1 2 5 I ] -IODOMELATONIN BINDING SITES IN THE CHICKEN RETINA 59

1. Kinetic study 2 . Specificity 3 • Saturation study 4 . Diumal variation

F . DEVELOPMENT OF [ 1 2 5 I]IODOMELATONIN BINDING SITES IN THE CHICKEN RETINA 6 2

1 • The binding characteristics of [^^1]iodomelatonin binding sites in embryonic chick retina 1). Kinetic studies 2). Specificity 3). Saturation study 4). Diumal variation of [^^I]icKiomelatonin binding

2 . The development of [^^I]iodomelatomn binding sites in chicken retinas

IV. DISCUSSION 6 5

A . CHARACTERIZATION OF [1 2 5I]IODOMELATONIN BINDING SITES IN THE AVIAN BRAIN 6 5

1 • The radioligand - [•'•^Ilxodomelatomn 2 . The identification and characterization of [ 1 ] iodomelatonin binding sites in avian brain 3 • The physiological significance of the [^^]iodomelatonin binding sites in chicken brain

B . C I R C A D I A N R H Y T H M AND E F F E C T S O F M E L A T O N I N MANIPULATIONS ON THE [125I]IODOMELATONIN BINDING SITES IN T H E CHICKEN BRAIN 73

1 • Diumal rhythms of serum melatonin levels and [^^jiodomelatonin binding capacities 2 . Effect of melatonin administration on serum melatonin levels and [ ] i o d o m e l a t o n i n

binding capacities 3 • Effect of pinealectomy on serum melatonin levels and [^^I]iodomelatonin binding in the

brain

C . THE DEVELOPMENT OF [1 2 5I]IODOMELATONIN BINDING SITES IN THE CHICKEN BRAIN 7 9

1. Characteristics of [^^I]iodomelatonin binding sites in the embryonic chicken brain 2 . The early appearance of [^*^1]iodomelatonin binding sites in the embryonic chicken brain 3 • The developmental pattern of [^^I]iodomelatonin binding sites in the perinatal chicken

brain 4 . The development of the diumal rhythm of iodomelatonin binding sites in the chicken

brain 5 . The significance of [^^]icxiomelatonin binding sites in the embryonic chicken brain

D. BINDING CHARACTERISTICS, PHARMACOLOGICAL PROFILE A N D DEVELOPMENT O F [ 1 2 S I ] IODOMELATONIN BINDING SITES IN THE CHICKEN RETINA 8 5

1. Characteristics of [^^1] icxiomelatonin binding sites in the embryo and chicken retina 2 . 丁he development of the [^I]iodomelatonin binding sites in "the chicken retina 3. The diumal variation of [125jjiodomelatonin binding sites in the chicken retina

V. REFERENCES 91

V L FIGURES 106

VII. TABLES 134

L INTRODUCTION

A . PHYLOGENY O F T H E PINEALOCYTES

The pineal organ (pineal gland or epiphysis) of most vertebrates plays a pivotal role in

the conversion of photoperiodic information to a rhythmic hormonal signal which affects major

physiological and behavioural adjustments to daily as well as seasonal fluctuations in the

environment (Cassone, 1990).

Vertebrate pineal systems manifest considerable morphological variation of

organization during the phylogenetic development at both cellular and whole-organ levels, (see

the reviews by Collin, 1971; Oskshe, 1971; Collin and Oksche, 1981; Collin et al., 1987;

Oksche, 1986). Embryologically, the pineal gland and lateral eyes both develop as evagination

of the diencephalon (Flight，1979). In mammals，the pineal gland develops into an indirectly

photosensitive secretory endocrine organ，whereas in lower vertebrates (fish，amphibians and

reptiles), the pineal develops into a photoendocrine transducer containing cellular elements that

are directly photosensitive. The structural and functional transfomation from a well developed

sensory pineal organ in lower vertebrates into the secretory pineal organ of mammals has been

demonstrated in avian pineal photoreceptor cells (Wurtman et al.，1968; Collin et al., 1987;

Pang et al.，1989).

1 . PINEAL PHOTORECEPTOR CELLS IN LOWER VERTEBRATES

The most prominent anatomical feature of pineal systems in lower vertebrates is the

presence of a two-component system consisting of both the highly differentiated eye-like

extracranial photosensory structures generally termed the parapineal component (third eye)，

including the parapineal organ in fish, frontal organ in amphibian and the parietal eye in reptile,

and an intracranial pineal organ proper portion (epiphysis cerebri) (Dodt, 1987; Underwood,

1982). In the fish (e.g. Agnatha) the eye-like parapineal organ is represented by a vesicle

consisting of two "retinas", a dorsal one, and a ventral one. The "retina" consists of an internal

epithelial layer and an external layer of ganglion cells and nerve fibres. In the reptile (e.g.

Anguis fragilis) the "retina" in the parietal eye contains synaptic junctions of photoreceptors

with ganglion cells very similar to the lateral eye synaptic zones. The axons of the ganglion

cells form the pineal nerve. Most of the pinealocytes in pineal organs of lower vertebrates are

also, in ultrastructural and functional terms，similar to the cone-like photoreceptor of the retina.

They are endowed with regularly lamellate outer segments (photoreceptor poles, receptive

poles). The basal processes (synaptic pedicles, transmission poles or effective poles) of the

photoreceptor cells are established by a basal pedicle containing clear synaptic vesicles and

synaptic ribbons. By means of these basal processes, typical pineal photoreceptor cells form

synapses with afferent, second-order neurons (ganglion cells) whose axons extend through the

pineal stalks. This afferent pineal tract (pineal nerve) projects to defined regions of the brain

(Collin and Oksche, 1981).

The functional response of the pineal photoreceptor to light stimulation was first

reported by Dodt and Heerd (1962) using electrophysiological records in the anuran frontal

organ and the lizard parietal eye. During the past 30 years electrical recordings from pineal

photoreceptors in lower vertebrates have demonstrated that the mode of action of pineal and

parietal photoreception resembles that of ocular photoreception in that the onset and offset of

light are followed by inhibitory and excitatory changes in neuronal activity respectively (Dodt,

1987). Dodt (1987)，Meissi (1986) and Underwood (1982) summarized these

electrophysiological studies and concluded that an intracranially located pineal was mainly a

luminance detector which encoded the quantity of light and functioned as an indicator of

daylength and dosimeter of solar radiation, and that an extracmnially located pineal principally

had chromatic (wavelength) response which encoded information about its quality.

Morphologically, the pineal photoreceptor cells of lower vertebrates have also given

ample evidence of secretory activity (Oskche et al.，1987). Thus, pinealocytes of lower

vertebrates can directly transduce the photoperiodic information both into neural impulses and

hormonal signals (Pang et al., 1989). Whether pineal of lower vertebrates have the ability to

generate circadian oscillations at the cellular levels is still an open question (Collin et al.,

1987).

2 . PINEAL PHOTORECEPTOR CELLS IN BIRDS

The avian pineal gland is superficially located beneath the skull. Photoreceptor cells in

the avian pineal gland are modified photoreceptor cells (rudimentary photoreceptor cells).

These cells morphologically resemble both typical sensory cells in lower vertebrates and the

endocrine cells in mammals (Collin and Oksche，1981).

Avian pinealocytes resemble pineal photoreceptors of poikilotherms in their basic

segmental zonation. These cells display a remarkable variety in the appearance of their outer

segments and basal pedicles. The outer segments of avian pinealocytes are less regularly

organized. Although the modified photoreceptor cells are not connected with afferent neurons

they still establish synaptic contacts with intrapineal neurons by "synaptic" ribbons, which

suggest the presence of intercellular communication by these organelles (Collin and Oksche，

1981; Collin et al.，1987). Parallel to these changes in the ultrastructure of the outer segments

there are an abundance of dense-core vesicles elaborated in the Golgi-complex (Collin, 1979;

Oksche, 1983) suggesting the secretory function of the pinealocytes.

Attempts to detect avian pineal electrical responses to direct stimulation with light have

failed (Morita 1966; Dodt 1987), although in some avian pineal parenchyma，the presence of

ganglion cells and a well-developed pineal tract have been demonstrated (See Ueck, 1979).

The possible direct photosensitivity of the bird pineal has been demonstrated by both in

vitro and in vivo studies. In vitro studies of NAT levels in the chicken pineal gland (Binkley et

al.，1978a; Deguchi，1979，Deguchi, 1981; Kasal et al” 1979; Kasal and Perez-polo, 1980;

Takahashi et al., 1980) have shown that the avian pineal is capable of responding to changes

in the L:D stimulus indicating direct photoreceptivity. In vivo，the chicken pineal melatonin

synthesis appears not to depend on an intact sympathetic innervation, as is the case in mammals

(Ralph et al., 1975; Binkley, 1981). It has been speculated that this direct response of chicken

pinealocytes to light is dependent on the presence of rhodopsin (Deguchi, 1981). Thus, the

chicken pineal gland can be considered to be a photoendocrine transducer (Zatz et al.，1988;

Pang et al., 1989).

In recent years, the concept of direct sensori-hormonal transduction in melatonin

secreting cells has been introduced (Pang et al., 1989). The criteria for the direct sensori-

hormonal transduction cells are: 1) It has to be a sensory receptor with characteristics of a

transducer and encoder to the adequate stimulus; 2) It has the ability to secrete a hormone; and

3) The secretory activity of the cell varies with changes in the quantity and/or quality of the

adequate stimulus (Pang et al., 1989). According to the above criteria, photoreceptors in the

retinas of fish and alligator and pinealocytes of the avian pineal gland are suggested to be

examples of sensori-hormonal cells with the adequate stimulus involved being light and the

hormone secreted，melatonin (Pang and Woo, 1987).

There is considerable evidence that the pineal gland of, at least, some birds contains a

circadian pacemaker which plays a major role in the overall temporal organization of the bird.

The control of circadian rhythms in avian locomotor activity by pineal melatonin has been well

established with the house sparrow where pinealectomy abolished and melatonin implantation

induced the rhythmic locomotor activity (Caston and Menaker，1968; Turek et al” 1976). The

presence of an endogenous pacemaker in the bird pineal gland was demonstrated

unambiguously by transplantation experiments (Pang 1974; Zimmerman and Menaker, 1979).

A series of arrhythmic, pinealectomized birds kept in the dark served as hosts for pineal glands

removed from donor birds, which had been kept under two different cycles of illumination.

Glands from the donors were transplanted into the anterior eye chamber of the hosts and the

latter were then returned to constant darkness. The hosts developed circadian rhythms in

locomotor activity with times of onset of activity corresponding closely to those previously

entrained in the donor. Moreover, the phase-response curve of the acquired rhythm of the host

birds when exposed to 6 hour pulses of light was indistinguishable from that of normal birds

(Zimmerman and Menaker, 1979).

The most extensive studies of control of N A T activity in avian pineal gland，both in

vitro and in vivo，have been made with domestic chicken，which suggest that the chick pineal

gland contains an endogenous oscillator (Binkley，1981).

In vivo, the chick pineal gland exhibits a marked diumal rhythm of NAT activity which

persists in constant darkness, is entrained by illumination and manifests a phase shift in

response to a phase-shift stimulus (Binkley, 1981; Binkley et a l , 1981), In vitro, chick pineal

gland explants continued to display daily rhythms in NAT activity under the normal light: dark

cycle or under constant darkness. When the photoperiod was reversed by 180® the shifted

NAT activity rhythm was in line with the new light:dark cycle (Deguchi, 1979; 1981). This

confirmed the photoreceptivity of chicken pineal and suggested that the chick pineal organ

contains an endogenous oscillator which controls the activity of the enzyme (Kasal and Perez-

Polo, 1980). Deguchi (1979), and Zatz et al. (1988) showed that dispersed cell cultures of

chick pineal expressed circadian rhythms of NAT activity and melatonin release as well as the

phase-shifting effect of single light pulses on the melatonin rhythm. These experiments

demonstrate that the dispersed pineal cells are photoreceptive in culture and that the melatonin

rhythm in dispersed pineal cell cultures can be entrained by light cycles in vitro. Thus，the

isolated pineal contains all of the components of a circadian system: a pacemaker or oscillator.

photoreceptive input，and an overt rhythmic output-melatonin. The key question remains: are

circadian oscillators contained in pinealocytes? Using microculture containing only one

pinealocyte，it has been demonstrated that the melatonin rhythm was still expressed (Takahashi

et al., 1989). This suggests that all three functions (photoreception, circadian oscillation, and

melatonin synthesis) might be a cellular property of chicken pinealocytes (Takahashi et a l ,

1989).

PINEALOCYTES IN MAMMALS

The last step in the evolution of pinealocytes is demonstrated in mammals. Mammalian

pinealocytes lack outer segments as well as typical inner segments. They contain dense-core

vesicles that are frequently concentrated in the processes of these cells. Their synaptic ribbons

are located close to the cell membrane and face adjacent pinealocytes (See Oksche et al” 1987;

Collin and Oksche 1981).

Pinealocytes of mammals are considered to be entirely secretory cells that are indirectly

influenced by photoperiodic stimuli (Wurtman et al., 1968). In adult mammals, pineal nerve

cells and their central projections are absent. However，its efferent innervation is well

developed. Light stimuli appear to be exclusively perceived by the retina of the lateral eyes and

conveyed to the pineal organ via a complex neuronal chain involving the retinohypothalamic

pathway (Moore，1987). The retinohypothalamic projection, probably originating in the

ganglion layer of the retina，terminates in the suprachiasmatic nucleus. The neural route by

which stimulatory signals from the suprachiasmatic nucleus reach the pineal gland starts with

efferent projections directed caudalward to the periventricular and ventral tuberal areas of the

hypothalamus, and then via the medial forebrain bundle and the midbrain reticular formation,

to the upper thoracic intermediolateral cell column. Cells in the thoracic intermediolateral cell

column provide preganglionic input to the superior cervical ganglia of the sympathetic nervous

system. Postganglionic sympathetic fibres from the superior cervical ganglion form the nervi

conari reaching the pineal organ with blood vessels (Moore, 1987). Noradrenaline from the

sympathetic fibres cause a receptor-mediated increase in the production of c A M P in

pinealocytes leading to a 70-100-fold increase in the activity of N A T within a few hours (see

Ueck，1979).

In mammals, neither the pacemaker nor photosensitivity reside in the gland itself (Pang

et al., 1989). Present evidence indicates that the pacemaker is in the suprachiasmatic nuclei of

the hypothalamus and the photoreceptors are in the retina. The mammalian pineal gland

produces melatonin in response to the neurotransmitter norepinephrine which is released by the

sympathetic nerves that innervate it. It is thus a "neuroendocrine transducer" driven by the

hypothalamic pacemaker through a multisynaptic neural pathway (Axelrod, 1974; Wurtman et

al•，1968). However，some mammalian pinealocytes undergo a photoreceptor-like

differentiation during a transient neonatal period (Clabough, 1973; Zimmerman and Tso,

1975)，and the presence of proteins in the pineal (Takahash et al., 1989) which are normally

involved in phototransduction in the retina, raises the possibility that direct photic events may

occur in the neonatal mammalian pineal gland This possibility awaits further studies.

MELATONIN RECEPTORS IN BRAIN AND RETINA

Melatonin was isolated and identified by Lemer and coworkers in the late fifties (1958，

1959). During the last three decades, pineal and retinal melatonin has been extensively studied

in vertebrates including fishes, amphibian, reptiles, birds and mammals. (Wiechmann 1986;

Pang 1985). The secretion of melatonin, influenced by the daily and seasonal environmental

light-dark cycle, has been implicated in the control of rhythmic adaptations to daily and

seasonal biological rhythms and neuroendocrine functions (Cassone, 1990). However, the

search for melatonin receptors in brain and retina has had limited success during this period.

1 . THE SPECULATION OF PUTATIVE MELATONIN RECEPTORS IN

BRAIN AND RETINA

1). Pharmacological study - the structure垂activity relationships of melatonin

The existence of putative melatonin receptors has been speculated long before it was

demonstrated by binding studies in recent years. Reward and Hankey (1975), using frog skin

bioassay, demonstrated that the physiological concentration of melatonin (10"^ M) could

lighten the skin of frogs. B y comparing the effects of melatonin and melatonin related

indoles, it was demonstrated that the presence of 汪 methoxy group on the 5th carbon atom was

essential for the biological activity (skin lightening activity) of the active compounds and that

the N-acetyl group on the 3rd carbon atom played an important role in the putative melatonin

receptor binding (Heward and Hankey, 1975). The data indicate that the intrinsic activity of

indole compounds on the melatonin receptor was determined primarily by the moiety

substituted on the 5th carbon atom whereas, the affinity for the binding site is determined

primarily by the moiety substituted on the 3rd carbon atom of the indole nucleus. The structure

and stereospecificity of melatonin in the bioassay strongly suggest the existence of melatonin

receptors in the target organ (Quay, 1986). Dubocovich (1985) demonstrated that melatonin at

picomolar concentrations (40 pmol/I) inhibited the calcium-dependent [3H]dopamine release

elicited by electrical stimulation from the rabbit and chicken retina through activation of a site

possessing the pharmacological and functional characteristics of a receptor. The results of the

structure-activity analysis of melatonin in rabbit and chicken retina correlated well with that of

the frog skin bioassay. These findings suggest that the efficacy of melatonin is determined by

the moiety substituted on carbon 5 (5-methoxy group), whereas the affinity for the receptor is

determined primarily by the moiety substituted on carbon 3 (i.e., ethyl N-acetyl group) of the

indole nucleus (Dubocovich, 1985).

2). Physiological study - localization of melatonin receptors in the brain and retina

It has been postulated that the brain is the target of the pineal hormone, melatonin.

Twenty years ago, Fraschini (1968a,b) observed that the implantation of micro amounts of

melatonin into the median eminence or into the reticular substance of the midbrain significantly

reduced plasma concentrations and pituitary levels of lutenizing hormone (LH) in castrated

male rats. Recently, it was shown that minute amounts of melatonin implanted into the

suprachiasmatic or the rostral tuberal areas of the hypothalamus significantly decreased the

weight and activity of the genital tract of the white-footed mice (Lynch, 1977). Thus, the

presence of melatonin receptors in these areas was implicated.

3). Biochemical study - The uneven distribution of exogenous and endogenous melatonin

in the brain

10

Anton-Tay (1971) and coworkers (Anton-Tay and Wurtman, 1969) demonstrated a

significant accumulation of [^H]melatonin in the hypothalamus and midbrain after intravenous

or intraventricular injection of the labelled compound. It is worth noting that these two brain

areas，with high accumulations of [^Hlmelatonin, correspond with those regions where

melatonin implantation inhibited the castration rise of L H release (Fraschini, 1968a,b). Not

only was the accumulation of exogenous melatonin by brain regions observed. Endogenous

melatonin was also described to have an uneven distribution in different brain areas of many

species (Pang and Brown，1983; Pang et al.，1974). The concentrations of immunoreactive

melatonin in different brain areas in the rat and chicken showed high levels in the

hypothalamus, intermediate levels in the mid-brain, cerebellum and pons-medulla and low

levels in the telencephalon (Pang and Brown, 1983; Pang et al.，1974). The immunoreactive

melatonin in the brain could reflect a local synthesis, or uptake of melatonin from blood or

cerebrospinal fluid (Sallanon et al., 1982). The findings that pinealectomy significaxitly reduced

melatonin in the rat and chicken brain (Pang et al., 1974; 1982) and that no measurable

HIOMT activity could be shown in the human brain (Kopp et al., 1980) favours the former

hypothesis. The above reports strongly suggest that the melatonin might act on some

structures of the central nervous system.

All the physiological, pharmacological and biochemical studies mentioned in this

section strongly suggest the existence of functional melatonin receptors in the brain and retina

2 . IDENTIFICATION O F MELATONIN RECEPTORS IN THE BRAIN

AND RETINA BY RECEPTOR BINDING STUDIES

1). The radioligands: Tritiated and iodinated melatonin in receptor identification

11

The choice of radioligand is of crucial importance in melatonin receptor binding

studies. Ideally, the radioligand should have high affinity for the binding site and exhibit a

high degree of pharmacological selectivity for this site. Specific activity is another important

consideration in the choice of a radioligand. Since a very small amount of radioligand is bound

to the biologically relevant site，only the radioligand with high specific activity can demonstrate

the high affinity and low capacity binding sites in the tissue studied. Another factor to be

considered in the choice of radioligand is its metabolic stability.

The tritium-labelled ligands are practically identical in molecular structure to their

unlabelled counterparts and are therefore pharmacologically indistinguishable. In contrast, the

substitution of a bulky iodine atom into a small molecule may significantly affect the

pharmacological properties of radioiodinated drugs. Thus，in many cases, radioiodination

decreases the affinity of the ligand for the site of interest and renders it less useful as a

radioreceptor probe (Miler，1988). However, in some cases, substitution of an iodine atom can

greatly increase the specific activity and even increase the specificity and affinity of a

radioligand for the site of interest (Miler, 1988). The 2-[-''^I]icxiomelatonin which was

synthesized (Vakkuri et al 1984a，b) with high specific activity (more than 2000 Ci/mmol) and

which retained a high specificity and affinity to the melatonin binding sites (Dubocovich et al.,

1988a) is one example. By studying the effect of melatonin and its analogs in the inhibition of

[^H]dopamine release f rom chicken and rabbit retina，Dubocovich et al. (1988a，b)

demonstrated that both 2-icxiomelatomn and 6,7-dichloro-2-methylmelatonin were more potent

than melatonin. Thus, the agonist activity on the melatonin receptors appears to be enhanced

when the melatonin molecule is substituted at position 2 by an iodine or methyl group.

2). Affinity and density of [3H]melatonin binding sites in the brain and retina.

12

In the late 1970s, several articles appeared which described [^H]melatonin binding sites

in membrane and/or cytosolic preparations from the rat brain (Niles et al., 1979; Vacas and

Cardinali, 1979), bovine brain (Cardinali et al.，1978; 1979) trout retina (Gem et al.，1981)

and frog retina (Weichmmann et al., 1986) (Table 1). These sites had low affinity binding and

did not agree in terms of the tissue distribution or biological effects of melatonin and related

indoles of the presumptive melatonin receptors. In part, the relatively low specific activity of

[^H]melatonin (50-80 Ci/mmol) might have hindered the detection of high-affinity binding

sites with low receptor density in tissues (Cardinali，1981; Dubocovich，1988a).

3). Affinity and density of [^^Ijiodomelatonin binding sites in the brain and retina

In 1984, Vakkuri synthesized 2-[^^I]iodomeIatonin which was used as a tracer in

melatonin radioimmunoassays. In 1987,2-[^^I]Iiodomelatomn was used as a radioligand for

biochemical (Dubocovich and Takahashi, 1987; Niles et al., 1987) as well as autoradiographic

(Vanecek et al., 1987) binding studies of melatonin receptors. 2-[-''^I]Iodomelatonin has

proven to be a selective and high affinity ligand with high specific activity. In vitro, 2-

[l2^I]iodomelatonin has also shown biological activity as it was a potent inhibitor of dopamine

release in the retina (Dubocovich and Takahashi，1987). In addition, 2-iodomelatonin

mimicked the inhibitory action of melatonin on testicular development and body weight gain

(Weaver et al., 1988; Sugden, 1989a). Thus, [125I]icxiomelatonin is a good radioligand for the

characterization and localization of the putative melatonin receptor in the nervous system

(Stankor and Reiter，1990).

3 . MULTIPLE [ 1 2 5I]IODOMELATONIN BINDING SITES IN THE BRAIN

AND RETINA

13

Based on kinetic studies revealing varying affinities and specificities of

[125j]iodomelatomn binding sites, the concept of multiple melatonin receptors, that is, the

high (picomolar) affinity binding sites (ML-1) and low (nanomolar) affinity binding sites (ML-

2) was first proposed by Dubocovich (1988a). Picomolar affinity (ML-1) binding sites for

[^^^1]icxiomelatonin have now been demonstrated in the whole brain tissue of lizard (Rivkees

et al 1989a) chicken (Dubocovich et al.，1989; Dubocovich, 1990; Rivkess et al•，1989b;

Stehle, 1990; Y ing and Niles, 1991; Yuan et al., 1990)，quail (Yuan and Pang, 1990) and

pigeon (Yuan and Pang, 1991). In mammals (rat，hamster, mouse, ovine and human),

picomolar affinity binding sites for [^^I]iodomelatomn have been localized to a relatively few

morphologically and functionally discrete brain areas including the hypothalamic

suprachiasmatic nuclei (SCN) of rat (Laitinen et al.，1989)，mouse (Fang et al.，1990) and

human (Reppert et al., 1988a), as well as the pars tuberalis/median eminence region of sheep

(Morgan et al.，1989)，rat (Vanecek et al., 1987) and hamster (Carlson et al., 1989; Duncan et

ai.，1989; Vanecek and Jansky, 1989). In addition [^^1]iodomelatonin binding has been

reported in the brain of the human fetus (Yuan et al., 1991) and the retina of chicken，rabbit

(Dubocovich, 1988a) and tree shrew (Lu et al., 1991) (Table 2).

The picomolar affinity [•'•^Iliodomelatonin binding sites are physiologically relevant to

the range of melatonin levels that are normally found in blood and brain (see Pang, 1985). The

pharmacological characterization of the high affinity [ iodomelatonin binding sites showed

that melatonin and related indoles inhibited binding with the same order of potency as that

found fo r the inhibition of [^H]dopamine release from chicken and rabbit retinas. The

pharmacological order of affinities is as follows: 2-iodomelatoniii > 6-chloromelatonin >

melatonin > N-acetyltryptamine. The reported antagonists for these binding sites are Luzindole

(N-0774; 2-benzyl-N-acetyltryptamine) (Dubocovich, 1988b) and N- [2,4-dinitrophenyl] -5-

methoxytryptamine (ML-23) (Zisapel and Laudon, 1987). The fact that GTP and GTP

analogues inhibited the high affinity melatonin receptor binding suggests that the melatonin

14

receptor belongs to the superfamily of G-protein linked receptors. However，it should be

noted that recent studies suggest that the high affinity melatonin receptors might exist in two

affinity states (Morgan et al” 1989; Latininen et al.，1990b; Dubocovich et a l , 1990)，a high

affinity form exhibiting an average Kd of about 4 5 pmol/1 and a second state with about a 10-

fold lower affinity (average K d of 400 pmol/1) (Krause and Duvocovich, 1991). The

significance of this remains to be determined.

Nanomolar affinity (ML-2) binding sites of [^^]icxlomelatonin were located in whole

brain tissues of hamster (Duncan et al., 1988; Niles et al.，1987) and rat (Zisapel and Anis,

1988) as well as in discrete brain regions of hamster (Pickering and Niles, 1989; Anis et al.,

1989) and rat (Laudon and Zisapel, 1986; Zisapel et al” 1988) (Table 3). These sites had rates

of association and dissociation higher than those found in high affinity binding sites (Duncan et

al., 1988，1989). The pharmacological profile of [12^I]iodomelatonin binding sites in the

hamster brain homogenate was different from that described for the ML-1 sites. The most

striking difference was the high potencies of N-acetyl-5-hydroxytryptamine and 6 -

methoxymelatonin in competing for [^^IJiodomelatonin binding in hamster brain membranes

(Duncan et al., 1988; 1989). It was reported that the two affinity binding states for

[•'•^Iliodomelatonin are present in the hamster cerebral cortex (Niles et al” 1987). The

curvilinear Scatchard plots yielded high affinity binding sites with Kd 0 3 2 土 0.14 nmol/1,

Bmax 5.6 土 1.7 fmol/mg protein, and low affinity binding sites with K d 10.5 土 3.2 nmol/1,

Bmax 123 土 33 fmol/mg protein. This study also suggests the coexistence of the two receptor

subtypes in the same tissue (Niles et al., 1987).

4. RECEPTOR AGONISTS AND ANTAGONISTS

15

Agonists: The agonists that are most effective in lightening the skin of frogs (Heward

and Hankey, 1975) and mimicking the inhibitory effect of melatonin on dopamine release

(Dubocovich, 1983; 1985) are compounds possessing a 5-methoxy group on carbon 5 of the

indole nucleus and an N-acetyl group in the same position as melatonin (Figure 1). The most

potent agonists tested in both the rabbit and chicken retina (Dubocovich，1985; Dubocovich,

1988a) were 2-iodomelatonin and 2-methyl-6>7-dichloromelatonin. 6»Chloromelatonin was

equipotent with melatonin in inhibiting dopamine release. In contrast, inhibitory potencies were

about 1000-fold less fo r N-acetylserotonin which lacks the 5-methoxy group and 5 -

methoxytryptamine which lacks the N-acetyl group (Dubocovich, 1983; Dubocovich, 1985).

Antagonists: Structure-activity relationship studies of the melatonin receptors on the

frog skin (Heward and Hankey，1975) and chicken retina (Dubocovich, 1985) showed that N-

acetyltryptamines lacking the 5-methoxy group were potential melatonin receptor antagonists

(Dubocovich，1988a). The development of melatonin receptor antagonists contributed greatly

to the study of the biological effects of melatonin. These antagonists, including the synthetic

derivatives of tryptaxnine, have been demonstrated to inhibit various effects of melatonin either

in vitro o r in vivo (Zisapel and Laudon, 1987; Dubocovich, 1988b; Laudon et al., 1988;

Nordio et al.，1989). Dubocovich (1988a，b) showed that one of these compounds, Luzindole,

an N-acetyltryptamine lacking the 5-methoxy group and possessing a 2-benzyl substitution,

was a competitive melatonin receptor antagonist (Dubocovich, 1988b). In vitro，Luzindole (1

mmol/1) completely antagonized the inhibition of the calcium-dependent release of

[^Hjdopamine elicited by melatonin. Luzindole also blocked the activation of melatonin

receptors in vivo，as shown by its reversal of the decrease in norepinephrine turnover in the

C3H/HeN mouse hypothalamus induced by endogenous melatonin or administration of the

melatonin agonist, 6-chloromelatonin (Fang and Dubocovich, 1990). In the Siberian hamster,

melatonin-induced inhibition of testicular weight and body weight could be antagonized by

luzindole. (Duncan et al., 1990). Further evidence for selectivity of Luzindole was provided

16

by binding studies, in which Luzindole did not affect the binding of specific radioligand to 04

and « 2 adrenergic, p j and P2 adrenergic, D j and D2 dopaminergic, 5-HT-l and 5-HT-2

serotonergic, muscarinic, adenosine-1 or bezodiazepine receptors (Dubocovich, 1988b). It was

therefore concluded that Luzindole was a novel competitive melatonin receptor antagonist with

high potency and selectivity that provides a new experimental tool to further investigate the

functional role of endogenous melatonin in mammals (Duvocovich, 1988b). Zisapel and

Laudon (1987) reported that ML-23 was also able to antagonize the inhibitory effects of

melatonin on [^Hjdopamine release in vitro and to prevent the melatonin mediated retardation

of postnatal development in immature male rats in vivo (Zisapel and Laudon, 1987). Prazosin,

an a^-adrenergic antagonist, was another potent inhibitor of binding in hamster cerebral cortex

membrane, which suggested that prazosin might be a potent antagonist at a unique binding site

for melatonin (Niles et al 1987). Structures of the melatonin receptor agonists and antagonists

are summarized in Figure 1.

5 . RECEPTOR REGULATION

Melatonin secretion in mammals has a very pronounced circadian rhythmicity which

has been observed in all species examined. The secretory pattern of melatonin changes with the

sex, age, and different environmental conditions (Pang, 1985). These changes are important

for the adaptation of neuroendocrine and behavioural functions. On the other hand, changes in

the affinity and/or density of melatonin receptors could also be crucial for these functions.

Recent studies have shown light-dark variations in I]iodomelatonin or

[3H]melatomn binding sites in the whole brain of rats (Vacas and Cardinali, 1979), hamster

(Vacas and Cardinali, 1979), quail (Yuan and Pang, 1990), chicken (Yuan et al” 1990) and in

discrete brain areas of rats (Zisapel et al” 1988; Laudon et al” 1988; Laitinen et al” 1989) and

17

hamsters (Anis et al.，1989). It was suggested that such changes in the density of binding sites

might be the result of receptor down regulation by melatonin (Pang et a l , 1990). Conversely,

the diumal rhythmicity of the [^^]icxiomelatonin binding sites might be endogenous in nature

(Stankov and Reiter，1990).

The circadian rhythmicities of [^^I]iodomelatonin binding sites in the various animal

brains reported was consistent with the diumal rhythms of biological responses to melatonin in

the hamster (Tamarkin et al., 1976), white-footed mouse (Glass and Luncy, 1982), rat (Reiter

et al.，1980)，and sheep (Karsch et al•，1984). Thus, it was suggested that the changed

sensitivity to melatonin might be related to alterations in the response of the melatonin

receptors (Reiter et a l , 1980).

Sex hormones are another important factor affecting melatonin receptors in the central

nervous system (CNS). In long-term ovariectomized (OVX) female rats, the in vitro inhibitory

effect of melatonin on dopamine release from the hypothalamus was abolished. After

implantation of estradiol capsules to O V X rats，the ability of melatonin to inhibit dopamine

release from the hypothalamus was reinstated. These estradiol-reversible changes in the ability

of melatonin to inhibit hypothalamic dopamine release were accompanied by estradiol-regulated

changes in melatonin receptors in the hypothalamus. In OVX rats, the density of

[12^I]iodomelatonin binding sites in hypothalamus was markedly reduced (Zisapel et al.，

1987). This finding suggests that the melatonin receptor could be regulated not only by its

homologous ligand-melatomn，but also by a heterologous ligand-estradiol. It was tempting to

speculate that a reciprocal interaction exists between estradiol and melatonin and their receptors

in the brain (Zisapel et al” 1987).

The biological significance of the ontogenesis of the melatonin receptor has not been

studied systematically. Martin and Sattler (1979) studied the effects of melatonin on the

pituitary L H and FSH responses to LHRH in culture with pituitary cells from female rats 5，

10，15, 20 and 3 0 days of age and from adult male and female rats. The results showed that

18

melatonin significantly inhibited LHRH-induced L H and FSH release by pituitary cells from S

，10- and 15-day-old animals. By contrast, pituitary cells from 20- and 30-day-old animals

showed no detectable response to melatonin even at micromolar concentrations. These findings

revealed that the inhibitory effect of melatonin on the pituitary response to L H R H was lost

during development in the rat between 15-20 days of age (Martin and Sattler, 1979). Vanecek

(1988a) reported that the specific binding of [^^Ijiodomelatonin in the pituitaries was highest

in 20-day-old fetuses (Bmax 31 fmol/mg protein) and it gradually decreased in the course of

postnatal development, until it reached 10% of that value in 29-day-old males. In contrast, the

density of melatonin receptors in the median eminence did not change markedly in the course of

development The marked decrease in the density of melatonin receptors in the pituitary might

account for the reported developmental loss of the melatonin inhibitory effect on LHRH-

induced L H release from the anterior pituitary (Vanecek, 1988a). Unlike the rat pituitary,

Carlson et al. (1991) demonstrated that the sites of high affinity [^^Ijiodomelatonin binding in

Siberian hamster brain was generally similar throughout the perinatal period. GTP inhibited

[•'"^Iliodomelatomn binding at each age suggesting that the melatonin receptor was associated

with the G protein during development (Carlson et al•，1991).

Furthermore, both the circulating melatonin levels and the melatonin receptor in the

brain were modified by aging. The pineal and circulating melatonin are reduced in aged animals

(Pang and Tang, 1983). Zisapel studied the distribution of melatonin receptors in 6 discrete

brain areas of mature and aged male rats. In the mature animals, the density of nanomolar

affinity binding sites in the hypothalamus, medulla, pons and hippocampus exhibited a clear

diurnal rhythm with a peak in the late photophase. In the aged rats，the density of these

melatonin binding sites in the hypothalamus was very similar throughout the 24h period. In

addition, the maximal binding capacities in the parietal cortex，hippocampus and medulla-pons

areas of aged rats were significantly lower than those in the mature rats (Zisapel et al.，1989).

19

T H E MECHANISM OF MELATONIN RECEPTOR ACTIONS

Hormones fall into two general chemical classes: lipid soluble hormones (steroids and

thyroxin) and water soluble hormones (catecholamines, peptides, and protein hormones). The

receptor localization and mechanism of action of hormones can also be categorized into two

classes. The l ipid soluble hormones act on intracellular receptors and change the gene

transcription and protein translation. 丁he receptors for water soluble homones are found in or

on the cell surface membrane. Three receptor-effector coupling systems have been

demonstrated for water soluble hormones. One class, typified by the nicotinic cholinergic

receptor, functions as a ligand-gated ion channel. Binding of agonist ligands alters

transmembrane ion flux by opening the channel. A second class of cell surface receptor

combines the signal detection function with the effector function in one macromolecule

(tyrosine kinase activity). The third type of cell surface receptor is coupled to a distinct effector

entity by a G-protein. Receptors in this category are activated by a diverse array of first

messengers including neurotransmitters, prostaglandins, polypeptides and photons of light

(Benidge, 1985),

Melatonin is a small and highly lipophilic molecule which easily passes through the cell

membrane. It may act on intracellular receptors. However, most studies demonstrate the

highly specific binding of [125I]iodomelatomn on crude membrane preparations (see review of

Stankov and Reiter, 1990; Pang et al, 1990). This suggests that the melatonin receptors may

have the same mechanism of action as water soluble hormone receptors. In addition,

accumulating experiments suggest that melatonin receptors are G protein linked receptors (See

Krause and Dubocovich，1991; Morgan, 1991).

1). Guanine nucleotide-binding proteins (G protein) related signal transduction systems.

20

G proteins function as intermediaries in transmembrane signalling pathways from the

receptor on the outer cell surface to the effectors (second-messenger systems) within cells

(Dohlman et al., 1987). Receptors that participate in such reactions are receptors of biogenic

amines, proteins, polypeptide hormones as well as neurotransmitters. The adenylyl cyclase

(AC) is the best known molecule that is controlled by G proteins. The regulation of

phosphoinositide phosphodiesterase (phospholipase C) activity and the function of ion

channels by G proteins are also strongly suspected (Oilman, 1987). Members of the G protein

family include Gs, Gi , Gt，Go and Gk which share certain common functional and structural

features. All function as receptor-effector couplers; bind guanine nucleotides with high affinity

and specificity; possess intrinsic GTPase activity; are substrates for covalent modification by

bacterial toxins; and share a common heterotrimeric structure (Spiegel et al., 1988).

Criteria for G protein action in signal transduction.

Several criteria have been employed to determine whether a G protein participates in

the transduction process: (a) GTP and hydrolysis-resistant analogues of GTP weaken the

binding of hormones to receptors coupled to G proteins; (b) Binding of GTP analogues and

GTP hydrolysis are stimulated by the hormone; (c) The activity of the putative effector is

altered upon the addition of hormone and GTP; (d) The effect of the hormone is altered by

cholera toxin or pertussis toxin; (e). The effects of the hormone are mimicked by the

introduction of GTPyS ； and (f) Functional reconstitution provides the most direct evidence for

the participation of a G protein in a transduction process (Stryer，1986).

Functions regulated by G proteins.

G proteins are a family of signal-coupling proteins that play key roles in many

hormonal and sensory transduction processes including: 1) Activation or inhibition of adenylyl

cyclase (AC): A C is reciprocally controlled by a stimulatory G protein (Gs) stimulating the AC

21

activity and an inhibitory G protein (Gi) inhibiting the AC activity in the regulation of cAMP

production (Stryer，1986) ； 2) Control of the phosphoinositide cascade: The hydrolysis of

phosphatidylinositol 4,5-bisphosphate (PIP2) by a specific membrane-bound

phosphodiesterase (phospholipase C，polyphosphoinositide phosphodiesterase) generates two

intracellular signal molecules, diacylglycerol and inositol 1,4,5-trisphosphate (IPS).

Diacylglycerol activates protein kinase C, whereas IP3 triggers the release of intracellular

calcium into the cytosol. G proteins control calcium-sensitive processes such as secretion and

chemotaxis (Berridge and Irvine, 1984); 3). Gating of potassium channels: Potassium

channels in cardiac pacemaker cells are opened by a G protein that is activated by the

muscarinic acetylcholine receptor. The change in K+ permeability slows the firing rate of

pacemaker cells. (Pfaffinger et al., 1985).

2). Melatonin receptors and G protein related signal transduction system

Radioreceptor binding studies have shown that the putative melatonin receptors reside

in the membranes (Stankov and Reiter, 1990) of lizard (Rivkees et al” 1989a) and chick brain

(Ying and Niles, 1991)，rat suprachiasmatic nuclei (Laitinen and Saavedra, 1990b), area

postrema (Laitinen et al” 1990), ovine pars tuberalis (Morgan et al., 1989), chicken retina

(Laitinen and Saavedra, 1990a) hamster brain (Niles et al” 1988) and hamster median

eminence/pars tuberalis (ME/PT) (Carlson et al., 1989). Treatment of crude membranes with

the nonhydrolyzable GTP analogue guanosine 5'-[y-thio]triphosphate (GTPyS), significantly

reduced the number of high-affinity receptors and/or increased the dissociation rate of

[^^1]iodomelatonin from its receptor. Thus, coupling of the melatonin receptor to G-proteins

across the species might be a rule rather than an exception.

22

In ovine pars tuberalis, dose-dependent inhibition of [ 125I]iodomelatonin binding by

GTP and hydrolysis-resistant analogues of GTP was observed, with an order of potency of

GTPys > GTP =GDP. GMP and ATP had negligible effects (Morgan et al” 1989). Analysis of

saturable binding revealed that GTPyS (1 |imol/l) promoted an apparent reduction in receptor

density of about 50%, without a concomitant change in receptor affinity. These results were

consistent with a melatonin receptor existing in equilibrium between high- and low-affinity

states, with GTP and related analogues able to cause a shift in the equilibrium in favour of the

lower-affinity form. The sensitivity of [^^1]iodomelatonin binding to guanine nucleotide

implied the action of the melatonin receptor on the ovine pars tuberalis was mediated via a G

protein (Morgan et al., 1989).

In the lizard brain (Rivkees et al•，1989a) GTPyS treatment led to a rapid dissociation

of iodomelatonin from solubilized ligand-receptor complexes. Gel filtration

chromatography of solubilized ligand-receptor complexes revealed two major peaks of

radioactivity corresponding to molecular weights of > 400 and 110 kDa. This elution profile

was markedly altered by pretreatment with GTPyS before solubilization as only the 110 kDa

peak was present in GTPYS-pretreated membranes. The results suggested that the

[^^Ijiodomelatonin binding sites in lizard brain were melatonin receptors, with agonist-

promoted G protein coupling and that the apparent molecular size of receptors uncoupled from

G proteins was about 110,000 kDa (Rivkees et al 1989b) • The size estimate of the free

melatonin receptor (110,000 kDa) compares favourably with size estimates for the well-

characterized G protein-coupled receptors (e.g. a-adrenergic and muscarinic acetylcholine) by

gel filtration (Caron and Lefkowitz 1976).

Laitinen et al.(1990b) demonstrated that the GTP-induced uncoupling of the melatonin

receptor from the G-protein was temperature dependent. Agonist binding decreased in the

presence of guanine nucleotide at 22° C. Similarly, decreased 2- [ 1 2 5I] iodomelatonin binding

at 250C as well as an increased dissociation rate for bound agonist was found in lizard brain

23

membranes in the presence of GTPyS (Rivkees et al.，1989b). On the contrary，studies

conducted at 0®C failed to show any effects of GTP on 2- [^^1]iodomelatonin binding in

chick and hamster brain or in chick retinal membranes (Dubocovich and Takahachi, 1987，

Dubocovich et al.，1989. Duncan et al” 1989). Thus, the uncoupling of the melatonin-G

protein complex was more readily detected at 22®C than at 0®C (Laitinen and Saavedra 1990b;

Laitinen et al” 1990). The negative heterotropic effect of guanine nucleotide on [ •'•^Iliodomelatonin binding

does not provide enough insight to determine the nature of the G protein transduction

mechanism linked to the melatonin receptor. However, one of the fundamental differences

which has been described for the regulation of the receptor by a stimulatory (Gs) and an

inhibitory (Gi) G protein is that GTP and GDP are equipotent in promoting the high- to low-

affinity shift for a receptor regulated by Gi, whereas GDP is more potent than GTP for a Gs-

regulated receptor (Bimbaumer et aL, 1985). If this generalization can be extrapolated to the

melatonin receptor then the equipotence of GTP and GDP at inhibiting [^%]iodomelatomn

binding implies that the melatonin receptor is linked to its second messenger system through a

Gi protein (Morgan et al 1989). This hypothesis was supported by experiments showing that

melatonin decreased cyclic AMP and increased cyclic GMP synthesis in rat hypothalamus in

vitro (Vacas et al” 1981). Vanecek and Vollrath (1989) reported that melatonin specifically

inhibited the cyclic AMP accumulation in the rat pituitary. Moreover, melatonin impaired the

p-adrenoceptor-induced activation of cAMP synthesis in rat astroglial cell cultures (Vacas et

al., 1987). In amphibian skin, melatonin reversed the melanocyte stimulating hormone induced

dispersion of melanosomes by preventing the formation of cAMP (Subgen, 1989b). In a

Xenopus dermal melanophore bioassay, melatonin reversed the pigment dispersion-induced by

forskolin, which was known to increase cAMP formation by activating adenylate cyclase. In

the latter preparation, melatonin appeared to cause pigment aggregation via activation of a

receptor negatively coupled to adenylate cyclase because pertussis toxin blocked the melatonin-

24

induced aggregation (White et al.，1987). Melatonin has been reported to decrease forskolin

stimulated cAMP accumulation in hamster median eminence/pars tuberalis explants (Carlson et

al., 1989). All together, the available evidence suggests that melatonin might exert its effect by

decreasing the formation of cAMP. The described inhibitory effect of melatonin on cyclic

nucleotides might be the key to understanding the mechanism of melatonin action on the

cellular level

It should also be noted that melatonin caused a dose-dependent relaxation of

precontracted vascular smooth muscle. However, pretreatment of vascular rings with low-

dose lithium sulfate (0.1 mmol/1) completely blocked the relaxation response to melatonin

suggesting that the inositol phosphate pathway might be involved in the relaxation effect of

melatonin (Weekley, 1991).

25

IL MATERIALS AND METHODS

A. MATERIALS

1. CHEMICALS

1). CHEMICALS FOR [ 125I]IODOMELATONIN BINDING ASSAY

Melatonin，N-acetylserotomn，3-acetylindole, l-acetylindole-3-carboxaldehyde,

acetylcholine chloride, harmaline, 5-hydroxyindole各acetic acid，5-hydroxytryptamine, 6-

hydroxymelatonin, 5-methoxytryptophol, 5-methoxytryptamine, S-methoxytrypothan,

tryptamine, norepinephrine, bovine albumin, Guanosine 5'- [r-thio]triphosphate (GTPyS) were

purchased from Sigma (St. Louis，MO); 6-chloromelatonin from Eli-Lily (Indianapolis, IN)；

Na[12^I] (3-7 GBq/ml，100 mCi/ml in NaOH solution with pH 7-11) from Amersham

(Buckinghamshire, England), [^%]iodomelatonin from Amersham and NEN (Mississauga,

Canada) with specific activity 2200 Ci/mmol. Iodogen (1,374y6-tetrachloro-3a, 6a-

diphenylglycoiluril) from Pierce Chemical Co” (Rockford, II” USA); and silica gel sheets

from Brinkmann Instruments (Palo Alto, CA). A l l other chemicals were from commercial

sources.

2) CHEMICALS FOR MELATONIN RADIOIMMUNOASSAY

H]melatonin were purchased from Amersham, (Buckinghamshire, England).

Melatonin antiserum was kindly donated by CIDtech Research Inc., (Hamilton, Canada).

ANIMALS

26

In these studies the brain tissues were obtained from male and female birds of different

species including chicken (4-12 weeks old and specified age as described in developmental

studies), pigeon (mature), duck (mature) and quail (mature). The development of

[125l]iodomelatonm binding sites in chicken brains was studied using different ages of

chickens from 6-day-old embryo to 1 year-old-chicken, (embryo: 6-day-old (E 6)，10-day-old

(E10)，13-day-old (E 13)，17-day-old (E 17), 18-day-old (E 18); newly hatched chick: 1-day-

old (H 1)，7-day-old (H 7); young chick: 20-day-old (H 20)，40-day-old (H 40); and adult

chicken: 6-month-old (M 6)，and 12-month-old (M 12), Retinas were obtained from chickens

of 4-8 weeks old and specified age as described in development studies. The development of

[^^I]iodomelatonin binding sites of the chicken retinas were studied using different ages of

embryo (10-day-old (E 10); 15- day-old (E 15); 18-day-old (E 18) and 2p-day-old (E 20); and

young chick of 21 day old (H21).

Chickens: The hybrid strain of "mini WM White Leghorn chickens {Gallus domesticuc)

both males and females of different ages were supplied by the Laboratory Animal Unit，

University of Hong Kong. They were housed three to five per cage in a temperature controlled

animal room (23 土 0.50 C) and fed chick feed twice a day. The lighting schedule was L:D

12h:12h. (lights on 0300h and off 1500h), unless otherwise stated. A l l animals were adapted

to the photoperiodic cycle of 12 h light and 12 h dark (light on 0300) for at least two weeks

prior to any experimental procedures, unless otherwise stated. Lighting was controlled

automatically. The intensity of illumination was approximately 200 Lux/cm at the centre of the

room during the light period. The middle of the light period (ML) was 0900 h and the middle

of the dark period (MD) was 2100 h.

Eggs of the hybrid strain of "mini W,r White Leghorn chickens {Gallus domesticuc)

were incubated at 38 ± J ) ^ C under a lighting regimen of 12 h light: 12 h dark with a light

intensity of 200 Lux/cm2 (lights on (B:(X)h and off 15:00h). The light source was a fluorescent

tube located inside the thermostatically controlled incubator with an automatic egg turner. After

27

hatching, the chicks remained in the incubator for 3-16 h and were then moved to a heated

brooder and maintained under the same light-dark cycle until they were two weeks old. During

this period the temperature decreased from 30oC to 230C gradually. They were then

transferred to a temperature controlled animal room (23 土 0.5oC).

Mature male and female quails (Halics sp) were purchased from a local market in Hong

Kong. They were housed 8 to 10 per cage and fed twice a day in a temperature controlled room

(23 土 O . f C). The lighting schedule was the same as that for the chickens, and the M L and

MD were 0900h and 21(X)h respectively.

Mature male and female Ducks (Anas Platyrhynchos) and pigeons {Columbia livid)

obtained from the market in Hong Kong were kept under a natural photoperiod (sunrise at

0700h and sunset at 17.30h). Water and food were available ad libitum. The middle of the

light period (ML) was 12.00-13.00h and the middle of the dark period (MD) was 2400-

OlOOh.

B. METHODS

L SAMPLE COLLECTION

The brains and/or retinas of birds were collected immediately after the animals were

decapitated. To obtain the brain, the skull was dissected by cutting bilaterally on the left and

right side of the skull through the interparietal bones, parietal bones and half way through the

frontal bones. Gripping the occipital bone above the foramen magnum with a rongeur，the cut

part could be lifted up easily to expose the brain, which was removed by a pair of hooked-end

forceps into a plastic vial and frozen, on dry ice prior to storage at -70oC. For the regional

28

distribution study of bird brain [ I]icxiomelatomn binding sites，the brains were dissected on

ice and divided into five regions: telencephalon, cerebellum, mid-brain, hypothalamus and

pons-medulla (Pang et al., 1974). To ensure a detectable number of melatonin-binding sites，

three to five pieces of the same region of brain tissue were pooled together in each of the five

samples. The samples were placed in plastic vials, frozen on dry ice and then stored at -7(PC

until assayed. Chickens were decapitated and the eyes were removed and hemidissected immediately

on ice. The retinas, including the pigment epithelial layer free from choroid，optic nerve and

ora serrata, were frozen on dry ice and stored at -70oC.

2 . TISSUE PREPARATION

1). REAGENT PREPARATION

Tris-HCL buffer (0.05 mol/1，with pH 7.4): Tris (M.W. 121.14) 12.114 g and 840 ml

of 0.1 N HC1 were added to 1 litre of distilled water. The pH was adjusted to 7.4 at room

temperature and made up to a total volume of 2 litres with distilled water.

Sucrose solution of 0 3 2 mol/1 (M,W. 342.2) was made by dissolving 54.75 g of

sucrose in 400 ml of 0.05 mol/1 Tris-HCl buffer ( pH 7.4) and made up to a total volume of

500 ml.

2). CRUDE BRAIN MEMBRANE PREPARATION

Brain: Fresh or thawed brain tissues of birds (chicken，pigeon，duck, quail) were

homogenized in 10 volumes (W/V) of ice cold Tris-HCL buffer (0.05 mol/1, pH 7.4) using a

glass homogenizer. The homogenate was centrifuged at 44,000g for 25 min at 40C. The

29

pellet was washed once by resuspension in the buffer and centrifuged a second time. The

crude membrane pellet was resuspended in Tris-HCl buffer (pH 7.4). Protein content was

determined (Lowry et al，1951) using bovine serum albumin as standard.

Retina: To prepare the crude retina membrane preparations, the retinal tissues were

homogenized in 10 volumes (W/V) of ice cold Tris-HCL buffer (0.05 mol/1, pH 7.4) using

a Kinematic Polytron (Lucerne, Switzerland) 3 x 5 second pulses at #6 setting. The samples

were centrifuged at 44,000 X g for 25 minutes at 4 0C and the supernatant decanted. The

pellet was then resuspended and recentrifuged. Prior to the assays，the crude membrane

pellets were resuspended in Tris-HCl buffer (pH 7.4, 40C) and homogenized at Polytron

setting #6 for 2 X 5 seconds. The protein content of the sample was determined as noted

earlier.

To study the intracellular distribution of melatonin binding，brain tissues of birds

(chicken, pigeon, quail) were homogenized in Tris-HCl buffer (pH 7.4, 40C) containing

032 mol/1 sucrose. The resulting homogenates were fractionated in refrigerated centrifuges

to yield a crude nuclear pellet (l，000g for 10 min), a crude mitochondrial pellet (27，000g for

10 min), a crude microsomal pellet (100，000g for 60 min), and a cytosol supernatant

(100，000g for 60 min) (Duncan et al., 1988). The pellets were resuspended in 2-8 volumes

of Tris-HCl buffer. The protein content of membrane and cytosol preparations were

determined as previously stated.

3. BINDING ASSAY

1). IODINATION OF MELATONIN

a). REAGENT PREPARATION

30

Phosphate Buffer (0.1 mol/1, pH 6.0): 44.0 ml of 0.2 mol/1 NaH2P04 and 6.2 ml

of 0.2 mol/1 Na2HP〇4 were mixed together and made up to 100 ml with distilled water.

The pH was adjusted to 6.0.

Melatonin (0.1 % W/V): 10 mg melatonin was dissolved in 0.5 ml absolute ethanol

and then made up to 10 ml in 0.1 mol/1 of Phosphate Buffer (pH 6.0).

Iodogen (0.01% W/V): 2 mg iodogen was dissolved in 20 ml chloroform. 20 yd

of this mixture was added into a 6 X 50 mm glass tube and allowed to evaporate under

nitrogen gas. It was then stored at 4 0C •

Iodine: 400 iCi Na1 2 5I (3-7 GBq/ml, 100 mCi/ml，in NaOH solution, pH 7-11) in

2-3 yil was diluted by 20 \i\ of 0.1 mol/1 Phosphate buffer (pH 6.0) in an Eppendorf tube.

Double distilled water，5% propanol in double distilled water and propanol were

prepared for use in the HPLC by filtering each through a 0.2 pore size nylon filter

paper by vacuum filtration • They were then degassed by blowing helium through each

liquid for 15 minutes.

b). RADIOIODINATION OF MELATONIN

[^^I]Iodomelatonin was prepared by modifing the method of Vakkuri et al. (1984a).

Iodogen (2 jxg in 20 yd of chloroform) in a 6 x 50 mm glass tube was dried under nitrogen.

Melatonin (20 \ig in 20 \il of 0.1 mol/1 Phosphate buffer, pH 6.0) was first added into the

iodogen tube (containing 2 iig of iodogen) and Na[^^I] (400 ixCi in 20 \i\ 0.1 mmol/1

Phosphate buffer, pH 6.0) was added immediately afterwards. The reaction was allowed to

proceed while vortexiiig for 1 min at room temperature and was then extracted twice in

chloroform (100 yd), and purified by TLC or HPLC.

c). PURIFICATION OF [1 2 5I]IODOMELATONIN BY THIN LAYER

CHROMOTOGRAPHY

31

Following extraction, the chloroform phase was subjected to TLC using silica gel

sheet (20x20) cm with fluorescent indicator and run for 3-4 hours to 18 cm distance using

30 ml of ethyl acetate as the solvent. Following TLC, the sheet was sectioned into 1 cm

segments and eluted with 10 ml of 2-propanol. The eluted sections were then counted in a

gamma counter to determine their radioactivity levels. To validate the separation of

[^5i]iodomelatonin from the melatonin, 10 ng of melatonin was run on the same TLC plate

and monitored by UV light. The Rf values of [^^I]icdomelatonin and melatonin were

calculated.

d). PURIFICATION OF [125I]IODOMELATONIN BY HPLC

The iodination product was dried under nitrogen and dissolved in 100 jxl of

methanoLThe mixture was injected into a HPLC (Bio-Rad 700 HPLC System) equipped

with a reverse-phase column (Bio-Rad Laboratories 250 x 4 mm Bio-Sil ODR-5S) and eluted

with a 2-propanol gradient from 5 to 45% during 90 min at a flow rate of 0.5 ml/min.

Elution of melatonin was monitored by a UV spectrophotometer at a wavelength of 220 nm.

Under these conditions, the retention times for melatonin and [•'•^Iliodomelatonin were

approximately 10 and 29 minutes respectively. Radioactivity levels of the fractions collected

were then counted on a gamma counter. To improve the syntheses, iodogen in amounts of 1-

20 jig was also used with different exposure times (1-10 min). The radioligand had a

specific activity of about 2200 Ci/mmol which remained stable for at least 60 days. The

iodination efficiency was 45-66%.

2). SATURATION STUDY.

Saturation binding assays were carried out by incubating 100 \x\ aliquots of the crude

brain or retina membrane preparations (protein contents were about 300-600 jig for the bird

brain membrane preparations and 150-300 pig for the chicken retina membrane preparations)

32

with 50 [xl of [125i]i0domelatonin (specific activity : 2200 Ci/mmol) in concentrations

ranging from 5-320 pmol/1 for the brain binding studies and 15-650 pmol/1 for the retina

binding studies. Non-specific binding was determined by adding 100 yd melatonin to

produce a final concentration of melatonin in the assay tube of either 1 or 10 imol/l. The

total assay volume was made up to 350 \x\ with Tris-HCl buffer. The binding of

[l^Ijiodomelatonin was measured routinely in duplicates or triplicates. The incubation

temperature was at 4 0C for 5 h or 370C for 1 h for the bird brain preparations and 370C for

1 h for the chicken retina preparations. To study the effect of GTP analogues on the

[^^I]iodomelatonin binding, the incubation was conducted at 250C for 1 h. Binding assays

were terminated by the addition of 3 ml of cold Tris-HCl buffer. The samples were then

immediately vacuum filtered through Whatman GF/B glass fibre filters (pore size 1.0 pm)

using a M24R Cell Harvester (Biomedical Research and Development Laboratories, Inc.,

Gaithersburg, Maryland) and washed with 2 X 3 ml aliquots of cold buffer. The filtration

was completed within 5 seconds. The filters containing bound [^^I]iodomelatonin were

measured for radioactivity by a LKB 1270 Bachgamma Gamma Counter (Turku, Finland)

with an efficiency of 70%.

3). KINETIC STUDY

Kinetic studies involved incubating 100 \il of either brain or retinal crude membrane

preparations with 10-30 pmol/1 or 40-60 pmol/1 of radioligand respectively. Non-specific

binding was determined by adding 100 yd melatonin to produce a final concentration of

melatonin in the assay tube of either 1 or 10 fxmol/1. The time course of [125I]iodomelatonin

binding association to the membrane preparations was conducted at designated times (5, 10，

15，20，30, 40, 60, 80, 210 minutes at incubation or 5， 10，30 minutes and 1，2，4，6，

8，10，12,14 h at 4 0 C incubation). Dissociation was initiated by the addition of 10 35 or

33

350 limol/l melatonin (to make the melatonin final concentration of 1 or 10 \xmolll) after a 60

minute incubation period at or 5 h incubation period at 40C.

4). SPECIHCITY STUDY

To determine the specificity of the [ 12^I]iodomelatonin binding sites, 100 (xl

membrane preparations were incubated with radioligand concentrations of 10-30 pmol/1 for

the chicken brain membrane preparations and 40-60 pmol/1 for the chicken retina membrane

preparations with or without varying concentrations of different indole analogues of

melatonin and neurotransmitters, including melatonin, 6-chloromelatonin and N-acetyl-5-

hydroxytiyptamine. The concentrations of these indole analogues ranged from 10" mol/1 to

10,13 mol/L IC5Q values were obtained from the analysis of the competition curves and the

inhibition constant (Ki) was calculated

5). CYTOSOL BINDING ASSAY

Bird brain cytosol fractions were used for the subcellular distribution study with the

same procedure as the membrane binding assay except for the separation of bound (B) and

free (F) radioligand. After incubation of 100 JA! of cytosol preparation (with protein 200-300

(xg), the binding assays were terminated by the addition of 3 ml of cold Tris-HCl buffer and

immediate vacuum filtration through HA filters (pore size 0.45 (xm. Micro Filtration

Systems，Dublin, CA). The filters were washed with 3 X 2 ml cold buffer. The filters

containing bound [ i o d o m e l a t o n i n were determined for radioactivity by a gamma

counter.

6). DATA ANALYSIS OF BINDING STUDIES

a). SCATCHARD ANALYSIS

34

The raw data from saturation studies was evaluated by Scatchard analysis

(Scatchard, 1949). Conventional linear regression techniques was used to calculate the line

of best fit which was computer generated. The equilibrium dissociation constant (Kd) was

estimated as the negative reciprocal of the slope of the line of best fit，and the apparent

maximum number of binding sites (Bmax) was estimated by the abscissa intercept. The

main calculation steps were as follows:

Specific binding (cpm) (SB) = total binding (cpm) (TB) - nonspecific binding (cpm)

(NSB)

The calculation of the bound (B), B (fmol/mg. protein)

CPM of SB X 1000

0.7 X 2220 X 2200 X Protein (mg)

The calculation of the free (F)，F (pmol/1):

CPM of total count X 1000

0.7 X 2220 X 2200 X 0.35

Where 70% was the efficiency of the y-counter; 2220 (dpm) was the decay constant

of 1 nCi radioactivity; 2200 was the specific activity of Ipmol [125I]iodomelatonin = 2200

nCi; 0.35 was the incubation volume (ml).

b). DETERMINATION OF HILL COEFFICIENT

The saturation data was used to fit into the Hill equation using logarithmic form:

LOG10(B/Bmax-B)=LOG10[L].

Where the Bmax (fmol/mg protein) was obtained from the Scatchard analysis; [L]

was the concentration of ligand; B was the bound (fmol/mg protein) under different

35

concentration of [125I]iodomelatonin. Conventional linear regression techniques were used

to calculate the slope of the line - the Hill coefficient

c). KINETIC STUDY

Data from kinetic experiments were analyzed using pseudo-first-order equations to

estimate the association rate constant (Kj) and first-order equation to estimate the

dissociation rate constant (Kq). The Kd value was calculated from the ratio of K

(Weiland and Molinoff, 1981).

丁he i q and was calculated by the following formula.

K i = In (LR)e/[(LR)e-(LR)a] = [K^t (L) (R)T] /(LR)e

K ^ I n ( L R h / a J ^ K h t

Where (L)x： The total concentration of ligand. (R)T; 丁he total concentration of

binding sites which was determined from the Bmax value of Scatchard analysis. (LR)e:

The concentration of the ligand-receptor complex at equilibrium. (LR)a: The concentration

of the ligand-receptor complex at time L (LR)0： The concentration of the ligand-receptor

complex just prior to the addition of the competing ligand. (LR)d: The concentration of the

complex at time t after initiation of dissociation.

d). SPECIHCITY STUDY

The inhibition constant (Ki) was calculated from the IC50 values as follows (Leslie, 1987):

1C50

1+ [L]/Kd

Where (L): The concentration of ligand. Kd: The equilibrium dissociation constant

determined from Scachard analysis.

4. PROTEIN DETERMINATION

1). REAGENT PREPARATION

Reagent 1: Mix 4% Na2C03 (in distilled water) and 0.2 N NaOH (in distilled

water) in equal volumes.

Reagent 2: Mix 1% CUSO4 (in distilled water) and 2% potassium sodium tartrate

(in distilled water) in equal volumes.

Reagent A was freshly prepared by mixing the reagents 1 and 2 in a volume ratio

50:1.

Dilute Folin's reagent with distilled water in a 1:1 ratio prior to the assay. Label as

reagent B.

Prepare the protein standard solution: 25 mg bovine serum albumin (BSA) was

dissolved in 100 ml distilled water (25 jxg protein /100 1). The standard tubes with the

following concentrations of protein (50, 100，150，200，250 jxg/1000 yd) were prepared

and stored at -20oC.

2). Procedure for protein determination: (all volumes in microlitres)

； blank standard sample

buffer 1000 一 980

standards 一 1000 _

sample 一 — 20

reagent A 5000 5000 5000

vortex and stand for 10 min in room temperature.

reagentB 500 500 500

vortex and stand for 30 min read in spectrophotometer at 650 nM.

37

The standard solutions were used to plot the standard curve from which the unknown

protein values were extrapolated.

5. RADIOIMMUNOASSAY OF MELATONIN

1). EXTRACTION OF MELATONIN

Serum melatonin was extracted with dichloromethane. 1-2 ml of serum was added

to 5 ml of dichloromethane，which was vortexed for 20 s then centrifuged at 20,000 X g

for 10 min. The serum layer was removed by aspiration to allow collection of the organic

phase. The organic phase was dried under nitrogen gas at room temperature and used in the

assay.

2). STANDARD SOLUTIONS

Standard solutions containing a known amount of melatonin (0 pg x 4 tubes, 5，10，

25，50, 100, 250，500 and 1000 pg x 2 tubes) in gelatin-phosphate buffer (NaaHPO#，

L42g; NaCl, 8.76 g; EDTA，9.3 g; merthiolate, 0.1 g; gelatin, 1 g; made up to 1 L with

distilled water) were prepared. The standard solutions were used to create a standard curve

from which unknown melatonin values could be determined. Two tubes containing 500 \i\

of the gelatin-phosphate buffer were designated for non-specific binding and two tubes

containing 50 \il [^H]melatomn only, were designated for total counts.

3). THE ASSAY

The radioimmunoassay for melatonin followed the procedure designed by Brown et

al. (1985). On the day of assay, 1 or 2 ml of gelatin-phosphate buffer was added to a dried

extracted sample in a glass tube. The mixture was vortexed for 20 s. According to the

predicted concentration of the melatonin, 100-400 pi of the sample was removed to a

plastic assay tube for assay. Tritiated melatonin (50 yd, about 2,000 c.p.m.) was added to

all the assay tubes. Melatonin antiserum was added to all but the non-specific and total

tubes. The samples to be assayed were made up to a total volume of 500 \x\ with the

gelatin-phosphate buffer. The tubes were then vortexed for 20 s before incubation at 40C

for 20 to 24 hours. Incubation was terminated by the addition of saturated ammonium

sulphate solution (0.65 ml, pH 7) and normal rabbit serum (50 fxl 1:20) to all tubes except

the totals. A further incubation period of 1 hour at 40C occurred which was terminated by

centrifugation at 4,000 g for 25 min. The supernatant was discarded and the remaining

pellet resuspended in 325 \xl distilled water (300 \il distilled water being added to the

totals). 300 fxl samples were removed from the assay tubes to plastic scintillation vials with

the addition of 100 fxl of distilled water used to flush out the unipump tubing between each

sample to avoid sample cross contamination. Four ml of scintillation cocktail (POPOP, 0.8

g; PPO, 11 g; triton-X, 455 ml; and toluene 1 L) was added and the vials capped and

handshaken prior to counting. The vials were counted for 10 min each in a liquid

scintillation counter (Beckman, model number 5801), and a computer was used to

determine the melatonin concentrations in the unknown samples.

39

PINEALECTOMY

Removal of the pineal was carried out on two days old chicks under ether

anaesthesia. Head feathers were clipped and the chick's head was placed in a head holder. A

mid line incision was made to expose the skull. The skull was cut open just anterior to the

position of the pineal using a pair of iridectomy scissors to allow a small inverted "U" shaped

flap to be lifted. A micropipette attached to a suction pump held at an angle to the skull was

used to suck away the pineal and its stalk. The skull flap was released and the skin wound

sutured closed. Control chicks (sham operation) were also anaesthetized with ether. The

head feathers were removed and the skin was cut. The wound was sutured closed (Pang et

al., 1974). A l l chicks were decapitated six weeks after pinealectomy.

7. MELATONIN ADMINISTRATION

Four weeks old chicks were kept under a 12h : 12 h light-dark cycle (lights on at

0900h and off at 21(X)h). 0.9% Saline (control) or melatonin (1 mg per kg of body weight)

was injected intraperitoneally two times a day, shortly after light onset and shortly before

light offset. After three weeks of injection the chicks were decapitated at midlight (1500h)

and middark (0300h).

8 • STATISTICS

All data is represented as mean 土 standard error of the mean (S. E. M). Statistical

significance between the two groups was tested by the one-way analysis of variance

(AVOVA) and/or Students t-test. Statistical significance between the various groups was

40

tested by the one-way analysis of variance (ANOVA), and followed by a Student-

Hewman-Keurs (NK) multiple range test. The level of significance was set at p<0.05.

Linear regression was employed to determine the correlation coefficient between two

variables.

41

III. RESULTS

A. METHODOLOGY

1 IODINATION OF MELATONIN

1). SEPARATION OF THE RADIOLIGAND

Separation of [ ^I]iodomelatonin by TLC and HPLC was studied. Figure 2 shows the

profile of purification of [ I]iodomelatonin by TLC. The first radioactivity peak was that of

[^^I]iodomelatonin which was demonstrated by its immunoreactivity with melatonin

antiserum and binding properties to chicken brain membrane preparations (Table 4). The

second radioactive peak was free iodide. The Rf values of melatonin and [^^I]icxiomelatonin

were 0.29 and 0.41 respectively. Figure 3 shows the purification profile of

[^I]iodomelatonin by HPLC. The radioactive and UV absorbency peaks were observed. The

first radioactive peak was demonstrated to be the free iodide. The main radioactive peak was

the second one which could bind both to the melatonin antiserum and chicken brain membrane

preparations (Table 4). The binding results of [125jjiodomelatonin separated by the TLC and

HPLC suggest that the effective separation of the [ ^1]iodomelatonin from the melatonin and

the free iodide is very important. The incomplete separation of [ 125jjiodomelatonin from the

free iodide by TLC was shown by the low signal to noise ratio (about 6:1, Figure 2)) and the

high nonspecific binding of the radioligand to both the antiserum and the membrane

preparations (Table 4). On the other hand, the incomplete separation of melatonin and

[125I]iodomelatonin by TLC revealed that the peak of [125I]iodomelatonin was close to the

peak of melatonin (Figure 2), indicating that a higher specific activity of [125I]iodomelatonin

could not be achieved by the TLC. This explains the poor binding of radioligand to the

42

antiserum and membrane preparations when purified by TLC. However, the

[125I]iodomelatonin separated by the HPLC had a high signal to noise ratio (more than 60:1，

Figure 3), and the peak of [^^jiodomelatonin was distinct from that of melatonin and free

iodide (Figure 3). Thus, the [^¾]iodomelatonin separated by HPLC had low nonspecific

binding and high specific binding compared with the binding results produced by the

radioligand purified by TLC (Table 4).,

2). [125]IODOMELATONIN STABILITY

The stability of [^^I]iodomelatonin synthesized in our laboratory was tested. The

binding properties of the [125jjiodomelatonin both to the antiserum and membrane

preparations showed no significant changes after one month of storage in -20^0.

We found that the reaction time and amount of iodogen used in the iodination procedure

was crucial for the quality of [ ^^iodomelatonin. Longer reaction time (more than 10 min)

and large amount of iodogen (> 40 fxg) tend to damage the melatonin molecule and postpone

the elution of [-‘-]iodomelatonin from the HPLC. Damaged [^^liodomelatonin does not

bind to either the melatonin antiserum or the chicken brain membrane preparations.

2 CONDITIONS FOR THE BINDING ASSAY

1). EFFECTS OF pH AND IONS ON [125I]IODOMELATONIN BINDING TO

CHICKEN BRAIN MEMBRANE PREPARATIONS

Effects of buffer pH, or addition of ions, on specific [125I]iodomelatonin binding in 4-

6 weeks old chicken brain membrane preparations were examined (Table 5). Under a 40C

43

incubation for 5 h，specific binding of [125I]iodomelatonin (26.2-3 L5 pmol/1) was 0.51-0.74

fmol/mg protein at pH 7 A Changes in the pH of the incubation buffer from 6.4 to 8.2 did not

significantly alter the amount of specific binding of [125I]iodomelatonin to chicken brain

membrane preparations. Lowering the pH below 6.0 resulted in a decrease in binding. Binding

dropped to only 5% of the maximum binding below a pH of 5.0. Addition of NaCl (100

mmol/1) to the incubation buffer slightly inhibited (about 20% 土 7.2 %) the specific binding of

[12^]iodomelatonin. In contrast, additions of CaCl2 (5 mmol/1), KC1 (100 mmol/1) and

MgCl2 (5 mmol/1) had almost no effect on the binding.

2). THE EFFECT OF TEMPERATURE ON [125I]IODOMELATOMN

BINDING TO CHICKEN BRAIN MEMBRANE PREPARATIONS

a). KINETIC STUDIES OF [125I]IODOMELATONIN BINDING AT 370C AND 40C

The effect of incubation temperature on the binding of [ I]iodomelatomn with respect

to association to and dissociation from the chicken membrane preparations was studied at 4° C

and 37° C. Figures 4 and 5 show the binding time courses of [125I]iodomelatonin with the

chicken brain membrane preparations at 37°。and 40C respectively. At 37° C the specific

binding increased during the first 30 minutes and equilibrated after approximately 50 minutes

with the association rate constant of (Kj) of 9.5 X 10 min 'K (Figure 4A). After the

equilibrium with 10 pmol/1 [-'•^^iodomelatonin for 60 min. at 370C, 10 \il of 35 [Jtmol/l of

melatonin was added to the assay (to make the melatonin final concentration of 1 fxmol/1).

Displacement of most of the specific binding of [125I]iodomelatonin occurred within 30 min

and nearly complete displacement occurred by 60 minutes, with the dissociation rate constant

(K^) of 6.7 X ICT2 min"1 (Fig.4B). The kinetically derived Kd value was 70.5 pmol/1 which

44

was obtained from the ratio of rate constants (K^/Kj). This was in good agreement with that

determined from equilibrium experiments (68.5 士 4.1 pmol/1) (Table 6).

The time course of 15.5 pmol/l [^^I]iodomelatonin binding with the chicken brain

membrane preparations conducted at 40C (Figure 5A and B) showed that the process of

[125i]iodomelatonin association to and dissociation from the brain membrane preparations

was slower than at (Figure 5A and B). The association of the specific binding of

[•'•^^iodomelatonin to the membrane preparations equilibrated after 5 hours of incubation.

Two hours after the initiation of the dissociation by addition of 10 \x\ of 350 pmol/l of

melatonin (to make the melatonin final concentration of 10 ^mol/1), only half of the bound

[125i]iodomelatonin was displaced. Total displacement was not achieved until 5 hours later

(Figure 5B). The K j and K- j at 40C was calculated to be 7.1 X 107 min “1. NT1 and 2.66 X

10" min"* respectively. Kinetic derivation of the dissociation constant from K j and K-^ give

Kd value of 37.5 pmol/1 which is consistent with that determined from equilibrium experiments

(42. 6 土 2.94). The results suggest that the binding affinity of [^^IJiodomelatonin with the

membrane preparations at 40C is higher than that at 370C.

b). SATURABILITY AND AFFINITY OF [125I]IODOMELATONIN BINDING AT

S^C, 25°。AND 40C

The effect of temperature on the binding capacity of [ iodomelatonin to the 10- to

12-week-old chicken brain membriie preparations was investigated by the saturation study and

the Scatchard analysis at 3 7 ° ^ 250C and 40C. At 370C the total and non-specific binding of

[125I]iodomelatomn increased over a range of 5320 pmol/1 [125I]iodomelatonin. Non-specific

binding was determined in the presence of 1 [imol/l melatonin. Specific binding of

[125I]iodomelatonin increased with increasing concentration of the radioligand (Figure 6A).

Scatchard analysis of the specific binding data yielded linear plots which gave a Bmax of 13.1

45

fmol/mg protein and a K d of 65.5 pmol/1 (Figure 6B). The Hill coefficient was 0.93. Figure 7

and Figure 8 show the saturation curves and the Scatchard plots at 4 0C and 25°C

respectively. A Bmax of 3.87 fmol/mg protein and Kd of 40 pmol/1 diminished at 4 0 C

(Figure 7B). These values increased to 8.8 fmol/mg protein (Bmax) and 57.2 pmol/1 (Kd) at

250C. The Hill coefficients were close to 1 in all cases. This demonstrates that

[125i]iodomelatonin binding with chicken brain membrane preparations at the 370C, 250C

and 4 0C were reversible and that they bound to a single class of sites under these incubation

conditions. Scatchard analyses showed that the Bmax and Kd values determined at 37°。and

were significantly higher than those at 40C (Table 6). Our results suggest that as the

incubation temperature decreases from 370C and 250C to 40C, the binding capacity decreases

and the affinity to the chicken brain membrane preparations increases.

3 • TISSUE CONCENTRATIONS

The linear regression between the specific binding of [ ^^I]iodonielatonin (11,250

cpm, 9.4 pmol/1) to pigeon brain membrane preparations with different protein concentrations

(70-820 mg) gave a straight line and the coefficient of correlation was 0.995 (Figure 9). In this

study, the NSB ranged from 221-279 cpm between protein concentrations of 70 jxg to 820

jxg. Subtracting the 丫-counter background counts (150 cpm) and the filter paper NSB (50 cpm)

results in a tissue NSB between 20 and 40 cpm at protein concentration ranging from 70 pg to

820 卩g. This clearly demonstrates that the specific binding of [125I]iodomelatomn to pigeon

brain membrane preparations is tissue dependent and suggests that within a certain range, the

contribution of tissue to the NSB is negligible especially when the radioligand is used at lower

concentrations.

T H E SAMPLE STABILITY

46

To study the tissue stability, quails were killed and the bodies were put in a 40C cold

room. The brains were collected after different post-mortem times (from 5 min to 14 hours).

The brain tissues were then kept in -70oC for 1 day. Binding studies was then conducted. The

binding results shown in Table 7 demonstrate that maintaining brain tissues at 40C for 14 h

does not significantly change [^ I]i(xiomelatomn binding. Avian brain tissues kept at -70oC

for 1-2 months were also stable as tested by binding assays.

47

B. [125I]IODOMELATONIN BINDING SITES IN THE BRAIN TISSUE

OF BIRDS: A GENERAL BINDING CHARACTERIZATION AND REGIONAL

DISTRIBUTION.

1. KINETIC STUDY

The time course of [^¾]icxiomelatonin binding to the membrane preparations

of the chicken (Figure 5)，quail (Figure 10)，pigeon (Figure 11) and duck (Figure

12) were studied at 40C. In all the animals studied, association of the radioligand

(concentration between 18-30 pmol/1) to crude brain membrane preparations at 40C

showed a gradually increase in specific binding during the first 3 hours and

equilibrated after approximately 5 hours.

After equilibrium was obtained at 5 hours, 10 yd of 350 [xmol/1 melatonin

was added (to make the melatonin final concentration of 10 |xmol/l) to initiate

dissociation. Most displacement of specific binding of [^^Ijiodomelatonin

occurred within 4-5 hours, indicating that specific binding of [^^I]icxiomelatonin

to bird brain membrane preparations is reversible. The data of the kinetic studies

from the different species of birds were very similar. The and the

kinetically derived Kd values are shown in Table 8.

2 . SATURATION STUDY

Saturation studies were conducted using the brain membrane preparations of

the birds collected at ML. Total and nonspecific binding of [125I]iodomelatonin

with the brain membrane preparations increased over the range of 5-120 pmol/1

[125j]i(xionielatonin. Nonspecific binding was determined in the presence of 10

pmol/l melatonin. Specific binding was determined as the total binding minus

nonspecific binding. Specific binding of [ ]iodomelatonin increased with

increasing concentrations of the radioligand and approached saturation at higher

radioligand concentrations. The saturability of the [125I]iodomelatonin binding

sites was shown by the representative experiments with the membrane preparations

from the brain of chicken (Figure 7)，pigeon (Figure 13)，duck (Figure 14) and

quail (Figure 15). The linearity of Scatchard plots of specific binding data from the

saturation studies in all the birds studied suggests that each brain has a single class

of binding sites for [^^I]iodomelatonin (Figure 7 and 13-15). The values of

Bmax and Kd from the Scatchard analysis demonstrates that [ ^1]iodomelatonin

binding sites are of high affinity and low capacity. The density of

[^^I]iodomelatonin binding in the brain of birds is as follows: duck > pigeon >

quail > chicken (Table 9).

3. SPECIFICITY

To ascertain the specificity of [12^]iodomelatonin binding to the membrane

preparation of the various avian brains, binding assays were carried out by adding

varying concentrations of unlabelled melatonin analoges and neurotransmitters, and

a constant amount of [^^I]iodoinelatonin (20-30 pmol/1) to membrane

preparations. The inhibition constant (Ki) values were calculated from 50%

inhibition concentration (IC5Q) values (Table 10). Both melatonin and 6-

chloromelatonin were found to be potent inhibitors of [125I]iodomelatoiiin binding

in bird brain membrane preparations, while N-acetylserotonin only showed minor

inhibition and the other compounds tested showed no significant inhibition of

[125I]iodomelatonin binding sites. The various concentrations of melatonin from

ICT to 10 ^ mol/1 in all the birds tested resulted in similar monophasic

competition curves (figure 16)，suggesting a single class of binding sites for

[12^I]icxiomelatonin in the birds brain membrane preparations.

4 . REGIONAL DISTRIBUTION

The regional distribution of [125I]iodomelatonin binding sites in the brain

membrane preparations of chicken and pigeon were studied. The Scatchard analysis

in the chicken and a one point analysis (using a [12^I]icxiomelatonin concentration

of 39.5 pmol/1 for the binding study) in the pigeon showed an uneven distribution

of binding sites. Specific binding was found in all brain regions examined with the

following order of densities in both the chicken and pigeon brain regions:

hypothalamus > midbrain > pons-medulla > telencephalon and cerebellum (Table

11). No significant changes of Kd values were recorded in the chicken brain

regional binding studies (Table 11).

5 . SUBCELLULAR DISTRIBUTION

Brain tissue homogenates of the chicken and pigeon were fractionated by

differential centrifugation. The subcellular distribution of [12^I]iodomelatonin

binding sites in the brain tissue of the birds is shown in Table 12.

[l^I]iodomelatonin binding was maximal in mitochondrial fractions followed by

the nuclear fractions (Table 12)，which were included in the crude membrane

preparations.

6. THE EFFECT OF GTP ANALOGUES ON THE CHICKEN

BRAIN [125I]IODOMELATONIN BINDING SITES

The addition of 10" mol/1 GTPyS significantly reduced the binding capacity

of [ ]iodomelatonin binding sites in 12 weeks old chicken brain membrane

preparations, without affecting the K d value (Table 13). The fact that GTPyS

exerted an inhibitory effect on [^]icxiomelatonin binding indicated that the

recognition sites of [^^jiodomelatonin are coupled to guanine nucleotide-binding

proteins.

C. DIURNAL VARIATION AND EFFECTS OF MELATONIN

MANIPULATIONS ON [125I]IODOMELATONIN BINDING SITES

IN THE CHICKEN BRAIN.

In this experiment, all the animals were sacrificed at the age of five to eight

weeks old. Animals were decapitated at midlight (0900-1000h) or middark (2100-

2200h), or at 4 h intervals throughout a 24 hour period (OlOOh，0500h，0900h,

1300h，ITOOh, 2100h).

1 . DIURNAL RHYTHMS OF SERUM MELATONIN LEVELS AND

BINDING CAPACITY OF [^^IJIODOMELATONIN BINDING

SITES

Serum melatonin levels and the iodomelatonin binding site capacities

of chicken brain membrane preparations measured at 4 h intervals throughout a 24

h L:D cycle showed clear light-dark variations (F = 3.18; df = 23/5; p < 0.05). The

well-established nocturnal peak of serum melatonin followed by an abrupt fall after

light onset was observed. The mean serum level of melatonin was lower in the

light period (39.9 士 6.9 pmol/1) compared to that in the dark period (764.0 土 226.7

pmol/1) (p < 0.05, Table 14).

Scatchard analyses of [ ^I]iodomelatomn binding sites in the chicken

brain membrane preparations revealed the presence of a single population of

binding sites at each time point of the 24 h cycle (Figure 17). The

[125I]iodomeIatonin binding capacities in the chicken brain membrane preparations

presented a light-dark variation (F = 9.35; df = 23/5; p < 0.05) with low levels

from middark (2100 h, 12.8 ± 0.82 fmol/mg protein) until the end of the dark

period (0100 h, 11.8 土 1.8 fmol/mg protein). The [ 125jjiodomelatonin binding

remained higher from the early light period to the early dark period with the

highest level recorded at the end of the light period (1300 h, 19.7 士 0.45 fmol/mg

protein) (Table 14). The mean values of the binding densities were 183 土 0.8

fmol/mg protein and 14.2 士 1.4 fmol/mg protein during the light and dark period

respectively (p<0.05). The binding was 48.4% higher at midlight than that at

middark (pcO.Ol). Values obtained at 2100 h and 0100 h were significantly lower

(p < 0.05) than those at 0500 h, 0900 h，and 1300 h.

Linear regression showed a significant inverse relationship between

[^^I]iodomelatonin binding capacities and serum levels of melatonin (correlation

coefficient = 0.823; p < 0.05). There were no significant changes (F = 0.63; df =

23/5; p >0.05) in the Kd values throughout the 24 h period studied (Table 14).

2 . EFFECTS OF MELATONIN ADMINISTRATION ON SERUM

MELATONIN LEVELS AND [1 2 SI]IODOMELATONIN BINDING

CAPACITIES

Four weeks old chicks were used. Saline (control) or melatonin (1 mg/kg;

i.p.) was injected intraperitoneally twice a day, shortly after the light onset and

shortly before the light offset . After three weeks of injection, the chicks were

decapitated at ML and MD. Melatonin injection greatly increased serum melatonin

levels at both midlight and middark, and abolished the rhythm of melatonin in the

serum (Table 15). Melatonin injection also abolished the light-dark difference of

the [125I]iodomelatonin binding to the membrane preparation of chicken brain by

53

decreasing the binding at mid-light (P < 0.01). There was no effect on the binding

at mid-dark nor effect on the Kd values at both mid-light and mid-dark (Table 15).

3, EFFECTS OF PINEALECTOMY ON SERUM MELATONIN

LEVELS AND [125I]IODOMELATONIN BINDING IN THE BRAIN

Pinealectomy almost eliminated the serum melatonin content and abolished

the daily rhythm of melatonin levels in the circulation (Table 16). It also increased

the [^^I]icdomelatomn binding capacities both at mid-light and mid-dark by 52.3

and 53.8% (p < 0.05) than their respective control groups (Table 16). There was,

however, no significant change in the Kd values (Table 16). The light-dark

variation of [^^I]iodomelatomn binding in the pinealectomized animals remained

significant (p <0.05) with a higher value at mid-light.

54

D. DEVELOPMENT OF [125I]IODOMELATONIN BINDING

SITES IN T H E CHICKEN BRAIN

1. COMPARISON OF [125I]IODOMELATONIN BINDING

CHARACTERISTICS BETWEEN THE 17-DAY-OLD EMBRYO AND

YOUNG CHICKEN BRAIN MEMBRANE PREPARATIONS

1). KINETIC STUDY

The time course of the [^^]icxlomelatonin binding to the 17-day-old

chicken embryo (Figure 18) and 10-week-old (Figure 4) chicken brain membrane

preparations were studied at 370C. Association of 10 pmol/1 of the radioligand to

the 17-day-old chicken embryo brain membrane preparations showed a rapid

increase in specific binding within 20 minutes and reached equilibrium after 40

minutes (Figure 18A)，with an association rate constant (Kj) of 5.1 X 10® min"

M-l (Figure 18A). Once equilibrium was reached, 10 |xl of 35 fxmol/1 melatonin

was added (to make the melatonin final concentration of 1 |Jimol/l) at 40 min to

induce the dissociation. Dissociation was complete within 60 minutes (Figure 18B),

with the dissociation rate constant ( K . j ) of 2.3 X 10" m i n ( F i g u r e 18B )• The

kinetically derived Kd value (K. j /k j ) was 45 pmol/1. A similar time course of

[^^I]iodomelatonin association with and dissociation from the 10-week-old

chicken brain membrane preparations at 370C was recorded (Figure 4). The Kd

value calculated from the kinetic study in 10-week-old chicken brain membrane

preparation was 70 pmol/1 from the ratio of K p 9 . 5 X 10^ min"-'". and K . j =

6,7 X 10"2 min"1- The kinetic studies demonstrated that the 17-day-old chicken

embryo and 10-week-old chicken brain membrane preparations had similar binding

time courses.

2) SATURATION STUDY

The brain membrane preparations of 17 days old embryo and 10 weeks

old chicken were incubated with [125I]iodomelatonin (concentrations of 10-320

pmol/1) at 37° C for 1 h. The saturation curves showed that the specific binding of

[^^I]iodomelatonin to both the 17 days old embryo (Figure 19) and 10 weeks old

chicken (Figtire 6) brain tissues were concentration dependent and reached

saturation towards the higher range of radioligand concentrations. The Scatchard

analysis of the specific binding data resulted in linear plots suggesting binding to a

single class of binding sites (Figure 19 and 6)

3). SPECIHCITY

The specificity of melatonin binding sites was studied using brain

membrane preparations of 17-day-old embryos and 10-week-old chickens. Figure

20 shows the representative competitive inhibition curves of melatonin, 6-

chloromelatonin and N-acetylserotonin, Table 17 shows the inhibition constant (Ki)

values which were calculated from 50% inhibition concentration (IC5Q) values. The

data demonstrate that the [•'•^Iliodomelatonin binding sites in the embryo and

young chick brain membrane preparations share the common pharmacological

profile.

D E V E L O P M E N T O F C H I C K E N B R A I N

[1 2 SI]IODOMELATONIN BINDING SITES

To study the development of chicken brain [125I]iodomelatonin binding

sites brains of chicken embryos (6 days; E6，10 days; E10，13 days; E13 and 17

days; E17), newly hatched chickens (1 day; HI , 7 days; H7), young chicken (20

days; H20, 40 days; H40) and adult chickens (6 months; M6, 12 months; M12)

collected at mid-light were used.

1). DEVELOPMENT OF CHICKEN BRAIN WEIGHT AND THE

PROTEIN CONTENT

The brain weight and protein content per gram brain wet tissue of the

embryo (E 10-17)，newly hatched (H 1-7), young (H 20-40) and adult (M 6-12)

chickens are shown in Table 18. A progressive increase in brain weight was noted

from the E 13 to newly hatched chicken. This increase in brain weight was

accompanied by a gradual increase in the brain protein content from E 17 to H 7 as

expressed by the protein content mg/g brain weight From the young to the mature

chicken，the brain weight increased continually and the protein content of brain

tissue was slightly higher in the adult animals (p< 0.01) compared to the embryo,

newly hatched and 20-day-old chicken (Table 18).

2). DEVELOPMENT OF CHICKEN BRAIN [125I]I〇DOMELATONIN

BINDING SITES

The specific binding of [125I]iodomelatomn with chicken brain

membrane preparations was not detectable before embryonic day 6. By embryonic

day 10, [ ^Iliodomelatonin binding could be repeatably measured but non-

specific binding was high and it was difficult to obtain acceptable Scatchard plots.

After embryonic day 13 the specific binding was clearly identifiable and Scatchard

analysis was performed to measure Kd values and the binding capacities of the

[125I]iodomelatonin binding sites during the development Table 19 and Figure 21

show the binding capacities as expressed in fmol/mg protein，fmol/g brain tissue

and fmol/brain. Between embryonic day 13 and posthatch day 7，there was a 14-

fold increase in [125i]iodomelatonin binding as expressed per whole brain (from

56.1 to 779.3 fmol/brain) and about 2.4-fold increase when expressed per

mg/protein (from 5.89 to 14.25 fmol/mg protein) (Figure 21). The

[l^I]iodomelatomn binding capacity reached its highest level in the 40 day-old

chicken as expressed by the fmol/mg protein (16.4 fmol/mg protein). The density

of the [•'•^Iliodomelatonin binding sites was slightly lower in the adult animals (M

6 and M 12) than in the 40-day-old chicken (P < 0.05) (Figure 21). The Scatchard

analysis showed that [ iodomelatonin binding sites in the chicken brain during

the development had high affinity. The apparent Kd values ranged between 46.9 -

89.2 pmol/1. Although the density of binding sites varied considerably during

development, Kd values did not change significantly from E 17 to adult (Table 19，

Figure 22). Only the embryonic 13 day group with the Kd value of 46.9 土 15.5

pmol/1 was slightly lower than the Kd values of 40-day-old chicken and 12-month-

old chicken (p< 0.05). The presence of high affinity [125I]iodomelatonin binding

sites in the early embryonic chicken brain suggest that melatonin might have a

central action in the early stages of development

The [125I]iodomelatonin binding capacity reached its highest level in

young chickens (H 40 with Bmax of 16.4 土 0.42 fmol/mg protein) and decreased

in the adult chicken ( M 12 with Bmax 12.2 ± 0.2 fmol/mg protein) (p<0.05)

suggesting that the binding capacity may be age-related (Table 19).

58

3- DEVELOPMENT OF THE DIURNAL RHYTHM OF

[125I]IODOMELATONIN BINDING SITES IN THE CHICKEN

BRAIN

The light-dark variation of [^^Iliodomelatonin binding sites in the

chicken brain membrane preparations was studied using the brains of 13 and 17

days old embryos; 1 and 7 days old posthatched chicks and the chickens of 20，40

days old and 6，12 months old. The light-dark variation of [•'•^Iliodomelatonin

binding sites was found in young chickens (20 and 40 day-old chicks) with high

binding at M L and low binding at MD. There was no significant change in Kd

values (Table 20，Figure 23).

4. THE EFFECT OF OTPYS ON THE [ 1 2 5]IODOMELATONIN

BINDING SITES IN EMBRYONIC CHICKEN BRAIN

Figure 24 shows typical saturation and Scatchard plot analysis of

iodomelatonin binding to brain membrane preparations at E 18. The data -C

demonstrates a single class of receptors with high affinity. The addition of 10"J

mol/1 GTPYS markedly reduced the maximal number of binding sites (from 10.6 to

5.7 fmol/mg protein) without affecting the Kd value (Table 21).

59

E . BINDING CHARACTERISTICS AND DIURNAL

VARIATION OF [125I]IODOMELATONIN BINDING SITES IN

CHICKEN RETINAS

To study the binding characteristics and light-dark variation of

[12^I] iodomel atonin binding sites in chicken retinas, 4- to 8-week-old chickens

were used. Chickens were killed at M L except in the diumal variation study where

chickens were killed at MD.

! • KINETIC STUDY

The time courses of [^^]icxlomelatonin association with and dissociation

from the binding sites on retina membrane preparations were studied. Eight-week-O

old chicken retina membrane preparations (140 jxg protein) were incubated at 37 C

with [^^I]iodomelatomn (60 pmol/1) in Tris-HCl buffer for the indicated times (5，

10，15，20，30，40，60，80 minutes) and the amount of specifically bound

[^^1] icxiomelatonin was determined following rapid filtration. Nonspecifically

bound [ 125jjjodomelatonin was defined as [ ^I]iodomelatonin bound in the

presence of 1 pmol/l melatonin. The association of the specific binding of

[125I]icxiomelatonin to the membrane preparation was rapid during the first 30 min

of incubation, reached equilibrium after 40 minutes and then decreased after 60

minutes (Figure 25 A). The association rate constant ( K ^ calculated from the

pseudo-first order equation was K j = 1.78 X 10^ min"^ M**-'- (Figure 25A). After

equilibrium was reached (at 50 min), 10 [il of 35 fxmol/l melatonin was added to

initiate dissociation and the amount of specifically bound [125I]iodomelatomn was

determined at the indicated times (5，10，15，20, 30，40，60，120 minutes). Rapid

displacement occurred during the first 20 minutes of incubation and complete

displacement occurred after 60 minutes of incubation (Figure 25B). The

dissociation rate constant (K.^) calculated from the first-order equation was =

4.46 X 10"2. min"1 (Figure 25B). The Kd value derived from the ratio of K^/K j

was 256 pmol/1.

2 • SPECIFICITY

The pharmacological profile of [125I]iodomelatonin binding sites was

determined by competition experiments. Ten chicken retinas were pooled and

membrane preparation aliquots with 100-150 \ig of protein were used for the

competition experiment in the presence of 10"13 to 10" mol/1 of various melatonin

analogues and neurotransmitters. Thirty pmol/1 [^^Ijiodomelatonin was used. The

Ki values are shown in Table 22. Comparison of Ki values for the inhibition of

binding by melatonin analogues and neurotransmitters showed that melatonin was

the most potent displacing agent (Ki 1 nmol/1), followed by 6-chloromelatonin with

an Ki value of 11.2 nmol/1. HAS was a weak displacing agent with Ki value of 50

nmol/1. [ "'•^^]Iodomelatomn binding was not substantially affected by the other

compounds tested.

3 • SATURATION STUDY

The specific binding of [^^I]iodomelatonin to chicken retina membrane

preparations at 37 0 C was a saturable process using radioligand concentrations

ranging from 10-650 pmol/1. Nonspecific binding, defined in the presence of 1

[xmol/l of melatonin, increased linearly with [125I]iodomelatonin concentrations. A

representative saturation isotherm and the Scatchard plot of [ 1 2 5 I ] iodomelatonin

binding to the membrane preparations of chicken retina is shown in Figure 26. The

Kd and Bmax values for iodomelatonin binding were 366.5 土 96.5 pmol/1

and 47.2 +. 1.26 fmol/mg protein respectively (Table 23).

4. DIURNAL VARIATION OF [ 1 2 5 I]IODOMELATONIN

BINDING

The [^iodomelaton in binding capacity and affinity to the retina

membrane preparations of 4-week-old chicken killed at M L and MD were studied

by the Scatchard analysis. The Bmax of [1 ^1]iodomelatonin binding sites was

47.1% higher at M L (47.2 土 1.26 fmol/mg protein) than at M D (32.1 土 4.55

fmol/mg protein). There was no significant change in the affinity (Table 23)

F . DEVELOPMENT OF [125I]IODOMELATONIN BINDING

SITES IN THE CHICKEN RETINA

1. T H E BINDING C H A R A C T E R I S T I C S O F

[125I]IODOMELATONIN BINDING SITES IN EMBRYONIC

CHICK RETINA

1). KINETIC STUDY

Fifteen-day-old embryonic retinas of 6 animals were pooled to perform

the kinetic studies. The membrane preparations (120 |xg protein) were incubated at

370C with 60 pmol/L of [^^I]icxlomelatonin in Tris-HCl buffer. The experiment

was conducted at the same time as the chicken retina kinetic studies and the same

procedure was used. The association of the specific binding of [必I]iodomelatonin

to the membrane preparation was rapid during the first 20 minutes of incubation and

reached equilibrium after 60 minutes (Figure 27A). K j calculated from the pseudo-

first order equation was 0.15 X 10 m i n " ( F i g u r e 27A). After equilibrium

was reached at 60 minutes, dissociation was initiated. Displacement occurred

rapidly at the first 40 minutes and complete displacement occurred after 60 minutes

(Figure 27B). The was 1.78 X 10"2 min"1 (Figure 27B). The K d value

derived from the ratio of K^ /K j was 118.6 pmol/L

2). SPECIFICITY

Fifteen-day-old embryonic retinas from 14 animals were pooled. Aliquots

of the membrane preparations with protein (110 \ig) were used for the competition

experiment in the presence of 30 pmol/1 [125I]iodomelatonin as well as various

melatonin analogues and neurotransmitters. The results showed the similar order of

pharmacological affinity of the compounds tested as that of the chicken retina

(Table 22).

3). SATURATION STUDY

The non-specific binding for [125I]iodomelatonin at embryonic day 10

was about 75%, therefore saturation curves and linear Scatchard plots were not

obtained from animals younger than this age. After embryonic day 15，the ratio of

specific binding to non-specific binding increased gradually. The Bmax values were

9.2 土 2.4 fmol/mg protein at embryonic day 15 (n=3), 15.2 fmol/mg protein at

embryonic day 18 (n=l) and 21.86 fmol/mg protein at day 20 (n=l). The K d

values were 102 土 21.3 pmol/1 at embryonic day 15 (n=3)，126.2 pmol/1 at

embryonic day 18 (n=l) and 181 pmol/1 at embryonic day 20 (n=l). Figure 28

shows the representative saturation and Scatchard plots of [ ^1 ] iodomelatonin

binding to the retina at embryonic day 15.

2 . THE DEVELOPMENT OF [ 1 2 5 I ] I O D O M E L A T O N I N

BINDING SITES IN CHICKEN RETINAS

The development of [ 12^I]iodomelatonin binding sites in chicken retinas

was studied by using embryonic chicks (7，10，15, 20 days old) and posthatched

chicks (four weeks old). Embryo and 4 week-old chicken retina weights, protein

contents, binding capacity expressed per milligram protein and the ratio of specific

binding are shown in Table 24. A progressive increase in the pair of retina weight

was noted from 1L7 mg from 10-day-old embryos to 1103 mg of posthatched 28-

day-old chicks. The protein content remained constant during this period. (Table

24). The binding study was performed by one-point analysis using 46.8 pmol/1

[125i]i0d0inelatomn. Specific binding was undetectable in animals less then

embryonic 10 days old. Even in 10 day old embryos non-specific binding

represented over 75% of the total binding. After embryonic day 15，the specific

binding increased greatly (Table 24). The results demonstrate the existence of

[1^1] iodomelatonin binding sites in the early development of chicken embryonic

retinas.

65

IV, DISCUSSION

A. CHARACTERIZATION OF [125I]IODOMELATONIN BINDING SITES

IN AVIAN BRAIN.

1 • THE RADIOLIGAND • [1 2 5I]IODOMELATONIN

The radioligands used in the melatonin receptor binding assay were either the iodinated

or tritiated form. Tritiated ligands have the same molecular structure, binding affinity,

biological activity and metabolic rate as melatonin. The principle disadvantage of the tritiated

radioligand is that it has a lower specific activity than the iodinated form. This disadvantage has

hindered the detection of melatonin receptors with very high affinity and low binding capacity

(Dubocovich, 1988a). On the contrary, iodinated melatonin has a much higher specific

activity than the tritiated compound. Unfortunately, the iodination of melatonin，a derivative

of tryptophan, has proven to be difficult Little progress had been made until 1984 when

Vakkkuri et al. (1984) successfully synthesized 2 - [^ iodomela ton in by using the mild

oxidase，iodo-gen. It has been demonstrated that 2- [^^jiodomelatonin has a high specific

activity (about 2200 Ci/mmol) and retains the high specificity and affinity for melatonin binding

sites (Dubocovich 1988a). According to the structure and activity relationship studies, 2-

[^^^1]iodomelatonin is a potent melatonin receptor agonist (Dubocovich 1988a). 2-

Iodomelatonin is more potent than melatonin in the reduction of dopamine release in the

chicken retina in vitro (Dubocovich and Takahashi, 1987)，and the inhibition of gonad

development in Djungarian hamsters in vivo (Weaver et al 1988; Sugden, 1989). The

development of [ 125I]icxiomelatonin has greatly facilitated the studies of melatonin receptor in

the last few years.

66

While our studies were in progress, [125I]iodomelatonin binding sites in homogenized

brain and retina tissues had also been studied in many species by radio-receptor assay (see

review of Stankov and Reiter 1990; Krause and Dubocovich 1991; Section IB2-3). The Bmax

and K d values determined by the Scatchard analysis varied greatly between species (see

review of Stankov and Reiter 1990; Krause and Dubocovich 1991; 丁able 2，3). Comparison of

the [ 1 1 ] iodomelatonin binding data in chicken brain shows there were some deviations

between laboratories (Dubocovich et al.，1989; Stehle, 1990; Rivkees et al.，1989b; Shankar

and Dubocovich, 1989; Yuan et al., 1990; Ying and Niles, 1991; Table 25). Apart from

different factors in the binding studies including the incubation temperature and time, tissue

preparations and methods of separating bound and free ligand，the specific activity and purity

of the 2 - i o d o m e l a t o n i n might be important factors. Thus, the iodomelatonin

prepared by different laboratories might have different specific activity and purity which might

result in the discrepancy of the binding data. It was demonstrated in our laboratory that the

separation of [•'•^Ijiodomelatomn by TLC and HPLC gave different binding results (Table 4).

In addition, the quality of commercially obtained [ ^I]iodomelatonin varied from one batch to

another. Taking these factors into consideration, it is not surprising that there were

discrepancies in the binding data reported from different laboratories. However，the

characteristics (time course, Kd and Bmax values calculated from Scatchard analysis and

specificity) of [125I]iodomelatomn binding sites reported within our laboratory is consistent in

the brain tissue of birds including chicken，quail，pigeon, duck (Table 8-10) and human fetus

(Yuan et al., 1991); in the spleen (Yu et al., 1991) and gonad (Ayres et al., 1992) of chicken,

in the gut of duck (Lee et al” 1992); as well as in the retina of chicken (Figure 25，26) and tree

shrew (Lu et al” 1991).

2. THE IDENTIFICATION AND BINDING CHARACTERIZATION OF

[1 2 S I]IODOMELATONIN BINDING SITES IN AVIAN BRAIN.

67

The identification and characterization of [125I]iodomelatomn binding sites in the brain

tissue of chicken has been reported since 1989 by several groups during the course of our

studies (Dubocovich et al., 1989; Rivkees et al.，1989b; Stehle, 1990). Comparable data for

the [ 125I]iodomelatonin binding studies in the chicken brain between our laboratory and the

other groups are shown in Table 25. Our results confirmed and extended the findings of

[123i]i0domelatonin binding sites in the brain tissue of birds. It showed that

[125I]iodomelatonin specifically bound to chicken, duck, pigeon and quail brain membrane

preparations. These sites satisfy all the criteria for hormone binding sites: They are saturable,

reversible, specific and of high affinity. The linearity of the Scatchard plot and the unity Hill

coefficient suggest that [1 2 5I] iodomelatonin binds to a single class of sites in membrane

preparations of the avian brain. The circulating levels of melatonin reported were 100-1000

pmol/1 in the chicken (Pelham et al, 1972), 300-700 pmol/1 in the pigeon (Pang et al” 1983)

and 100-1000 pmol/1 in the quail (Pang et al•，1983; Underwood et aL, 1984). The high

affinity of [ 125I]iodomelatonin binding sites in avian brain as was demonstrated by the K d

value calculated from Scatchard analysis (30-50.6 pmol/1. Table 9) and kinetic analysis (26.9-

72.2 pmol/1. Table 8) indicates that these binding sites are physiologically relevant (Pang et al”

1990). The present results demonstrated that the binding characteristics were similar in the

brains of different avian species (chicken, pigeon, quail and duck). The Kd and Bmax values

for the [125I]iodomelatonin binding sites in chicken brain membrane preparations obtained in

our laboratory were consistent with those reported by other groups (Table 25). In addition, the

observed order of potencies for inhibiting [^ ] iodomela ton in binding with various

indoleamines and monoamines in avian brain was also similar to that observed by other groups

in chicken brains (Table 25)，chick retinas (Dubocovich and Takahashi., 1987; Table 22) and

the other high affinity binding sites in neural tissues (Section IB2-3，Table 2). Some minor

68

differences could be due to differences in methodologies used such as the preparation of the

[12¾]iodomelatonin，incubation, time and temperature as well as the age and species of animals.

Our present studies demonstrated that the [125I]iodomelatonin binding sites were

similar in the brain of different avian species (Table 8-10). Dubocovich (1988a) suggested that

[^^1]iodomelatonin binding sites in the nervous system could be classified into two different

subtypes, the ML-1 (picomolar affinity binding sites) and ML-2 receptors (nanomolar affinity

binding sites) (see introduction section Table 2 and 3). The ML-1 binding sites were found in

the whole brain tissue of reptile and birds as well as in the discrete brain areas of birds. The Kd

and Bmax of high affinity [125I]iodomelatonin binding sites were less than 350 pmol/1 and 100

fmol/mg protein respectively. The phannacological order of affinities was 2-iodomelatoiiin > 6-

chloromelatonin > melatonin > N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine > 5-

hydroxytryptamine (Dubocovich, 1988a). Our results demonstrated that the

[125I]iodomelatonin binding in the membrane preparations of chicken, quail, pigeon and duck

(Table 8-10)，possessed similar binding characteristics with the ML-1 [^^I]iodomelatonin

binding sites proposed by Dubocovich (1988a).

Present studies showed that the icxiomelatonin binding sites in the membrane

preparations were found in all brain regions of chicken and pigeon examined: The order of

density of specific binding in those region was: hypothalamus > midbrain > pons-medulla >

telencephalon > cerebellum (Table 11). Similar findings in chicken brain regions were

recorded by other groups using autoradiography (Rivkees et al., 1989b; Stehle 1990) or

receptor binding studies (Dubocovich et al” 1989). These data were, in part，consistent with

the regional distribution of endogenous melatonin levels in the chicken (Pang et al” 1974) and

rat brain (Pang and Brown, 1983), and the uptake of exogenous [3H]melatonin in the rat brain

(Anton-Tay and Wurtman, 1969). Earlier studies have suggested the involvement of the

hypothalamus in the melatonin action. Implantation of melatonin in the hypothalamus reduced

pituitary and plasma LH in castrated rats (Fraschini et al” 1968a), as well as the weight and

69

activity of the genital tract in white-footed mice (Glass and Lynch, 1982). Melatonin treatment

increased hypothalamic 5-hydroxyindole acetic acid and serotonin levels in the chicken

(Cassone et al” 1983)，and hypothalamic norepinephrine levels in the mouse (Fang and

Dubocovich, 1988). Furthermore, pinealectomy decreased 5-hydroxyindole acetic acid in the

hypothalamus and midbrain in rats (Niles et al•，1983). In vitro, melatonin decreased cyclic

AMP, prostaglandins, and E2 synthesis (Vacas et al., 1981)，but increased somatostatin release

(Richardson et al.，1981)，and cyclic GMP synthesis (Vacas et al., 1981) in the rat

hypothalamus. The above findings together with the melatonin binding studies are in line with

the suggestion that the hypothalamus is an important site of pineal melatonin action.

In birds, the high affinity [^^I]iodomelatonin binding sites are widely distributed

throughout the whole brain. This is in marked contrast to the very restricted distribution of

melatonin receptors in mammalian brains (Table 2). Autoradiography revealed that

[125I]iodomelatonin binding sites only occurred in the suprachiasmatic nuclei and the median

eminence of the hypothalamus from rats (Vanecek et al.，1988b; Williams，1989)，

suprachiasmatic nuclei from human adults and fetuses (Reppert et aL, 1988), and the median

eminence and anterior pituitary from hamsters (Vanecek and Jansky, 1989). Rivkees et aL，

(1989b) reported a 125-fold difference in the [125I]iodomelatonin binding sites in whole brain

of chicks as compared to rats using the same binding assay conditions. Similarly melatonin

concentrations in tissues of birds were higher than in mammals (Pang, 1985). The distribution

of [125i]i0domelatonin binding sites in spleens of birds were also higher than those in

mammals (Yu et al” 1991). These seemingly well correlated phenomena suggest differences in

the activities and/or functions of pineal melatonin between birds and mammals. In this study, the distribution of [125I]iodomelatonin binding sites among subcellular

fractions of chicken and pigeon brain was demonstrated (Table 12). Our results showed that

binding was higher in the mitochondrial pellet (P2) than in the nuclear pellet (PI) (Table 12).

This is consistent with the subcellular distribution of [^^Iliodomelatonin binding in the

70

chicken brain (Dubocovich et al” 1989), hamster hypothalamus (Duncan et al” 1988) and

bovine brain (Cardinali ct al” 1979). It should be noted that specific iodomelatonin

binding sites were reported in the nucleus of a tumour cell line (Stankor and Reiter，1990).

Stankov and Reiter (1990) suggested the possible existence of intracellular melatonin

receptors, since melatonin is a small and highly lipophilic molecule which passes through the

cell membrane easily. Pang et al., (1991) also speculated that the radioligand-binding site

complex might undergo internalization for its physiological action and/or degradation.

However, the subcellular fractions prepared by the differential centrifugation had a tendency to

be cross-contaminated and the above data has to be treated with caution. Further studies with

more precisely refined subcellular fractionation is needed for the better understanding of the

sites and mechanism of melatonin action.

In the present study GTPyS of 10"^ mol/1 significantly decreased the

[^^Ijiodomelatonin binding to chicken brain membrane preparations (Table 13)，suggesting

that these receptors are linked to a G-protein. This is in line with previous studies (Dubocovich

et al., 1990; Ying et al., 1991; Niles et aL, 1991) which demonstrated that

[^^1]iodomelatonin binding sites in chicken brain were G-protein related binding sites. Niles

et al. (1991) further suggested that the G-protein linked melatonin receptor was a pertussis

toxin-sensitive Gi-protein.

The [125I]iodomelatomn binding capacity and affinity was affected by the assay

conditions including the presence of ions, guanine nucleotide and incubation temperature. In

the present study, it was demonstrated that Na+ (100 mmol/1) decreased the binding density by

20% while neither Mg+ + nor Ca++ had any effects on the binding in chicken brain membrane

preparations (Table 5). These results were consistent with those obtained from chicken brain

and retina (Dubocovich and Takahashi•，1987; Dubocovich et al., 1989)，and rat brain

(Laitinen and Saavedra, 1990b; Laitinen et al.，1990). The mechanism by which sodium

71

modulaties [125I]iodomelatomn binding is still unclear but it is suggested that NaCl may affect

the conformation of the receptor molecule ((Laitinen and Saavedra, 1990b).

In the present study, neither binding density nor affinity had any significant changes

between 370C and 250C incubation temperature. However, binding density was significantly

decreased while the affinity increased as incubation temperature decreased from 370C or 250C

to 40C (Table 6). The kinetic studies showed that both association and dissociation of

[^^I]iodomelatonin from the chicken brain membrane preparations were slower at 40C than

that at 370C (Figure 4 and 5) and that binding to the membrane preparations was temperature

dependent. Dubocovich et al. (1990) also reported that binding to chicken brain membrane

preparations showed no significant difference between 370C and 250C. However they found

that the binding affinity but not density of [ ^]iodomelatonin binding sites studied at 40C

decreased when compared to the affinity at 370C. The discrepancy of these results needs to be

studied further.

3. T H E P H Y S I O L O G I C A L SIGNIFICANCE O F T H E

[125I]IODOMELATONIN BINDING SITES IN CHICKEN BRAIN

The possible physiological significance of the high affinity [•'•^Ijiodomelatonin

binding sites in the chicken brain is supported by: 1) the Kd values of [^^1] iodomelatonin

binding sites in the chicken brain had the same order of magnitude as melatonin concentrations

in the chicken circulation; 2) the regional distribution pattern of endogenous melatonin

paralleled that of [125I]iodomelatomn binding sites in the chicken brain (Pang et al” 1990);

3) melatonin and related indoles had the same order of potency in the inhibition of

[125I]iodomelatonin binding in the chicken brain membrane preparations and in the inhibition

of H] dopamine release from the chicken retina (Dubocovich, 1988a); 4).

72

[125i]iodomelatonin binding was inversely related to the rhythm of serum melatonin levels

(Table 14) and melatonin manipulations changed the binding capacity ((Table 15,16); 5). GTP

and its analogues decreased the binding capacity of [^^Ijicxiomelatonin in the chicken brain

(Dubocovich et al” 1990a; Table 13) and in solubilized chicken brain membrane preparations

(Ying and Niles, 1991) indicating that these binding sites belong to a family of G-protein

linked receptors. The functional significance of chicken brain melatonin receptor remains to be

established.

73

B. CIRCADIAN RHYTHM AND THE EFFECT OF MELATONIN

MANIPULATION ON THE [ 125I]IODOMELATONIN BINDING SITES

IN THE CHICKEN BRAIN.

Diumal variations of both high affinity binding sites in the brain of chickens (Yuan

et al., 1990), pars tuberalis area of rams (Pelletier et al，1990), suprachiasmatic nuclei

of rats (Laitinen et al” 1989) and low affinity binding sites in discrete brain areas of

hamsters (Anis et al., 1989) and rats (Zisapel et al., 1988) have been reported. It should

be noted that the high and low affinity binding sites had a difference in their respective

Kd of about 100 to 1,000 fold. Thus they may have different mechanisms and functions

in the diumal variation and regulation of these receptors.

In the present study, we tried to explore the possible explanation for the diumal

variation of the [125j]iodomelatonin binding sites in chicken brain by observing the

changes of melatonin binding sites under light and dark photoperiod and following

manipulations of circulating melatonin levels by melatonin injections and pinealectomy.

1 , DIURNAL RHYTHMS OF SERUM MELATONIN LEVELS AND

[125I]IODOMELATONIN BINDING CAPACITIES

Our results showed that the rhythm (the wave-form, phase, and amplitude) of

[^^1] iodomelatonin binding sites in the brain was inversely related to the rhythm of

semm melatonin levels (Table 14). It was demonstrated that melatonin concentrations in

the chicken brain had similar diumal variations as that of melatonin concentrations in the

blood both in the bird (Pang et al” 1983) and the rat (Cardinali et aL，1991). It is

74

suggested that the density of [125I]iodomelatonin binding site in the brain tissue was

saturated by the endogenous melatonin when the melatonin levels in blood and brain are

high during the night time.

Barsano and Baumann (1989) suggested that hormone receptor saturation could be

calculated by the receptor Bmax, K d value and hormone concentration. Brain melatonin

receptor saturation calculated by [^^I]iodomelatomn binding capacity (Bmax), apparent

affinity (Kd)，and brain melatonin concentration (Pang et al” 1991) showed that

[125I]iodomelatonin binding sites were about half (57%) saturated at midlight and close

to completely saturated at middark (Table 26). This may partly explain why there was

about 50 % higher [^^Ijiodomelatonin binding capacity at ML and during the late light

period than that at the MD period, assuming that the 50% decrease of

[1 ^1] iodomelatonin binding at the MD was due to the saturation of the melatonin

receptors by endogenous melatonin.

It is still unknown whether this receptor saturation can initiate the receptor down-

regulation, and/or receptor desensitization. The hypothesis of receptor down regulation

by endogenous melatonin (Reiter, 1981) is consistent with the diumal rhythms of

biological responses to melatonin during certain periods of the day in hamsters (Tamakin

et aL, 1976)，white-footed mice (Glass and Lynch，1982)，rats (Reiter et al., 1980) and

sheep (Karsh，1984). In vitro study showed that the concentrations of melatonin need to

decrease the elicited Ca"1"4" influx by hypothalamic synaptosomes fractions of rats

killed in the morning were higher (1 fxmol/1) than that in the late evening (0.01-1 pmol/l)

(Cardinali et al., 1991). It is probable that the decrease of [125I]iodomelatomn binding

during the dark period was the result of receptor down-regulation by endogenous

melatonin levels.

The physiological implication of the density change in [•'•^liodomelatonin binding

sites is still an open question. Weaver et al., (1990) showed that both the melatonin

75

sensitive and non-sensitive mice had the same density of [125I]iodomdatomn binding

sites in brain tissue. Such a condition was found in rats and hamsters which had a

similar number of [125I]iodomelatomn binding sites but different sensitivity to melatonin

(Vanecek and Jansky, 1989). These findings suggest that increased sensitivity to

melatonin in animals is probably due to a difference in a post-receptor mechanism

(second messenger transduction system) rather than to a difference in the density or

location of the receptors (Vanecek and Jansky, 1989). Measurement of the cellular

effects of melatonin receptors is required before definite conclusions can be drawn.

A similar change in high affinity [•'•^]iodomelatonin binding density was reported

in pars tuberalis/median eminence of ram (Pelletier et aL, 1990). When the animal was

exposed to light throughout the night prior to slaughter there was a significant increase in

the apparent number of [^^1]iodomelatonin binding sites in comparison to animals

maintained under darkness, while the Kd values were similar in both groups (Pelletier et

al., 1990). The authors suggested that the number of binding sites was light-dependent.

An opposite variation was reported by (Laitinen et al., 1989). In the rat suprachiasmatic

nucleus the binding density increased towards the end of the dark period and decreased at

the end of light period. The authors suggested that this rhythm might be due to a shift of

the putative melatonin receptor from a high affinity (40 pmol/) to a low affinity state (50.8

nmol/1) (Laitinen et al., 1989). As this low affinity was in a concentration range outside

the range of [ 12^I]icdomelatonin used in our assays, this lower affinity form could not

be measured in the present study. In order to demonstrate the possible change of the

high affinity (picomolar) to low affinity [^^liodomelatonin binding sites (nanomolar) in

chicken brain at different times in the photoperiod, higher concentrations of the

radioligand would be necessary for further studies.

76

2. EFFECTS OF MELATONIN ADMINISTRATION ON SERUM

MELATONIN LEVELS AND [125I]IODOMELATONIN BINDING

CAPACITIES

Following the administration of exogenous melatonin, serum melatonin levels

increased greatly with the abolition of the diurnal variation. In these animals,

[125I]icxiomelatomn binding capacities decreased 68% during midlight, with no change

during middark (Table 15). This suggests that during midlight the high concentration of

exogenous melatonin could saturate the brain melatonin receptors and reduce the binding

densities.

3 • EFFECTS OF PINEALECTOMY ON SERUM MELATONIN LEVELS

AND [1 2 5I]IODOMELATONIN BINDING IN THE BRAIN

Six weeks following pinealectomy, serum melatonin levels were significantly lower

during the dark period without any significant change during the light period (Table 16).

There was a 523% and 53.8% increase of [^^I]icxiomelatomn binding capacities in the

pinealectomized animal killed at midlight and middark respectively. This could be due to

the removal of the main source of circulating melatonin. The fact that pinealectomy did

not abolish the rhythm of brain [ ^IJiodomelatonin binding sites suggests that the brain

melatonin receptor rhythm is not only dependent on pineal melatonin. The importance of

melatonin secretion from extra-pineal sources such as the retina, Haderian gland, gut

and lacrimal gland (Pang and Allen, 1985) in regulating binding sites in the brain should

also be investigated.

It is speculated from our studies that [^^IJiodomelatonin binding sites in the

chicken brain might have an endogenous rhythm with the peak occurring in the light

77

period and the nadir in the dark period，which is independent of pineal melatonin

secretion. However，the binding capacities are thought to be regulated by endogenous

and exogenous melatonin. The possibility of re-establishment of the [125I]icdomelatomn

binding rhythm a few weeks after pinealectomy needs to be studied.

The hypothesis that the melatonin receptors have an endogenous rhythm correlates

well with studies of diumal variation of the low affinity (60-230 nmol/1)

[125I]Iodomelatonin binding sites in discrete brain areas of hamster (Anis et al., 1989)

and rat (Zisapel et aL, 1988). It has been demonstrated that diurnal variation of low

affinity [^^Hlcxiomelatonin binding sites was unlikely to be merely the consequence of

changes in content of the hormone or of down-regulation by elevated nocturnal melatonin

(Anis et a l , 1989). This speculation is supported by 1). The Kd value of the low affinity

binding sites (Zisapel et al., 1988; Anis et al•，1989) was several hundred folds higher

than the circulatory and brain melatonin concentration (Pang and Brown, 1983); 2). The

distribution of the binding site in the brain of rats (Zisapel et al., 1988) was not parallel to

that of the distribution of endogenous brain melatonin (Pang et al., 1983) nor the brain

uptake of exogenous [^H]melatomn as reported by Anton-Tay (1971); 3). The profile of

24 h rhythms of the Iodomelatonin binding sites in discrete brain areas of the

hamster or the rat was shown to be regionally specific, with different patterns and phases

in the hypothalamus, hippocampus, medulla-pons and midbrain, but no variation in the

parietal cortex throughout a 24 h period (Anis et al” 1989; Zisapel et al” 1988); 4) The

timed daily melatonin injection for four weeks had no effect on the density and diumal

variation of the [^^I]iodomelatonin binding in most of the discrete brain area of the rats

(Zisapel et al.，1991) and hamsters (Anis et al” 1989) despite prolonged duration of

elevated melatonin (the exception being the hippocampus); 5). In young male rats，

pinealectomy did not affect the amplitude or shape of daily variations of

[^^I]iodomelatonin binding in the hypothalamus and hippocampus (Zisapel et al.,

78

1991). A l l these studies demonstrated that the fluctuation of melatonin levels during day

and night or the manipulation of melatonin levels by exogenous melatonin injections and

pinealectomy had no key effect on the melatonin binding- This suggests strongly that the

density of low affinity binding sites have their own endogenous rhythm and are not

related to either the circulatory or brain melatonin levels.

It should be noted that in the experiment conducted by Anis and coworkers (1989)

the dosage of melatonin administrated only raised the semm melatonin concentration to

>8 nmol/1 in the hamster which was far lower than the Kd value of [ iodomelatonin

binding sites in the hamster brain synaptsome preparations (60-230 nmol/1). Under this

condition of melatonin administration, the extent of receptor saturation might not have

changed enough to affect the low affinity binding sites. To demonstrate the effect of

melatonin on the nanomolar affinity site，higher dosages of melatonin administration may

be needed.

The other possible explanation for the diumal variation of low affinity binding sites

may be due to a change in affinity. The diumal variations both in melatonin binding

capacities and apparent affinities in synaptosomal preparations of discrete brain areas of

the hamster (Anis et a l , 1989)，and in discrete brain areas of ovariectomized and

oestradiol-treated female rats (Zisapel et al” 1987) have been reported. Anis et al. (1989)

proposed the existence of two types of [^^1]icxiomelatonin binding sites in the hamster

brain: low affinity1 (i.e. Kd value of 230 nmol/1) and 丨high affinity' (i.e. Kd value of 60

nmol/1), with diumal variations in the density of the "low affinity"sites only. Further

insight into the mechanism of diumal variation of melatonin receptor is necessary to

understand the rhythmicity of melatonin actions.

79

C. THE DEVELOPMENT OF [125I]IODOMELATONIN BINDING

SITES IN THE CHICKEN BRAIN

As the degree of biological response from melatonin stimulation can be influenced

by the number and affinity of their receptors along with postreceptor events (Pang et al”

1991; Laitinen et al., 1989; Weaver et al” 1990; Zisapel et al” 1989; Stankov and Reiter,

1990), we studied the characteristics of [125I]iodomelatonin binding sites at the chicken

embryonic stage. To assess further the possible physiological significance of the

melatonin receptor, the developmental pattern and the ontogeny of diumal variation of

[^^Ijiodomelatonin binding sites were also investigated.

1 . CHARACTERISTIC OF [125I]IODOMELATONIN BINDING

SITES IN EMBRYONIC CHICKEN BRAIN

Our studies provide the first detailed characterization of [^^1]iodomelatonin

binding sites in the brain of the chicken during development. Binding of

[^^Ijiodomelatonin to membrane preparations of chicken embryonic brain was

saturable, reversible, specific and of high affinity. At embryonic day 17，the Kd value

obtained by Scatchard analysis was 57.5 土 5.26 pmol/1 (Table 19) which was comparable

to the K d value of 45.1 pmol/1 calculated by the kinetic studies (Figure 18). In addition,

within the range of [^^I]iodomelatonin concentrations used, Scatchard analysis and Hill

plots of saturation data suggest a single class of binding sites in the embryonic chicken

brain (Figure 19). Of all the indoles tested，only melatonin, 6-chloromelatonin, and N-

acetylserotonin showed significant inhibition on the [^^I]iodomelatoniii binding (Table

17). No other indoles showed any significant affinity for these binding sites. Thus, the

binding characteristics of [125I]iodomelatomn in the brain of embryonic day 17 chicken

80

were similar to that of the young chicken. The fact that GTPyS exerts an inhibitory effect

on [125I]iodomelatonin binding at embryonic day 18 indicates that these sites are coupled

to a nucleotide binding protein as early as E 18. These results strongly suggest that

functional melatonin receptors appear early in the embryonic life.

2. T H E EARLY APPEARANCE OF [ 1 2 5 I ] IODOMELATONIN

BINDING SITES IN EMBRYONIC CHICKEN BRAIN

The present results demonstrated clearly the appearance of specific binding of

[125I]iodomelatomn at an early embryonic stage (detectable at embryonic day 10 and with

Kd 46.9 + 15.5 pmol/1 and Bmax 5.89 + 0.52 fmol/mg protein at embryonic day 13，

(Table 19). The data of binding studies were comparable with the development of

chicken pineal gland (Collin et al., 1987), pineal HIOMT (Bernard et al” 1991)，NAT

activity，and melatonin production (Binkley and Geller, 1975; Zeman, 1990). In the

chicken pineal, photoreceptor like cells (rudimentary photoreceptor cells) display an

inner segment at embryonic day 10. By day 14，most of cytological characteristics of the

photoreceptor like cell compartments are acquired (Collin et al” 1987). Sympathetic

nerve fibres appear in the pineal gland of the chicken during the final stages of embryonic

development (17 days) (Collin et al.，1987). Bernard et al. (1991) reported that HIOMT

in the chicken pineal first appeared 4 days before hatching and rose linearly until the 7th

day posthatch. Immunocytochemistry reveals a growing number of HIOMT-positive cells

during the period of 2 days before hatching and 15 days posthatch. The NAT activity

was first detectable at embryonic day 14 (Fraser and Wainwright, 1976; Tanabe et al.，

1983) and increased progressively, then rising rapidly after the time of hatching. NAT

activity reached adult levels around the 20th day after hatching (Binkley and Geller,

81

1975; Zeman, 1990). The earliest detectable level of melatonin in the pineal was reported

in 18-days-old chick embryos with concentration of 85 土 8 pg/mg tissue which was

much lower than in adult chickens (Zeman, 1990).

The early appearance of the [125I]iodomelatonin binding sites at E 10 to E 13 in

the chicken brain was also comparable with the [125I]iodomelatomn binding sites in

mammalian and human brains studied by autoradiography (Weaver et al” 1991) or

receptor binding assays ( Weaver et al” 1988; Williams et al.，1991; Vanecek, 1988a;

Carlson et al.，1991; Reppert at al.，1988a; Yuan et al., 1991). In vitro autoradiography

revealed that the earliest appearance of [ -‘- I]iodomelatomn binding sites in pituitary and

paraventricular nucleus of the thalamus were in the fetal rat from day 17 of gestation

(Williams et aL, 1991). The present study showed that the appearance of

j-125jjjocjomejatonjn binding sites in chicken brain was earlier than the melatonin

production in embryonic chicken pineal. These binding sites also appeared earlier than

[^^Ijiodomelatonin binding sites in rats (Williams et al., 1991) and hamster (Carlson et

al.，1991). The physiological significance of the early development of the

[^^I]iodomelatonin binding sites in the chicken brain should be considered

3 . THE DEVELOPMENTAL PATTERN OF [125I]IODOMELATONIN

BINDING SITES IN PERINATAL CHICKEN

The varying patterns of ontogenetic changes in the concentration of hormone and

neurotransmitter receptors among the different species during the perinatal period have

been well demonstrated (See Scaba, 1987). It was our hypothesis that if the melatonin

receptor represents a physiological binding site, then, as has been shown for other

neurotransmitter receptors, it should also display a distinct developmental profile* Our

82

binding studies revealed that major modification in the number of melatonin binding sites

occur during ontogenesis. An overall increase in the number of melatonin receptors was

observed from embryonic day 13 to posthatch day 7. During this period, there was a 14-

fold increase in the [ AZ^I]iodomelatonin binding numbers as expressed per whole brain

(from 56.1 to 779.3 fmol/brain) and about a 2.4-fold increase when expressed per mg

protein (from 5.89 to 14.25 fmol/mg protein) (Table 19).

Species or tissue differences in the pattern of [125I]iodomelatonin binding site

development have been demonstrated. In the rats, the existence of [^^I]iodomelatonin

binding sites in the anterior pituitary was reported in the 21st day of gestation and then

gradually declined to about 10% by the 29th day (Vanecek, 1988a). The decrease in

density of pituitary melatonin receptors correlated closely with the developmental loss of

the melatonin inhibitory effect on pituitary L H release induced by LHRH in neonatal rats

(Martin and Sattler, 1979). It has been suggested that the marked decrease in the number

of the pituitary melatonin receptors might be the cause of the reported developmental loss

of the melatonin inhibitory effect on LHRH-induced L H release from the anterior

pituitary (Vanecek, 1988a).

In Siberian hamsters, melatonin (10 nmol/1) inhibited forskolin-stimulated cAMP

accumulation in median eminence/pars tuberalis (ME/PT) explants 4 days before birth,

but was ineffective in explants collected on the day of birth. Thus，physiological

sensitivity to melatonin appears to be depressed around the day of birth (Weaver et al.，

1987). However, [125I]iodomelatonin binding sites remained constant throughout the

perinatal period (Carlson et al” 1991). This suggested that the alteration of sensitivity to

melatonin resulted from the altered responsiveness of cAMP regulatory system during

the perinatal period (Carlson et al” 1991).

The different patterns of development of [125I]iodomelatonin binding sites were

also demonstrated in different brain areas. In rats, the concentration of melatonin

83

receptors in pituitary gradually decreased while the concentration of melatonin receptors

in median eminence did not change greatly during the course of postnatal development

(Vanecek, 1988a).

4. T H E DEVELOPMENT OF THE DIURNAL RHYTHM OF

[125I]IODOMELATONIN BINDING SITES IN CHICKEN BRAIN

In all animals studied so far，a diurnal rhythm of melatonin secretion has been

shown (Pang, 1985). The diumal variation of [^2^I]icxiomelatomn binding sites has been

reported in the brains of rats (Zisapel et al., 1988; Laudon et al., 1988; Laitinen et al•，

1989)，hamsters (Anis et al” 1989) and birds (Yuan et al，1990). The mechanism of the

diumal variation of [ ] iodomelatonin binding site in the nervous system is yet to be

revealed. To assess further the possible physiological significance of the melatonin

receptor, we have examined the ontogeny of diumal variation of [^2^]iodomelatonin

binding sites in the chicken brain. Our results showed that a light-dark difference in

[^^]i(xiomelatomn binding was first recorded in 20-days-old chick brain (Table 20).

The development of a diumal variation of [ ^I]iodomelatonin binding sites in chicken

brain correlates well with the diumal variation of pineal NAT activity and melatonin

content reported elsewhere (Binkley and Geller, 1975; Zeman, 1990). During embryonic

development of the chicken, a significant light-dark difference of N-acetyltransferase

(NAT) activity in chicks was observed by the second day after hatching and increased to

a maximum amplitude of over 20-fbId after posthatch day 15 up to adult (Binkley and

Geller, 1975). A significant light-dark difference in pineal melatonin content was

recorded only 10 days posthatching, with an increase in dark-time melatonin content

(Binkley and Geller, 1975).

84

The profile of [^iodomelatonin binding sites during development showed

that there was a tendency toward a reduction in [^^I]iodomelatomii binding sites with

aging (Table 19)，this being associated with a diminishing of the difference of light-dark

binding in adult chicken brain (Table 20). The age-associated reduction in pineal and/or

serum melatonin levels in rats (Pang et al., 1984; Reiter et al” 1981) and hamsters (Pang

and Tang, 1983) have already been demonstrated. Zisapel et aL (1989) reported age-

related decrease in melatonin receptor densities in discrete rat brain areas. The age-

associated decreases of both serum melatonin and brain [^^]iodomelatonin binding

sites might have significant effects on the animals.

5 . THE SIGNIFICANCE OF [125I]IODOMELATONIN BINDING

SITES IN EMBRYONIC CHICKEN BRAIN

The functional significance of [125I]iodomelatonin binding sites in chicken brain

has been well studied (see Discussion A-3)). The functional significance of

I* 125j]iodomelatonin in the early stage of development needs to be demonstrated.

However，the similarity of [^^Ijiodomelatonin binding characteristics between the

embryo and young chicken and the similarity between the developmental pattern of

[•'•^IJiodomelatomii binding sites and that of the pineal NAT activity and melatonin

content suggest that the [^^^I]iodomelatonin binding sites in the early stage of

development may have physiological significance. Putative melatonin receptors in the

early stage of development was also demonstrated in hamster brains by the high binding

affinity and the existence of a functional receptor-G-protein-cffector pathway as early as 4

days before birth (Carlson et aL, 1991).

A major difference between melatonin production in chicken embryo and

mammals is that in avian embryos, the maternal melatonin effects are excluded.

85

In rats, no pineal melatonin is produced during the fetal stage. Rhythmic melatonin

production in the pup appears only two weeks after birth (Tang and Pang, 1984). A

large body of evidence exists which reports that in mammals there is a maternal-fetal

communication of circadian phase (Reppert et al., 1988b). The maternal transfer of

circadian rhythmicity and photoperiodic infoimation to the fetus and pup has been clearly

demonstrated in several species (see review of Reppert et al, 1988b). Perhaps the

melatonin receptor during the mammalian fetal development is necessary to accept the

maternal melatonin signal.

The early appearance of melatonin and melatonin synthesis enzyme in chicken

embiyos at E 14 to E 18 (Zeman, 1990), and [ ]iodomelatonin binding sites at E 10 to

E 13 showed that the expression of the binding sites in the chicken embryonic brain was

earlier than that reported in brains of hamsters (Carlson et al.，1991), and rats (Williams

et al., 1991). The appearance of iodomelatonin binding sites at E 10 to E 13 also

preceded that of NAT activity and pineal melatonin which were detected at embryonic day

14 and 18 respectively (Zeman, 1990), suggesting that the development of melatonin

receptor is earlier than that of the hormone. Further study of the melatonin receptor

functions and the possible effects of melatonin imprinting on its receptors during the early

embryonic stages is needed.

D. BINDING CHARACTERISTICS, PHARMACOLOGICAL PROFILE

AND DEVELOPMENT OF [125I]IODOMELATONIN BINDING SITES IN

THE CHICKEN RETINA

The precursors and synthesizing enzymes in the melatonin biosynthetic pathway

have been demonstrated in the retina of vertebrates, hence substantiating the postulation

86

that melatonin may be synthesized in situ (Wiechmann, 1986; Wiechmann et al., 1988).

In birds and mamnials, retinal melatonin may function as a paracrine secretion and

regulate photomechanical changes, disk shedding of the photoreceptor, retinal dopamine

release, and intraocular pressure (see review of Pang and Allen, 1986). In some lower

vertebrates, in addition to the paracrine action, retinal melatonin may also be released into

the circulation and be responsible for endocrine functions (Pang et al., 1989). The

melatonin effects mentioned above suggest that the retina, whilst a site for melatonin

synthesis and release, is also a target organ for melatonin. The presence of melatonin

receptors in the retina has been speculated by the pharmacological study of Dubocovich

(1985). Picomolar concentrations of melatonin were shown to selectively inhibit Ca++

dependent release of dopamine from rabbit and chicken retina in vitro through the

activation of a site possessing functional and pharmacological characteristics of a receptor

for the indole (Dubocovich，1983). Melatonin receptors (labelled by [3H]melatonin) have

been reported in the retinal tissue of trout (Gern et al” 1981) and frog (Wiechmann,

1986). Up until now, the melatonin binding sites labelled by [^^I]iodomelatonin in

retinal tissue have only been reported in chick (Dubocovich, 1987; Laitinen and

Saavedra, 1990a)，rabbit (Blazynski et al., 1989) and tree shrew (Lu et al.，1991). In the

present study, we identified the binding characteristics of [^%]icxiomelatomn binding

sites in the chicken retina. The diurnal variation of the binding capacity and the

development of [ ^Ijiodomelatonin binding sites from embiyonic 10 day to posthatch 4

weeks were also studied.

1 • CHARACTERISTICS OF [12SI]IODOMELATONIN BINDING

SITES IN CHICK EMBRYO AND CHICK RETINA

87

The [l Sfjjodomelatonin binding characteristics both in embryo and young chick

are comparable. The results showed that the specific binding of [125I]iodomelatomn to

both the embryo and young chick retinal membrane preparations belonged to an identical

binding site and satisfied all the criteria for a hormone binding site: i.e. it was saturable,

reversible, specific and of high affinity. Both in the embryo and young chick, the

linearity of the Scatchard plot and Hill coefficient suggest that [^^Jicxiomelatomn binds

to a single class of sites under the present experimental conditions (Figure 26 and 28).

Kinetic studies showed that they had a similar time course of association and dissociation

of the binding (Figure 25 and 27)，The pharmacological affinity of [ ^Ijiodomelatonin

binding sites in chicken retina (both embryo and young chicken) was in a similar order to

that of chicken brain: melatonin > 6-chloromelatomn > N-acetylserotonin (Table 22).

The [^^Ijiodomelatonin binding affinity of the chick retina in the present study

(with Kd value of 366.5 土 96.5 Pmol/1) was consistent with that reported by Dubocovich

et al. (1987) (Table 27) The binding parameters from kinetic and competitive studies are

comparable to those reported by Dubocovich and Takahashi (1987).

Our results suggest that the chicken brain and retina [ I]iodomelatonin binding

sites belong to the same class of high affinity binding sites (ML-1 receptor, Dubocovich,

1988a). In addition, we demonstrated that binding sites in the brain and retina of

embryonic and young chicks showed similar binding association and dissociation

processes and the same order of pharmacological profile: melatonin > 6-chloronielatonin

> N-acetylserotonin. The main difference between the [125I]iodomelatonin binding

characteristics in the chick retina and brain was the binding affinity. The Kd value in

young chicken retina (350-450 pmol/1) reported by Dubocovich and Takahashi (1987)

and the present study (Table 27) was slightly higher than the Kd values in the chicken

brain (about 50-80 pmol/1) in the present study and other high affinity binding sites

reported including the brain of lizard (Rivkees et al” 1989a) and chicken (Rivkees et al”

88

1989b), the pars tuberalis of sheep (Morgan et al” 1989) and suprachiasmatic nuclei of

rat (Laitinen et al.，1990b). Whether these discrepancies reflect differences in

methodology or in receptor characteristics has not been addressed. In the present study,

the high Kd value obtained from the chicken retina could be partly explained by the

higher non-specific binding which was caused by the presence of the pigment epithelium

in the retina membrane preparations. Using in vitro autoradiographic techniques, Laitinen

and Saavedra (1990a) demonstrated two bands of radioactivity in 2-day-chick eyeball

section, one restricted to the retina (inner plexiform layer) with a single class of high

affinity binding site (Kd 19.8 pmol/1) and the other to the pigment epithelium. The retina

(inner plexiform layer) binding was displaceable with an excess of unlabelled melatonin

while binding to the pigment epithelium was not. The authors suggested that the binding

of [^I]iodomelatomn to the pigment epithelium was nonsaturable and with low affinity

(Laitinen and Saavedra, 1990a). It has been demonstrated that melanin bind non-

specifically to several small molecules in the pigment epithelium, e.g. the benzazapines

(Aqui et al.，1987). It is probable that there was high nonspecific binding to the pigment

epithelium which resulted in high binding capacity and low affinity.

2 . THE DEVELOPMENT OF THE [ 1 2 5 I ] IODOMEL ATONIN

BINDING SITES IN CHICKEN RETINA

Detectable [125I]iodomelatonin binding first appeared at embryonic day 10.

Specific binding increased quickly during the embryonic stage and thereafter gradually

throughout all the perinatal period (Table 24). The time sequence and developmental

pattern of [^^1]iodomelatonin binding sites in chicken retina was similar to that in the

brain. The development of [ ^^I]iodonielatoniii binding sites in chicken retina was

89

consistent with the ontogenic development of chicken retina NAT activity and melatonin

synthesis (luvone, 1990). During embryonic development, daytime NAT activity was

consistently detectable by embryonic day 6 when most retinal photoreceptor and neuron

precursors were postmitotic, and primitive photoreceptors were first observed (Gnm，

1982). NAT activity began to increase progressively on E 17，reaching a maximum

daytime value on E 20 and a maximum light time value on posthatch day 6 (luvone,

1990).

3 . THE DIURNAL VARIATION OF [ 1 2 5 I ] I O D O M E L A T O N I N

BINDING SITES IN CHICKEN RETINA

In the present study, the diumal variation of [ ^1] iodomelatonin binding sites in 4-

week-old chick retinal membrane preparations was determined. The binding density

during the light period was significantly higher than during the night time. There were no

significant changes in the Kd values (Table 23). Comparison with the binding amplitude

of the diumal variation in chick brain，showed a light time increase of [^^Ijiodomelatonin

in the chick retina of 47% which was in the same order of that in chick brains. The

developmental diumal variation of chick retina NAT activity was studied by luvone

(1990). During embryonic development, significant light-dark differences of retina NAT

activity were first observed on embryo day 20，and increased to 迁 maximum amplitude of

six-fold by posthatch day 3. Circadian rhythmicity of NAT activity appeared to develop at

or prior to hatching (luvone, 1990). In a two point investigation, Reppert and Sagar

(1983) studied the developmental pattern of the day:night melatonin variation in the

chicken retinal from 16 days of embryo to adulthood. A significantly higher retinal

melatonin level in the dark period was first detected in the 19-day-old embryo. Retinal

90

melatonin contents at mid-dark increased with age until adulthood. Whether the light-dark

binding difference represents a similar mechanism of the receptor saturation by

endogenous melatonin and/or down-regulation needs to be studied.

91

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Agonists and antagonists of putative melatonin receptors

AGON(STS ANTAGONISTS

FIGURE 1

107

Figure 2. A representative experiment of melatonin iodination showing the T L C

separation profile. Iodogen (2 (xg in 20 jil of chloroform) in a 6 x 50 mm glass tube was dried

down under nitrogen. Melatonin (20 ig in 20 of 0.1 mol/1 phosphate buffer, pH 6.0) was

first added to the Iodogen tube (containing 2 jxg of Iodogen) and Na[125I] (400 jxCi in 20 fxl

0.1 mol/1 phosphate buffer，pH 6.0) was added immediately afterwards. The reaction was

allowed to proceed for 1 min at room temperature while vortexing. The reaction product was

then extracted twice in chloroform (100 pi) and subjected to TLC using silica gel sheet 20x20

cm (with fluorescent indicator) and 30 ml of ethyl acetate was used as solvent One cm sections

of silica gel were cut and extracted in 2-propanol. Radioactivity levels were measured with a

gamma counter. Rf values for melatonin and [ 125 j j iodomelatonin were 0.29 and 0.41

respectively. M，melatonin; 1 2 5I-M，f1 2 51]iodomelatonin; 1251，free N a f ^ ^ I ]

o

40000 1

30000

20000

10000

125

125

10 15 20 Fraction Number

FIGURE 2

108

Figure 3. A representative experiment of melatonin iodination showing the H P L C

separation profile. Iodogen (2 \ig in 20 \il of chloroform) in a 6 x 50 mm glass tube was dried

down under nitrogen. Melatonin (20 \xg in 20 \i\ of 0.1 mol/1 phosphate buffer, pH 6.0) was

first added to the Iodogen tube (containing 2 jxg of Iodogen) and Na[125I] (400 [id in 20 il

0.1 mol/1 phosphate buffer, pH 6.0) was added immediately afterwards. The reaction was

allowed to proceed for 1 min at room temperature while vortexing. 丁he reaction product was

then extracted twice in chloroform (100 \i\) and blown to dryness under nitrogen gas,

dissolved in 100 \il methanol and subjected to HPLC (Bio-Rad 700 HPLC system) using

revers-phase column (Bio-Rad Laboratories 250 X 4 mm Bio-Sil ODR-5S) and eluted with a

2-propanal gradient from 5-45 % in 90 min at a flow rate of 0.5 ml/min. Elution of melatonin

was monitored by U V spectrophotometrically (220 nm). Radioactivity of the fractions collected

were counted. The retention times was 10 min for melatonin, 29 min for [125I]iodomelatomn

(fraction 28-30), and 3 min for free Na[125i] (fraction 2-3). M, melatonin;

[125I]iodomelatonin; 125I, free Na[^^I]

10 20 30 40 50 60 70 Fraction number

FIGURE 3

o X

G L o

109

Figure 4. Time courses of [ ^]icxiomelatonin (A) association to and (B) dissociation from

brain membrane preparations of 10-week-old chicken incubated at 370C for 5，10，15，20，30，

40 60 and 80 min. (A) Inset, pseudo-first-order plot of the association data with an association

rate constant ( k i ) 9 .5 X 10 8 (LR)e, the concentration of specific

[125i]iodomelatonin binding at equilibrium; (LR)a, the concentration of [125I]iodomelatonin

binding at time t Dissociation was initiated by addition of melatonin 1 iimolll and incubated

for the indicated time (5，10, 15，20，30，40，60,80 minutes). (B) Inset, first-order plot of the

dissociation data with an dissociation rate constant (k-i) 6.7 X 10 -2 min - 1 • (LR)o, the

concentration of specific [ i o d o m e l s i t o n i i i binding just before the addition of competing

melatonin; (LR)d，the concentration of specific [125I]iodomelatonin binding at time t after

initiation of dissociation. The kinetically derived Kd value (K4/K1) was 70.5 pmol/L Data

shown are from one experiment done in duplicate.

10 20 30 40

Time (min.)

20 40 60 Time (min.)

10 20 30 40 5C Time (min.)

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110

Figure 5. Time courses of [ ^I]iodomelatonin (A) association to and (B) dissociation from

brain membrane preparations of 10 week-old chicken after incubation at 4 0 C for 15,30，60，

120，180，240，300, 360 minutes. (A) inset，pseudo-first-order plot of the association data

with an association rate constant (ki) 7.1 X 107 milri.M"1. (LR)e, the concentration of specific

[l^ijiodomelatonin binding at equilibrium; (LR)a，the concentration of [^^I]iodomelatonin

binding at time t Dissociation was initiated by addition of melatonin 10 and incubated

for the indicated time (15, 30，60, 120, 180，240，360 minutes). (B) Inset, first-order plot of

the dissociation data with an dissociation rate constant ( L i ) 2.66 X lO'3 min-1. (LR)o, the

0.7，

100 200 300 400 Time (min.)

u.u 0 100 200 300 400

Time (min.)

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100 200 300 400

Time (min.)

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Figure 6A. A representative experiment showing saturation isotherm of [^^Ijiodomelatonin

binding to membrane preparations from the brain of 10 weeks old chicken killed in the middle

of the light period. Membrane preparations were incubated at S ^ C with [^^I]iodomelatonin

concentrations from 8-310 pmol/L Nonspecific binding was defined as binding in the presence

of 1 |xmol/l melatonin. TB，total binding; SB, specific binding; NSB, non-specific binding.

Figure 6B. A representative experiment showing a Scatchard plot of the above binding data

at 37 0 C with dissociation constant (Kd) 65.5 pmol/1, a maximal number of binding sites

(Bmax) 13.1 fmol/mg of protein and a correlation coefficient (r) 0.96. Data are means of

duplicate values from one experiment B, bound; B/F, bound/free.

40000 丁 3

NSB SB

30000

20000

10000

0 100 200 300

[ 1 25 j j iodomelatonin (pmol/ll)

4 0 0

4 6 8 10

Bound (fmol / mg protein)

12 1 4

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Figure 7A . A representative experiment showing the saturation isotherm of

f l^Sj^odomelatonin binding to membrane preparations from the brain of 10 weeks old chicken

100001

8000

6000

4000

2000

50 100 150

[ 1 25 i 】iodomelatonin (pmol / 1 )

200

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FTCrURE 7

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113

Figure 8A. A representative experiment showing the saturation isotherm of

[125i]iodomelatonin binding to membrane preparations from the brain of 10 weeks old chicken

killed in the middle of the light period. Membrane preparations were incubated at 250C with

[125i]iodomdatomn concentrations from 9-120 pmol/1. Nonspecific binding was defined as

binding in the presence of 1 |xmol/l melatonin. TB, total binding; SB, specific binding; NSB,

non-specific binding.

Figure 8B. A representative experiment showing Scatchard plot of the above binding data at

250C with dissociation constant (Kd) 88.4 pmol/1，a maximal number of binding sites

(Bmax) 8.2 fmol/mg of protein and a correlation coefficient (r) 0.973. Data are means of

duplicate values from one experiment B, bound; B/F, bound/free.

25 50 75 100 125 150 [ 1 2 5 | ] iodomelatonin (pmol /1)

Bound (fmol mg protein)

10

FIGURE 8

TB NSB SB

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Figure 9. Relationship between specific binding of [^^I]iodomelatonin to pigeon brain

membrane preparations and protein concentration in the membrane preparations. The linear

regression between the specific binding of [^^I]iodomelatx)nin (9.4 pmol/1) to the membrane

preparations with protein concentrations from 70 \ig to 820 \ig gave a straight line, the

coefficient of correlation being 0.995.

1200

200 400 600 800 1000 Protein concentration (jtg)

FIGUTRTR q

2 1000-sX 3 800-m

•！ 600-s • pa t • • • pii

o 2 0 0 • o a W A

115

Figure 10. Time courses of iodomelatonin (A) association to and (B) dissociation

from quail brain membrane preparations. Membrane preparations were incubated with 19.8

pmol/1 [125I]iodomelatonin at 4 0 C for the indicated time (15，30，60, 120，180，240, 420,

540 minutes). (A) inset, pseudofirst-order plot of the association data with an association rate

constant (ki) 4 1 X 107 mirr iM - 1 . (LR)e, the concentration of specific [l^I]iodomelatomn

binding at equilibrium; (LR)a, the concentration of iodomelatonin binding at time t.

Dissociation was initiated by addition of melatonin 10 jxmol/1 and incubated for the indicated

time (15，30，60, 120，180，240, 300 minutes). (B) Inset, first-order plot of the dissociation

data with an dissociation rate constant (k_i) 2.96 X 10*3 min"1. (LR)o, the concentration of

specific [ i c x i o m e l a t o n i n binding just before the addition of competing melatonin; (LR)d,

the concentration of specific [^^I]iodomelatonin binding at time t after imtiation of

dissociation. The kinetically derived Kd value (K-i/Ki/) was 72.2 pmol/1. Data shown are

from one experiment done in duplicate.

0.81

100 200 300 Time (min.)

400 500 600

0 150 300 450 Time (min.)

100 200 300 400 Time (min.)

u.u* 0 100 200 300 400 500

Time (min.)

FIGURE 10

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Figure 11. Time courses of [125I]iodomelatonin (A) association to and (B) dissociation

from pigeon brain membrane preparations. Membrane preparations were incubated with 18.2

pmol/1 [125I]iodomelatonin at 4 0 C for the indicated time (15，30，60，120，180，240，300，

420, 540 minutes). (A) inset, pseudo-first-order plot of the association data with an

association rate constant (ki) 1.1 X 108 (LR)e, the concentration of specific

[12^I]icxiomelatonin binding at equilibrium; (LR)a, the concentration of [12^I]iodomelatonin

binding at time t. Dissociation was initiated by addition of melatonin 10 iimoVl and incubated

for the indicated time (30，60» 120，180，240, 300，420 540 minutes). (B) Inset, first-order

plot of the dissociation data with an dissociation rate constant (lei) 2.9 X 10-3 min*1. (LR)o,

the concentration of specific [^^]iodomelatonin binding just before the addition of competing

melatonin; (LR)d, the concentration of specific [125I]iodomelatonin binding at time t after

initiation of dissociation. The kinetically derived Kd value (K4/K1/) was 26.9 pmol/L Data

shown are from one experiment done in duplicate.

150 300 450 Time (min.)

0.0J

0 100 200 300 400 500 600 Time (min.)

FTG imE 11

G 主

I 'c' 」

1.0

0.8

0.6

0 .4 .

0:2,

"O r-5 o c 三 §1 ra 2 a5 CX E朵 o c *n 今 £ g

tn CM

in CM

Figure 12. Time courses of [^^I]iodomelatonin (A) association to and (B) dissociation

from duck brain membrane preparations. Membrane preparations were incubated with 30.2

pmol/1 [12^]iodomelatonin at 4 0C temperature for the indicated time (15，30，60, 120, 180，

240, 300，360, 420，540 minutes). (A) inset, pseudo-first-order plot of the association data

with an association rate constant (ki) 3.6 X 107 (LR)e, the concentration of specific

[125i]io(iomelatonin binding at equilibrium; (LR)a，the concentration of [^^I]iodomelatomn

binding at time t. Dissociation was initiated by addition of melatonin 10 pmol/l and incubated

for the indicated time (30，60, 120，180, 240, 300，360, 420，540 minutes). (B) Inset, first-

order plot of the dissociation data with an dissociation rate constant (k-i) 2.6 X 10"3 min"1.

(LR)o, the concentration of specific [^^]iodomelatonin binding just before the addition of

competing melatonin; (LR)d，the concentration of specific [ ^I]iodomelatonin binding at time

t after initiation of dissociation. The kinetically derived Kd value (Kli/Ki/) was 71.8 pmol/L

Data shown are from one experiment done in duplicate.

100 200 300 400 500 600

Time (min.)

FTGIJRF 1 7

100 200 300 400 Time (min.)

100 200 300 400 Time (min.)

500

旦 0.0-

5 -0.5-

I-1.0« a: zL -1.5. c 」-2.0-

X3 C 3 5 •s芎 § 3 1 1 号？

ZT兰 to 04

J - J

T35 C g JD C c § 1 ？5 2 O CL E o E -o ^ o o

CO C\i

118

Figure 13A. A representative experiment showing the saturation isotherm of

[l^ijicxiomelatonin binding to membrane preparations from the brain of pigeon killed in the

middle of the light period. The membrane preparations were incubated at 4 0 C with

[125i] iodomelatonin concentrations from 8-90 pmol/1. Nonspecific binding was defined as

binding in the presence of 10 fxmol/1 melatonin. TB, total binding; SB, specific binding; NSB,

non-specific binding.

Figure 13B* A representative experiment showing Scatchard plot of the above binding data

with a dissociation constant (Kd) 25.9 pmol/1, a maximal number of binding sites (Bmax)

5.65 fmol/mg of protein and 汪 correlation coefficient (r) 0.986. Data are means of duplicate

values from one experiment. B, bound; B/F, bound/free.

12000

10000

8000

6000

4000

Z000

0 20 40 SO 80 1 cc [125I]iodome!atonin (pmol/I)

Bound (fmol/mg prote in)

ao^h/TJcnoal

(

rd8iotl.£JJ2iop.2

【

JS<N二

s

BBS

rSN

FIGURE 13

Figure 14A. A representative experiment showing the saturation isotherm of

[125i]iodomelatonin binding to membrane preparations from the brain of duck killed in the

middle of the light period. The membrane preparations were incubated at 4 0 C with

[^^Ijiodomelatonin concentrations from 15-114 pmol/1. Nonspecific binding was defined as

binding in the presence of 10 fxmol/l melatonin. TB，total binding; SB, specific binding; NSB，

non-specific binding.

Figure 14B. A representative experiment showing Scatchard plot of the above binding data

with a dissociation constant (Kd) 28.6 pmol/1, a maximal number of binding sites (Bmax)

11.8 fmol/mg of protein and a correlation coefficient (r) 0.995. Data are means of duplicate

values from one experiment. B, bound; B/F, bound/free.

15000

10000-

5000

〇 T B SB

20 40 60 80 100 120 [ 1 2 5 | ] iodomelatonin (pmol / 1 )

Bound (fmol / mg protein)

HGUUE14

120

figure ISA. A representative experiment showing the saturation isotherm o f

[125i]iodomelatonin binding to membrane preparations from the brain of quail killed in the

middle of the light period. The membrane preparations were incubated at 4®C with

[^^Ijicxiomelatonin concentrations from 10-125 pmol/L Nonspecific binding was defined as

binding in the presence of 10 \xmo\ll melatonin. TB，total binding; SB, specific binding; NSB,

non-specific binding.

Figure 15B. A representative experiment showing Scatchard plot of the above binding data

with a dissociation constant (Kd) 24.4 pmol/1, a maximal number of binding sites (Bmax)

5.86 fmol/mg of protein and a correlation coefficient (r) 0.977. Data are means of duplicate

values from one experiment. B, bound; B/F，bound/free.

12000

10000

2 8000 -

6000

4000

2000

• NSB o SB

50 100 150 [1251]iodomelatonin (pmol/I)

Bound (fmol/mg protein)

FIGURE 15

121

Figure 16. Representative experiments showing competition curves for iaMbition of

[125i]i0<iomelatomn binding by various melatonin agonists ( ICfU .moj/i 動 10* md/l) ia

chicken (A), duck (B), pigeon (C) and quail (D) brain membrai« prejaratioim The mcinh^ne

preparations were incubated with 25.2 pmol/1 [125I3iodomelatooiii aiKl various coiKrcntrations

of melatonin (curve a , ) , 6-chioromelatonin (curve b , ) and N-acetyl-Shydroxytrypiamific

(curves c, )• Values are means of duplicates.

o — melatonin m 6-chioromeL a NAS

14 -12 -10 - 8 -6 - 4 ^ Log [concentration^ mol/1

-12 -10 -8 -6 Log [concentration] (moi/l)

120

1 0 0 -

60

4 0

20

-12 •10 -14 8 - 4 Log [concentration] (mol/1)

FIGURE If i

4 2 -10 - 8 - 6 Log [concentration] (moi/l)

c o • _

• M X： c Vi o c 44 o u 知 a.

c o tm

c O 4*# c 4i 0 w 由 a.

c a • _ •M* <«•* c

« * • — 0 c 4t O U <b a.

Figure 17. The representative Scatchard isotherms of [125I]iodomelatonin binding to brain

membrane preparations of chicken killed at lh，5h，9h, 13h，17h and 21h. The animals were

kept under a 12h: 12h light-dark cycle (lights on at 03(X)h). Note the lower binding capacities

of chickens killed at 2100h and OlOOh in the dark period Data are means of duplicate values

from one experiment. The dissociation constant (Kd), maximal number of binding sites

(Bmax), and 汪 correlation coefficient (r) of the above Scatchard isotherms are as following:

1 h: Kd 94.0 pmol/1, Bmax 9.8 fmol/mg protein, r 0,948.

5 h: Kd 79.9 pmol/1, Bmax 15.4 fmol/mg protein, r 0.962

9 h: Kd 70 pmol/1，Bmax 18.8 fmol/mg protein, r 0.931.

13 h: Kd 69.9 pmol/1, Bmax 2 L 0 fmol/mg protein, r 0.991

17 h: Kd 54.7 pmol/1，Bmax 18.9 fmol/mg protein, r 0.996.

21 h: Kd 90.9 pmol/1, Bmax 1 0 3 fmol/mg protein, r 0.977

OSOOh 090011 1300h

8 12 18 Bound (fmol/mg protein)

Dark Time 1700h 2 1 0 0 h OlOOh

B 12 16 Bound (fmol/mg protein)

2C/TCI90Q.

OE woloEj) osJ/TDunog

350

o

o

0

5

1

《

T2C/T uooJd 6

uj/

s9

oe

o

ealLL/

pu

no

Q

3 0 0

2 5 0

2 0 0

1 5 0

FIGURE 17

Figure 18. Time courses of [ ^I]icxlomelatonin (A) association to and (B) dissociation from

17 days old chicken embryo brain membrane preparations. Membrane preparations were

incubated with 10 pmol/1 [^2%]iodomelatonin at 37° C for the indicated time (5，10，15，20,

30，40，60，80 minutes). (A) inset，pseudo-first-order plot of the association data with an

association rate constant (ki) 5.1 X 108 (LR)e，the concentration of specific

[l^Ijiodomelatonin binding at equilibrium; (LR)a, the concentration of [^^Ijiodomelatonin

binding at time t. Dissociation was initiated by addition of melatonin 1 (imol/1 and incubated

for the indicated time (5，10，15，20，30，40，60, 80 minutes). (B) inset, first-order plot of the

dissociation data with an dissociation rate constant (k_i) 2.3 X 10 -2 min - 1 . (LR)o, the

concentration of specific [^^]iodomelatomn binding just before the addition of competing

melatonin; (LR)d, the concentration of specific [125I]iodomelatomn binding at time t after

initiation of dissociation. The kinetically derived Kd value (K.1/K1/) was 45 pmol/L Data

shown are from one experiment done in duplicate.

in CM "T-*

to OJ

Figure 19A. A representative experiment showing saturation isotherm of

[125I]iodomelatonin binding to membrane preparations from the brain of 17-days old chicken

embryo. The membrane preparations were incubated at 37 0C with [125I]iodomelatonin

concentrations from 10-310 pmol/1. Nonspecific binding was defined as binding in the

presence of 1 [imol/1 melatonin. TB, total binding; SB, specific binding; NSB, non-specific

binding.

Figure 19B. A representative experiment showing Scatchard plot of the above binding data

with a dissociation constant (Kd) 33.3 pmol/1, a maximal number of binding sites (Bmax)

10.6 fmol/mg of protein and a correlation coefficient (r) 0.987. Data are means of duplicate

values from one experiment. B，bound; B/F，bound/free.

30000

20000

10000

100 200 300 40C

I ' 2 5 I ] Iodomelatonin (pmol/1}

u.u* 0 2 4 • 6

Bound (fmoi/mq protein)

2 OL o CO c E c

•E 〇 "c5 "a5 E o "O «9

LO OJ

CD CD v— U--a c o

CQ

• NS 〇 T B

鑤SB

FIGURE 19

125

Figure 20. Competitive inhibition curves of [125I]iodomelatonin binding in 17 days old

chicken embryo brain membrane preparations by various melatonin analogues (ICT12 mol/1 to

10"5 mol/1). The membrane preparations were incubated with 15.2 pmol/1 [ 125I]iodomelatonin

and various concentrations of melatonin, 6-chloromelatonin (6-chloromeL) and N-acetyl-5-

hydroxytryptamine (NAS). Values are means of duplicates.

120 n

c 100 -o “

| 80 : •i* • j S -

•S 6 0 -v* o -•v 4 0 攀 c 由 0 “ 1 20 _ 由 •

^ 0:_ . 1

FIGURE 20

126

Figure 21. Development of [125I]iodomelatonin binding sites (expressed fmol/mg protein)

in chicken brain at the ages of embryonic day 13，17; posthatch day 1，7，20, 40; and 6

months old, 12 irionths old chicken. Data are means of S.E.M of 4 animals. The statistics are

shown in Table 19.

FIGURE 21

127

Figure 22. Development of the affinity of [125I]iodomelatomn binding sites in the chicken

brain at the ages of embryonic day 13，17; posthatch day 1，7，20，40; and 6 months old，12

months old chicken. Kd (pmol/l) values are means (土 S.E.M) of 4 animals. The statistics are

shown in Table 19.

20.

00.

8 0

6 0

4 0

20 J/IO

Ed

§

FIGURE 22

128

Figure 23. Ontogeny of the diumal variation of [^^I]iodomelatonin binding capacity in the

chicken brains of indicated ages measured by Scatchard analysis. Values are means (± S.E.M)

of 4 animals. The differences of ML vs MD binding within the same age group were analysized

by one way analysis of variance and student's t-test. *p<0.05.

爾

1

1

cjeoJdD5JJ)

pu

no

m

FIGURE 23

129

Figure 24. A representative experiment showing the effect of GTPyS (ICT5 mol/1) on the

[125I]iodomelatonm binding to membrane preparations from the brain of 18-days old chicken

embryos. The membrane preparations were incubated at 25° C for 1 h with

[12^I]iodomelatonin concentrations ranging from 10-310 pmol/L Nonspecific binding was

defined as binding in the presence of 1 [xmol/1 melatonin. Scatchard plots of the binding data

was linear. It was demonstrated that the GTPyS (10"5 mol/1) decreased the Bmax from 10.7

fmol/mg protein to 5.9 fmol/mg protein without changing the Kd values. Data are means of

duplicate values from one experiment

0.4-1

Without GTPyS GTPyS

iLJL/

pu

no

m

ao

130

Figure 25, Time courses of [ i o d o m e l a t o n i n (A) association to and (B) dissociation from

chicken retinal membrane preparations. Retina membrane preparations of 8-week-old chicken

were incubated with 60 pmol/1 [ ^1] iodomelatonin at 370C for the indicated time (5，10，15,

20, 30, 40，60，80，120 minutes). (A) inset, pseudo-first-order plot of the association data with

an association rate constant (ki) 1.78 X 108 (LR)e，the concentration of specific

[12^I]iodomelatonin binding at equilibrium; (LR)a, the concentration of [125I]icxiomelatomn

binding at time t (B) Dissociation was initiated by adding melatonin to final concentration of 1

pmol/l and incubated for the indicated time (5，10，15，20，30，40，60, 80，120 minutes). (B)

inset, first-order plot of the dissociation data with an dissociation rate constant (k-i) 4.46 X l O

2 min"1. (LR)o, the concentration of specific [^^^1]iodomelatonin binding just before the

addition of competing melatonin; (LR)d，the concentration of specific [^^1]iodomelatonin

binding at time t after initiation of dissociation. The kinetically derived Kd value (Ki/Ki/) was

25.6 pmol/L Data shown are from one experiment done in duplicate.

20 40 60 Time (min.)

8 0 10C

80 120 (min.)

20 40 60 Time (min.)

80 100 120

旦o-| -2*

-4-

5 -6-

*a

L c © B o

i f o •go 一 c

t n CM

£* - j 主 cc j §

cc ci

5 -cooaa J oue

XJunoq

UC-0«I91U0T30I

一

一

gca产

FIGURE 25

131

F i gu re 26A . A representative experiment showing saturation isotherm of

j-^SjjjiQdomelatonin binding to membrane preparations from the retina of B-week-old chicken

killed in the middle of the light period. Membrane preparations were incubated at 31°。with

[125i]i0do melatomn concentrations ranging from 16-607 pmol/L Nonspecific binding was

defined as binding in the presence of 1 jxmol/1 melatonin. TB，total binding; SB, specific

binding; MSB, non-specific binding.

j r jgure 26B. A representative experiment showing Scatchard plot of the above binding data

with a dissociation constant (Kd) 421.9 pmol/1, a maximal number of binding sites (Bmax)

6 L 9 f m o l / m g of protein and a correlation coefficient (r) 0.972. Data are means of duplicate

values from one experiment B，bound; B/F, bound/free.

80000 1

200 r 125

4 0 0 6 0 0

1 ] Iodomelatonin (pmol /1)

60000

40000

20000

SB TB NSB

800

• < -—i i— »—* 1 1 0

2 0 3 0 4 0 5 0 6 0

Bound (fmol / mg protein)

P IGUiyE26

<D £ i t T3 C ZJ o QQ

Figure 27. Time courses of [125I]iodomelatonin (A) association to and (B) dissociation

from chicken embryo retinal membrane preparations. Retina membrane preparations of ISday-

old embryo were incubated with 60 pmol/1 [125I]iodomelatonin at 370C temperature for the

indicated time (5，10，15，20，30，40，60, 80，120 minutes). (A) inset, pseudo-first-order plot

of the association data with an association rate constant (ki) 1.5 X 108 (LR)e, the

concentration of specific [^^I]iodomelatonin binding at equilibrium; (LR)a, the concentration

of [•'•^Iliodomelatomn binding at time t. (B) Dissociation was initiated by adding melatonin

to final concentration of l|imol/l and incubated for the indicated time (5，10, 15，20，30，40，

60，80，120 minutes). (B) inset, first-order plot of the dissociation data with an dissociation

rate constant (k-i) 1.78 X 10-2 min"1. (LR)o, the concentration of specific [-'•^^icxiomelatomn

binding just before the addition of competing melatonin; (LR)d, the concentration of specific

[ i o d o m e l a t o n i n binding at time t after initiation of dissociation. The kinetically derived

Kd value (K.i/Ki/) was 118.6 pmol/L Data shown are from one experiment done in duplicate.

Z4 i

20 40 60 80 100 Time (min.)

0 10 20 30 40 50 Time (min.)

U.U 0 20 40 60 80 100 120 140

Time (min.)

FIGURE 27

£ * •

5 ‘

"D =3

-S

O® 3 2 CD Q .

231 Jc to 04

s 主 s

^

8'

6'

4;

2 CvJ

1

1

1

《U

j0ojd B

JOEJ)

punoqcco

一iseEOPO

二 I

gOJ-1

133

Figure 28A. A representative experiment showing saturation isotherm o f

[l^Sijiodomelatonin binding to membrane preparations from the retina of 15-day-old embryos.

The membrane preparations were incubated at370C with [^^Ijicxiomelatoiiin concentrations

ranging from 16-620 pmol/L Nonspecific binding was defined as binding in the presence of 1

melatonin. TB，total binding; SB, specific binding; NSB, non-specific binding.

Figure 28B. A representative experiment showing Scatchard plot of the above binding data

with a dissociation constant (Kd) 77.7 pmd/1，a maximal number of binding sites (Bmax)

10.94 fmol/mg of protein and a correlation coefficient (r) 0.918. Data are means of duplicate

values from one experiment. B，bound; B/F, bound/free.

20000

10000

NSB 丁 B SB

200 400 600 800

[ 1 2 S I ] I o d o m e l a t o n i n ( pmo l /1 )

0,16-r

0,12

0.08

0 .04

0.00 10 12

Bound (fmol/mg protein)

FIGURE 28

XI c *H c o 4J OS rH 0) H O TS O M r-™i M

t A CM

««1

44 由 L. U. "S. Tf C s> o

CQ

134

Table 1. [3H]melatonin binding sites with nanomolar affinity in the brain and retina.

Animal Region Fractio n

Method K d pmol/1

Bmax fmol/m g protein

Ref

Rat whole brain C M B A 73,000 62.334 Vacas & Cardinali., 1979

Rat hypothalams CY B A 8,650 78.36 Niles et al.，1979

hippocampus CM BA 11,300 166.13

striatum 28,060 61.1

midbrain 302,43 1370.3

Bovine Occipital CM BA u

6.5 Cardinali et al., 1979

cortex 4 .8

amygdala 2.2

striatum <0.5

pons <0.5

RPEC <0.5

Trout retina CY BA 1,250 45 Gem et al., 1981

Frog CM BA 200,00 0

8 .5 Wiechmann et al” 1986

CM, crude membrane preparations. CY，Cytosol preparations. BA, Receptor binding assay. RPEC, Rgmented-epithelium choroid body.

135

Animals Region Fraction Method Kd pmol/1

Bmax fmol/mg protein

Ref

Chickens whole brain CM BA 344 57.6 Dubocovich et al.，1989

Chickens telencephalon

hypothalamus

mid-brain

pons-medulla

cerebellum

CM BA 189

172.6

232.5

137.5

123.5

13.1

23.1

18.2

18.9

2.15

Yuan etal., 1990

Lizard whole brain 23

206

82

118

Rivkees et aL, 1989 (high and low affinity binding sites

Mice SCN, ME/PT ARG Fang et al，1990

Rat ME CM BA 21 8.5 Vanecek et al., 1987

SCN

A P

ARG

ARG

52.8

45.9

16.5

30.8

Laitinen and Saavedra 1990b Laitinen et al.J990

Ram PT CM BA 70 2.31 Pelletier et al” 1990

Ovine PT CM BA 16.5 98.5 Morgan et al., 1989

Human SCN ARG 150 Reppert, 1988a

Human fetus

SCN ARG 110

Human fetus

hypothalamus BA 26.1 5.4 Yuan etal.，1991

Chicken retina IPL ARG 19.8 97.6 Laitinen & Saaved r a , IQOOa

retina CM BA 434 74.0 Dubocovich et al.，1987

Rabbit retina 353 85 Dubocovich et al., 1988a

Tree shrew

retian CM BA 51 1.97 Lu et al., 1991

Ovine

Human

Human fetus

Human fetus

Chicken

Rabbit

Tree shrew

SCN

A P

SCN

SCN

hypothalamus

retina IPL

retina

retina

retian

Method

BA

ARG

ARG

BA

BA

ARG

ARG

BA

ARG

BA

BA

Kd pmol/1

344

189

172.6

232.5

137.5

123.5

23

206

21

52.8

45.9

70

16.5

150

110

26.1

19.8

434

353

51

Bmax fmol/mg protein

57.6

13.1

23.1

18.2

18.9

2.15

82

118

8.5

16.5

30.8

2.31

98.5

5.4

97.6

74.0

85

1.97

Ref

Dubocovich et al.，1989

Yuan etal.，1990

Rivkees et aL, 1989 (high and low affinity binding sites

Fang et al，1990

Vanecek et al., 1987

Laitinen and Saavedra 1990b Laitinen et al.J990

Pelletier et al” 1990

Morgan et al., 1989

Reppert, 1988a

Yuan etal.，1991

Laitinen & Saavedra . 1990a Dubocovich et al.，1987

Dubocovich et al., 1988a

Lu et al., 1991

BA, receptor binding assay. CM, crude membrane preparatioi^i. A . ARG, autoradiography. SCN，supmchiasmatic nuclei. ME, median eminence. ME/PT，median eminence and pars tuberalis. AP, area postrema. IPL, inner plexiform layer

Table 3. ^1]Iodomelatonin binding sites with nanomolar affinity

136

Animals Region Fraction Method Kd nmol/l

Bmax {moling protein

Ref

Rat whole brain

medulla

SS BA 40

229

120

380

Zisapel & A n i s , 1988 Laudon et al., 1987

hypothalamus 290 550

striatum 172 229

cerebellum 74 135

parietal cortex 117 146

hippocampus 104 149

Syrian hamster

whole brain

hypothalamus

CM BA 3.7

1.8

99.3

7 5

P icke r ing a n d Niles, 1990

Syrian hamster*

whole brain SS BA 0.32 10.5

5.6 123

Niles etal., 1987

Syrian hamster

hippocampus

mid-brain

hypothalamus

medulla-pons

SS BA 65

91

230

110

650

430

1040

580

Anis et al., 1989

Djungarian hamste

parietal cortex

whole brian C.M BA

230

1.48

850

293 Duncan et al., 1989

CM，crude membrane preparations. SS, crude synaptosomal preparations. BA，receptor

* T h ^ L ^ i l i n n e a r Scatchard plots yielded high (Kd=0.32 nmol/1, Bmax=5.6 fmol/mg protein) and low (Kd=10.5 nmol/1，Bmax=123 fmol/mg protein) -affinity binding sites.

137

Table 4. The comparison of the binding property of [ ^]icxiomelatonin separated by TLC

and HPLC

TLC

HPLC

Binding with antiserum

(1: 3000 dilution)

T NSB SB B%

5009 309 1047 20.9

4260 108 2569 60.3

Binding with chick brain membrane

preparations (protein 300 [ig/tube)

T NSB SB B%

5009 20 513 10.2

4260 52 1418 33.3

The binding studies were carried out at 40C incubation for 5 hours. Melatonin antiserum

(1:3000 dilution) o r chicken brain membrane preparations 300 \ig/tube) were incubated with

[1 巧I]iodomelatonin . The data show the means of duplicate (cpm). T, total count. NSB,

nonspecific binding. SB, specific binding. B%9 percent of specific binding/total count

Table 5 . The effect of pH or ions on the [1 2 5

I] iodomelatonin binding to the chicken brain

membrane preparations.

Rinding condition

NaCl 100 m M

KC1 100 m M

CaCl2 5 m M

MgCl 2 5 m M

EDTA I m M

pH 2.1

pH 5.0

pH 6.0

pH 6.4

pH 6.6

pH 6.9

pH 7.04

pH 7.15

p H 7 .4

pH 7 .5

pH 7.7

pH 8.2

pH 8.7

PH 8.9

% Binding

8 1 土 7 . 2

9 6 + 3 .8

9 0 + 5 3

1 0 8 土 2 . 9

9 5 + 3 .2

0

5.4 土 7 . 4 ”

5 4 土 6.8**

9 9 + 5 . 5

104 + 6.3

103 ± 4 . 2

103 土 5.8

101 ± 3 . 2

100 + 4 9

9 5 ± 3 . 2

9 0 ± 7 3

91 ± 6.3

72 土 3 . 6 #

67 + 5.2**

T h e experiments were conducted at 40C incubation for 5 h. Specific binding of

[1 2 5

I ] iodomela tonin (26.2 pmol/1) was 0.51-0.74 fmol/mg protein at pH 7 . 4 which was

expressed a s 100%. Data points are the means + S.E.M of 3 experiments. ** pcO.Ol

139

Table 6. The affinity and binding capacity of [1 2 5

I]iodomelatonin binding sites in chicken

brain membrane preparations at 370C, 25

0C and 4

0C.

Temperature Kd (pmol/D Bmax(fmol/mg protein、

40C (3) 42.6 土 2.94 3.87 土 0 3 1

250C (4) 70.1 土 16.2* 11.1 土 1.88**

370C (4) 68.5 土 41** 10.8 土 0.58朱*

Data are the means 土 S.E.M. * ’p<0,05，**p< 0.01 Student't test compared to 40C The

numbers in the parentheses are the numbers of experiments.

140

Table 7 . [-‘-Iodomelatonin binding in the quail brain membrane preparations collected at

different postmortem times.

5 min lOmin 30min 1 h 2 h 4 h 6 h 8 h 10 h 12 h 14 h

B 1.96

+

1.97

+

1.97

+

1.79

+

1.5

u.

1.86

丄

1.94

i

1.76 2.1 2.0 2.0

0.66 0.58 0.58 0.5

iT

0.3

十

0.5 0.49 0.5 0.5 0.5

+

0.9

The quails were killed and the bodies were put in a 40C cold room. The brains were collected

after different postmortem times (5，10，30 minutes and 1，2, 4，6，8，10，12，14 hours). The

brain tissues were then kept in -70oC for 1 day, and then binding studies were conducted. The

membrane preparations (protein content of 640-720 |ig/tube) were incubated with 22 pmol/1

[125i]iodomelatonin at 3 70C for 1 h. The data are the means + S.E.M of 4 brains each in

duplicate. The binding results showed no significant difference at each postmortem time. B,

bound per fmol/mg protein.

141

Table 8. Results of the kinetic study of [1 2 5

I] iodomelatonin binding association to and

dissociation from the brain membrane preparations of chicken，duck，pigeon and quail

chicken duck pigeon

JilXJL

quail

K | ([mol/1]"1 min

1)

K _ i (min"1)

K d (pmol/1)

7.1 X 1 07

2.66 X 10"3

37 .5

3.62 X 1 07

2.6 X 10-3

71.8

1.1 X 108

2.9 X 10 '3

26.9

4.1 X 107

2.96 X lO"3

72.2

Membrane preparations were incubated with 18-30 pmol/1 [l^I]iodomelatonin at 40C for the

indicated time. The was calculated by pseudofirst-order plot of the association. The

was calculated by first-order plot of the dissociation data. Kd values were obtained by the ratio

of Data shown are from one experiment done in duplicate.

142

Table 9. [ 1 2 5I ] iodomela tonin binding capacity and affinity in chicken, duck, pigeon，quail

brain membrane preparations.

chicken quail pigeon duck

Bmax (fmol/mg

protein)

Kd (pmol/1)

3 .87 + 0.13

42.6 ± 2.95

5,6 土 0.34

42.2 土 16

6.65 ± 0.54

50.6 ± 15

10 .4+1 .8

30 ± 2 3

Data are the means 土 S.E.M of 4 animals killed in the middle of the light period. Membrane

preparations were incubated at 40C with [ ^Ijiodomelatonin concentrations from 5-120

pmol/1 fo r 5 h . Nonspecific binding was defined as binding in the presence of 10 ixmol/l

melatonin.

Table 10.

Specificity

of【lboutIJiodom

elatonin binding

to chicken, duck, pig

eon and

quail b

rain m

embran

e preparations.

Ki

(nm

oi/1)

Drugs

chicken

pigeon

quail

duck

selatonin

6-chloromelatoB

.n

N-acetylserotonin

5-hydroxytryptamine

tryptam

ine

5-3

ethoxytry

pto

phol

1

丨 tryptophan

5-h

ydro

xyin

dole

丨3la

cetic

acid

1

丨 acetyJindole-3-carboxaldehyde 5-hydroxytryptophan

3-acetylindole

norep

inep

hrin

e

acetylcholine

L4±0.4

4 (4

)

33

±1

00

(4

)

looo±320 (3

)

V5000

V5000

V5000

V5000

V5000

>5,0

00

>5,0

00

V5000

>5000

V5000

P42±

11 (3

)

5±

L8 (3

)

1,5

00±§

(3)

V5000

V5000

V5000

>5,0

00

V5000

V5000

>5000

>5,0

00

>5,0

00

>5,0

00

14.4

±4

(3)

1L

2±2.6

s

1,5

00±400

(3)

V5,§

V5000

>5,0

00

V5000

>5,0

00

V5000

V5,§

>5,0

00

V5000

V5§

LI

±

P6

(3)

2.3

± L

6 (3

)

1,500±

§ s

V5000

>5,0

00

>5,0

00

V5000

>5,0

00

V5000

V5000

>5,0

00

V5000

V5000

Mem

bran

e preparations w

ere incubated at

40c fo

r 5 h

for

specifc bindinOQ

o『【

1255Romelaonin in

the presence

various

目E-oges raginoo

^33

10—

G30S&s.u 301/1.

IC50

values (nm

oyl) w

ere obtained from

analysis of co

mpetitio

n curves and the K

i was

calculated. T

he num

bers in the

parentheses are

the n

um

ber

of experim

ents. T

he

meanDQ.

屮 gmse.0 show

n in

the

table.

143 Table 11. Regional distribution of [

1 2 5I]iodomelatonin binding in the brain of chicken and

pigeon.

chicken** pigeon*

Bmax Kd B

Resion ffmol/ms protein) Cpmol/D Cfmol/ms protein)

Telencephalon 2.5 土 0.42 52.4 土 18 1.04 + 0.1

Hypothalamus 6.65 土 1.8 38.4 ± 3 3.1 土 0.11

Mid-brain 5.15 ± 0 . 95 59.6 土 0.1 1.78 土0.11

Pons-medulla 4.89 ±0 . 01 56.1 ±11 .4 1.56 ± 0 . 1

Cerebellum 0.39 + 0.035 31 .4+16 0.59 + 0.013

"Regiona l distribution of [^^I]iodomelatomn binding in membrane preparations from brain

of chicken. Values are means 土 S.E.M of Maximal number of binding sites (Bmax) and

dissociation constants (Kd) in Scatchard analysis in each chicken brain region. n= 3 groups of

animals.

木 Regional distribution of [125

I]iodomelatonin binding in membrane preparations from brain

of pigeons. Values are means 土 S.E.M of the binding sites in one-point analysis using

[1 2 5

I ] iodomela tonin concentrations of 39.5 pmol/1 in each brain region. n=3 groups of

animals.

144

Table 12. Subcellular distribution of specific [1 2 5

I]iodomelatonin binding in chicken and

pigeon brain.

Chicken Pigeon

Fraction fmol/mg protein % of total fmol/mg protein % of total

Nuclear 1.65 2 7 0.625 34

Mitochondrial 2.43 40 0.72 39

Microsomal 1.63 26 0.387 21

Cytosol OA 6 OJ 5.6

Brain homogenate were fractionated by differential centrifugation, as described in Materials and

Methods, and the fractions were tested for [125

I]iodomelatonin binding. 丁he values were one

determination in duplicate. The [^^I]iodomelatonin concentrations used for the studies were

28 pmol/1 and 7 3 pmol/1 for the chicken and pigeon binding studies respectively.

145

Table 13. The effect of GTPyS on [1 2 5

I]iodomelatomn binding in the chicken brain.

Control With GTPyS

Bmax (4) 11.8 土 0.88* 8.0 土 0.55

Kd(4) 70.1 + 16.2 77.9 + 7.6

* p< 0.05

Chicken bra in membrane preparations were incubated a t 2 5 ° C for 60 minutes with

[ l^ IJ iodomela tonin ranging from 8-150 pmol/1 with or without GTPyS (10"^ mol/1). The

parentheses show the numbers of animals. Data are the means 土 S.E.M Significant

d i f f e r ence s b e t w e e n each groups were determined b y Student 's t- test .

146 Table 14. Twenty- four hour variation of serum melatonin level, density of brain

[1 2 5 I

] iodomela ton in binding sites (Bmax), and affinity [1 2 5 I

]iodomelatonin binding sites

(Kd) in the chicken brain.

Light period Dark period

time OSOOh 0900h 1300h 1700h 2100h OlOOh

serum

melatonin.

fpmol/1)

30.7±5.6 32.2+21.1 56.8±18.5 398.1土51.6 585.4+159.2 1310.6+68.9

Bmax

(fmol/mg

protein)

16.0±1.1 19.0+0.28 19.7+0.45 18.0+0.82 12.8土 0,82 1L8±1.8

Kd

Jpmol/I)

77 .8+53 91.5+8.2 73.1 土 5,2 68.4±5.8 82.4+5.3 84.6±6.5

Data points a re the means 士 S .E .M of 4 animals. The lighting schedule was L:D 12h:12h

(lights o n OSOOh off 1500h). The data were analyzed by analyses of variance. There were

significant t ime effects in serum melatonin and Bmax, and pair comparisons by student-

Newman-Kuhlfs multiple range tests were conducted. Serum melatonin: (1700h，21(X)h,

OlOOh) v s (OSOOh，0900h, 13(X)h); Bmax of [125

I]iodomelatonin binding: (21(X)h, OlOOh) vs

(OSOOh, OSOOh, 1300h); The above groups within single parentheses are not significantly

different (p>0.05) from each other; groups in separate parentheses are significantly different

(p<0.05) f rom each other.

147 Table 15. Effects o f melatonin injection on the serum melatonin concentration and brain

[1 2 5

I]icxiomelatonin binding sites in the chicken.

serum

melatonin

(pmol/1)

Bmax

(fmol/mg

protein)

Kd

(pmol/1)

Control ML 121.1 ± 45.6

392.2 ± 25.8*

17.7 ± 0.96

10.7 ± 0.8

97.7 ± 16.7

93.0 ± 16.4

Melatonin

injected

2146.5 ±741#

1922 ± 224.1#

10.5 ± 0.7#

11.9 ± 0 . 7

98.5 ± 8.9

99.3 ± 10.6

n = number o f samples; Bmax = density of brain [^2 5

I]iodomelatomn binding sites; Kd =

affinity of [ iodomelatonin binding sites; ML = Mid-light; MD = mid-dark. Data shown are

means 土 S . E . M * p<0.05 between M D vs ML within the same group; # p<0.05 between the

melatonin injected and control group.

148 Table 16. Effects of pinealectomy on the serum melatonin concentration and brain

[125i]iodomelatonin binding sites in the chicken.

serum

melatonin

(pmol/1)

Bmax

(fmol/mg

protein)

Kd

(pmol/1)

Control ML

MD

37.5 士 193

357.7 土 112术*

17.2 士 1.2

10.4 土 0.9**

94.4 土 10.6

98.0 土 17.8

Pineal-

ectomized

28.4 土 15.9

75.8 ± 24.5#

26.2 土 2.7#

16.0 ± 1.5*宋#

130 ± 13.2

98.0 ±17.8

n = number o f samples; Bmax = density of brain [1 2

^I]iodomelatonin binding sites; Kd =

affinity of [1 2 5

I]icxiomelatonin binding sites; ML = Mid-light; MD = mid-dark. Data presented

are means 土 S.E.M.; ** p<0.05 between MD vs M L within the same group; # p<0.05 between

the pinealectomized and control group.

149 Table 17. Specificity of [

1 2 5I]iodomelatonin binding to young chicken (10-week-old) and

embryo (17-day-old) brain membrane preparations.

embryo young chicken

Drugs Ki (nmoVY)

melatonin 0.4 土 0.12 0 3 5 ± 0.21

6-chloromelatonin 0.5 + 0.16 0.45 土 0.32

N-acetylserotonin 550 1,000

5-hydroxytryptamine >5,000 >5,000

tryptamine >5,000 >5,000

5-methoxytryptophol >5,000 >5,000

1-tryptophan >5,000 >5,000

5-hydroxyindole-3-acetic acid >5,000 >5,000

l-acetylindole-3-carboxaldehyde >5.000 >5,000

5-hydroxytryptDphan >5,000 >5,000

3-acetylindole >5,000 >5,000

norepinephrine >5,000 >5,000

acetylcholine >5,000 >5,000

epinepherin >5,000 >5,000

hydroxymelatonin >5,000 >5,000

harmaline >5.000 >5,000

Ki was calculated from IC50 values which were obtained from competitive inhibition curves

of [1 2 5

I] iodomelatonin (30-40 pmol/1) binding by various indoles and neurotransmitters (10"

13 mol/1 t o 10"5 mol/1) in young chicken and embryo brian membrane preparations. The

experiments were conducted a t 3 7 ° C incubation for 1 h. Data for melatonin and 6-

chloromelatonin are the mean of three experiments. Data for the other indoles are one

experiment done in duplicate.

g i HA

！=r Q CL Q < o*

3 0 53 r-t-0

r-n — bO Ol •—4 ImmmA

1 3 o

5 . » 2： J3 O-3 *

00 «5

B CO

D 0 Sf o"

1 I p

s

3 2 -

| | 1 1 l i 5

1 1 | 4

1 31 f v S ^ ^ ^ 8： 、- CTQ

戋 2

i+ — Ui In

^ g § h-* ^

^ J ^ f t o^ P s ^ ^

u t m

0 i < H-Ui 0\

i— U> vo 00 VO 00

^ 00 o ^ w OS H- KV 00

S S On hh 00

U> 各 vo g s “ U On i+ I t l l

^ ^ S — '\o

00 H-t- p

S t o CO o H-00

<1 00 ^ 、 i ON 谷 ^ ！0 to U) <1 Ot 屮 i+ 十

H-4 二 to d ^

= 8 8 S

^ l i g H- <1 ^ oo bo Ux

00 X

to o H-H-k bO On

in ！t"

H-4 ^

萃 g Os to H-h-* tn In

^ 5 5； 汊 8S ‘

H- H- ^ \o P

5$ O s » ^ ^ t o

t o 05 5 -H-* "-"J O 92 H bo J 0 0 H-rr l l a s r： P ^ N U\

^ vo

to K 00 P *N>

^ s ^ 5n J : p g s "

All

data are

represented as

Sean

dt S

.E.M

. of

4- anim

als. Statistical significance betw

een the

varioujscrQroups

was

tested by the one

丨 way

analysis

。『

variance (A

Z6V

A)

and sc

ltiple

comparisons tested by the

New

2an

丨Ksrs

analysis. *

and

**

within

single parentheses

P3 sig

nifican

tly d

ifferent

at p<

pow

目Q-ool

J.。gpoJ=r.<。

The sig

nifican

t differen

ce o

f【R

ulso

dom

elatonin

bin

din

g capacity

expressed p

er gram

brain

tissue in

the chicken brain mem

brane preparations

E

F

n 10.76; df

n 7/2

4;

p

A P

ol,

(E13

vs E

17,

35, H

20=^

H40S-

M6,

M12**)；

(E17

vs H

7

J H20**,

H40**,

M6, M

12**)； (H

I vs H7**,

moj

i*，Mltoy

(

Mchvs M

40

錾)

The sig

nifican

t differen

ce o

f【lto

5so

dom

elatonin

bin

din

g sp

acity

expressed per

mOQP32.

3&Qchicken

brain mem

brane preparations

had F

n

30.8

9;

df =

7/2

4;

p <

0.0

1, (E

13 vs

H7**,

mon,

H40=,

M6**,

M12**)；

(HI vs

H7, H

40**)； (E

17 vs H

7, H

40**)； (M

6 v

s H40**)；

(M12

vs H

40*)

The sig

nifican

t differen

ce o

f【1255odom

elatonin

bin

din

g capacity

expressed in

per

brain

in the chicken brain m

embrane preparations had

F =

30009; d

f =

7/2

4;

j?

A

Pol,

(E13

s, H

20**,

H40**, M

6**, M

12**)； (E

17

vs H

7,

H20**,

u M

6**,

M12**)；

(HI vs

H20,

u

I.AM12 j; (H

7 V

SH

4C

M6**M

12J;

(H20 v

s H40**,

M6JM

12

J

The sig

nifican

t differen

ce of【125J】

iRom

elatonin bindinoo

p32.

^expressed in K

d values in

the chicken brain mem

brane

pspiions had

F

“

2.5

9;

df

N 7

/24;

p <

005, (E

13 v

s M

12*, H

40*)

——OO QO 7 O JO On 00 VO t o ON t o 1+1+1+1+ — O VO 广

I p

a c 3 £L < ？S3 a . B. 疔 t3 S, r-«n —

•—4 Ummmi a

i £L p 1

o 5. S3 a 4

5* o -5*

00 c«

m £3 & O O c r

| 0 ！=J

1

衾 2 VO ^

00 >—* t o

W W 3 3

in bo vo H-o In t o

oo ,, a \ u 1+ 0 H- H-H-* W Ul

U M ON H- : 1 + 1+

s 云 如 5 U k> bo H- ^1+ H-

《 0 0 “

s 啡 s …j+ o o o o o ^ OH- H-O ^ P 0 ? 0

00

— H oo m t o a \ ^ y t S bo t o o + + + + ！T™4 o ^ —

^ t o Ul On *

h-* H-* ON tA 1

O O 00 ’

00 00 1+ Ul ^1+ h-H-i + o 》 P l r t

0 0

沒VO

P 2 . ^ w 2. o 5.

t 2¾ D . o 目 % o o

2 I l § | + B w

• * § •

3 八gL p 平 0 H

g & p jri

- 3 ^ 口 | 8 2 , w 3 ? r

a o 穸 ^ o | §

1 2

CL ^

l i o c r S S ' “ a .

5* 00

h**.* g » 0 CO 1 o

00 §

"O

3 0

1 £ CO

5* n> CL

l l

151

Table 21. The effect of GTPyS on embryo brain [^^I]iodomelatonin binding.

Control With GTPyS

Bmax (5) 10.6 土 0.64** 5.7 土 0.33

K d (5) 31.5 + 3.1 39.2 + 4.1

O The 18-day-old chicken embryo brain membrane preparations were incubated at 25 C for 60

min with [ -‘- iodomelatonin concentrations ranging from 10-310 pmol/1 with or without

GTPyS (10"5 mol/1). Data are the means 土 S.E.M of five animals. Significant differences

between groups were determined by Student's t-test. ** p<0.01.

152

Table 22 . Specificity of [1 2 5

I]iodomelatonin binding to chicken (8-week-old) and embryo

(15-day-old) retina membrane preparations

Drugs 8-week-old 15-day-embryo

Ki (nmol/l)

melatonin LO 1.8

6-chloromelatonin 11.2 5.1

N-acetylserotonin 50 45

5-hydroxytryptamine >5,000 >5,000

tryptamine >5,000 >5,000

Smethoxytryptophol >5,000 >5,000

3-acetylindole >5,000 >5,000

norepinephrine >5,000 >5,000

acetylcholine >5,000 >5,000

epinepherine >5,000 >5,000

hydroxymelatonin >5,000 >5,000

harmaline >5,000 >5,000

Ki (nmol/1) was calculated from IC50 values whish was obtained from competitive inhibition

curves of [1 2 5

I] iodomelatomn (30 pmol/1) binding by various indoles and neurotransmitters

( ICT1 3

mol/1 to ICT5 mol/1) in 8 weeks old chicken and 15-day-old chicken embryo retina

membrane preparations. Data are the mean of two experiments.

153

Table 2 3 . Diumal variation of [^^I]icxiomelatonin binding to chicken retina membrane

preparations.

Bmax (fmol/mg protein) Kd (pM)

M L (n=5) 47.2 土 1.26* 366.5 ± 96.5

MD(n=4) 3 2 . 1 + 4 5 5 498.3 + 112.5

0.05.

Four-week-old chickens were used for binding affinity determinations, n = the number of the

experiments. Data are the means 土 S,EM. Significant differences between each groups were

determined b y student11 test.

154

Table 24. [ 125j j iodomelatonin binding in the development of chicken retina.

age weight o f protein (mg/g B (fmol/mg r a t i o o f

retina (mg) retina protein) SB/TB

E 10 (4) 1 L 7 + 2.5 28.5 + 4 3 0.28 + 0.19 2 3 . 2 + 1 . 8

E 1 5 (6) 14.2 + 2.4 23.7 + 2.8 2 . 4 + 0 3 6 48 + 2.2

E 2 0 ( 5 ) 56.4 + 4.9 2 8 . 2 + 1 . 9 3.92 ± 0.62 4 8 ± 2 . 8

H 2 8 ( 8 ) 110.3 ± 1 6 33.1 ± 12.5 5.77 土 0.6 59.5 土 2.8

The parenthesis show the numbers of the animals used. Data are the means 土 S.E.M. The

b ind ing s tudies were performed by one-point analysis using of 46 .8 pmol/l

[1 2 5

I] iodomelatomn SB，specific binding; TB, total binding.

Tab?r

泛 cosp

ariso

n of【

口olIjiodomelatonin

binding

一口

chicken

brain

.

§

Tissu

e M

embrane

Incu

batio

n

Dubocovich et al

(1990)

35-42

丨 day

丨

OE

whole

brain

44,0

00

g p

ellet

40P 2h.

Stehle

Rivkocs ct

al Y

c

目sE-

(1989b)

(1990

and

piessudy)

2

丨 day-old

12

丨 day

丨。Id

tectum opticum

w

hole brain 5a§

pellet

280P 40

min.

250P30

min

6-1

2l

-week

-oQ

: w

hole brian

44,0

00

pellet

%c, 5h

目

d

370P lh

.

Ions

Sodium

K+M

gt, c

at,

GT1P

inhib

ition

no effect no effect

inhib

ition

no effect in

hib

ition

Affm

it、

Saturation K

d K

inetics

s

344 pM

407

pM

Density of

sites w

max

fmol/m

g p

rotein

57.6

Specificity m

elatonin 20

nM

-¾)

6-c

hlo

roila

to3.n

9

nM (

扭) N

AS

in

8H2 p

M

16」

Regional d

istributio

n

hypothalamus

pons-midbrain

med

ulk

oblonOQ

ata cortex cerebellum

Subcellular d

istributin

nuclear (P

I) m

itochondrial (P2)

microsom

al

Bin

din

g study

A

RG

4.8

8 fmol/m

OQ『

offs-

4.5

2

3.1

8

443

33払

1.87 fmol/m

cro

^offs-

2.2

3

47.2

pM

37.8

3.7

I (K

i) 7.3

nM

(Ki)

840nM

(Ki)

AR

G

nuclei of visual, au

dito

ry and lim

bic

system

42.6

(40c)，

68.5

(370c)

pM

12.4

(4。

c)，70

(37。

c)

呈

3007 (40c)，

1000Qs)

035

1 (Ki)

P45

i (K

i) r§nM

(K

i)

tdindincroMHcuy

6.6

5

f30y3g protein

5.1

5

489

2.5

(telencephalon) 039

1.65

fmol/3cro

oa

口

2.4

5

L63

Table 26. Calculation of percentage of melatonin binding to the putative melatonin receptor

the chicken brain at midlight and mid-dark

mid- mid- Remark

light dark

A. Total hormone (Ht)

(nmol/1)

B. Free hormone (Hf)

(nmol/1)

0.81 3 .2

0.03 1.86

calculated from brain

melatonin concentration

reported [41]

calculated according to

[(Barsano and Baumann 1989)

see 1

C. Bound hormone (Hb) 0.78

(nmol/1)

1.34 Hb = H t -

D. Dissociation

(Kd) (nmol/1)

0.091 0.082 The mid-light and mid-

dark Kd in Table 1

E. Binding capacity

(nmol/1)

136 0.89 see 2

F. % of melatonin-receptor

complex 57% 100% Bound hormone/Binding

capacity C/E

- Ht + Kd) 士 V(Bmax - Ht + K d )2 - 4(-Ht/Ka)

1: f ree hormone (Hf) 2 一

(Barsano and Baumann. 1989). where Ka = 1/Kd

156

2: In our laboratory, one gram of wet brain tissue had 50 mg of protein in the crude

membrane preparations and the crude membrane preparations have about 70% of the brain total

binding (Yuan et al 1991). Assuming that 1 g of wet brain tissue is 1 ml in volume, then

Binding capacity (fmol/1): Bmax (fmol/mg protein) x 50 mg x lCX)%/70% x 1000 ml

157

Table 27. Comparison of [ ^I]iodomelatonin binding sites in the chicken retina

between laboratories.

embryo retina

Yuan

chick retina

Yuan

chick retina

Dubocovich

Age E 15 days or 18 days 4 weeks or 8 weeks 4-6 weeks

Incubation 370C，1 h 370C, 1 h 0

0C，2 h

Kinetic study

K I 1.5 x 1 08

1.78 X 108

5.5 x 107

K 4 0.0178 0.0446 0.018

KAIK1 (Kd) 118.6 256 342

Saturation study

Bmax fmol/mg pro 10.94 47.2 ± 1.26 74+13 . 6

Kd pmol/1 77.7 366.5 + 96.5 434 ± 5 6

Compitition study (Ki

dM)

Melatonin 3.43 2.0 6.3

6-Chloromelatomn 10.2 22.5 4.0

NAS 90 100 3,000

sm >100,000 >100,000 >100,000