Clinical Endocrinology (1997) 47, 349–355

Anisomycin and verapamil influence the action ofprogesterone on regulation of term humanmyometrial contractile activity

Xin Fu*, Christian Moberg*, Torbjo ¨ rn Ba ckstro m*,Ulf Ulmsten* and Erik Gylfe†*Department of Obstetrics and Gynaecology and†Department of Medical Cell Biology,Uppsala University, Sweden

(Received 26 November 1996; returned for revision 6 February1997; finally revised 12 March 1997; accepted 2 June 1997)

Summary

OBJECTIVE Progesterone has been shown to haveboth stimulatory and inhibitory effects on termhuman myometrial contractile activity. The mecha-nisms involved in this action of progesterone are stillpoorly understood.DESIGN Myometrial tissues were obtained from thelower uterine segment at elective Caesarean sectionof 30 term pregnant women. The contractile activity ofmuscle strips was measured by a superfusion tech-nique and protein synthesis evaluated by [ 3H]-leucineincorporation.RESULTS [3H]-leucine incorporation into term myo-metrial strips was not affected by progesterone(10 mmol/l), but was markedly reduced by the proteinsynthesis inhibitor anisomycin ( P < 0.05). However,progesterone increased frequency and tonus ofcontractions and reduced the activity-area of con-tractions ( P < 0.01). Anisomycin (100 mmol/l) did notchange these effects or the spontaneous contractileactivity. Removal or extracellular Ca 2þ or addition ofthe L-type calcium channel blocker verapamil pre-vented the spontaneous as well as the progesterone-induced contractions, but had less pronouncedeffects on contractions initiated by oxytocin.CONCLUSION The results indicate that the actions ofprogesterone on term myometrial contractile activityoccur without protein synthesis and that increasedCa2þ influx or decreased outward transport of Ca 2þ

may play a possible role.

It is well known that decreased progesterone level andincreased oestrogen production play a key role in the initiationof labour in certain animal species, e.g. sheep, cow, goat andpig (Liggins, 1973; Puriet al., 1982; Ligginset al., 1983; Fuchset al., 1992). In humans, however, the term uterus isprogesterone dominated (Khan-Dawoodet al., 1984; Kofinaset al., 1990) and the concentration of progesterone in theperipheral circulation does not decline at onset of or duringlabour (Tulchinskyet al., 1972; Boroditskyet al., 1977). Inaddition, the progesterone concentration in term uterus issignificantly higher during labour than at elective Caesareandelivery (Haukkamaaet al., 1977). Moreover, the progester-one concentrations in the umbilical cord and maternalperipheral blood are significantly higher in patients who deliverspontaneously than in those with functional dystocia and poorcontractions (Lo¨fgrenet al., 1992). It seems that progesterone isimportant for uterine contractions during human parturition. Onthe other hand, the antiprogesterone RU 486 has been shown toincrease uterine activity and to mature and dilate the pregnantcervix uteri. Recently RU 486 has also been used successfullyfor induction of labour in women at term (Frydmanet al., 1992;Laffargueet al., 1988). The possible role for progesterone in themechanism of parturition in humans remains undetermined.

The effects of progesterone and its metabolites on themyometrial contractile activity in different species has beenstudied intensively bothin vitro and in vivo. In nonpregnantwomen (Becket al., 1982), pregnant women (Lo¨fgren et al.,1992) and rats (Kubli-Garfiaset al., 1983) progesterone andits metabolites inhibit myometrial contractile activity. How-ever, in these studies only the results of intensity (amplitude)or activity-area of contractions have been reported. In ourprevious study,in vitro, we found that progesterone has bothstimulatory and inhibitory effects on term human myometrialcontractile activity, e.g. it increases the contractile frequency,tonus and decreases the amplitude and activity-area ofcontractions (Fuet al., 1993). The mechanisms underlyingthis action are not understood fully.

The classical effect of progesterone and other steroidhormones is to initiate specific cellular functions bymodulating transcription of target genes after interactionwith cytoplasmic receptors which bind in the nucleus (Jensenet al., 1979). Recently considerable interest has also beenfocused on nongenomic actions of steroids (Wassermanet al.,1980; Blackmoreet al., 1990; Rendtet al., 1992), e.g.

349q 1997 Blackwell Science Ltd

Correspondence: Dr. Xin Fu, Department of Obstetrics andGynaecology, Uppsala University, 751 85 Uppsala, Sweden.Fax:þ 46 018 559775.

progesterone has been reported to increase intracellular freecalcium of human spermatozoa and to enhance calciumcurrents in rat myometrium.

Myometrial smooth muscle contraction is initiated by anincreased intracellular free calcium in sarcoplasm which mayresult from the entry of extracellular Ca2þ and/or the release ofCa2þ from internal stores (Wray, 1993). The most importantsource of calcium for long-term and repeated contractions isthat which enters the cell from the external environment(Kawarabayashiet al., 1989; Poliet al., 1990). The calciumchannels of plasma membrane are thought to regulate thiscalcium influx (Miller 1987; Catterall 1988). The purpose of thepresent study was to examine whether the action of progester-one on term human myometrial contractile activity is mediatedby nuclear receptor of progesterone or by calcium channels inthe myometrium. We employed a protein synthesis inhibitor,anisomycin and L-type voltage-dependent calcium channelblocker, verapamil, to block the transcriptional events or theactivity of calcium channels and examine the myometrialcontractile response pattern.

It has been well demonstrated that oxytocin inducedmyometrial contractions are caused by both calcium influxand release of Ca2þ from intracellular stores (Izumiet al.,1990). In the present study we also tested the effects of a

calcium channel blocker on oxytocin-induced contractions andcompared this with progesterone-induced contractions.

Material and methods

Myometrial tissue was obtained from 30 women aged 20–41years who presented normal uncomplicated pregnancies andwere terminated by elective Caesarean section before the onsetof labour (between weeks 38–41 of pregnancy). The indica-tions for Caesarean section included previous Caesareansection (10), breech presentation (6), feto-pelvic disproportion(5), fear of labour (7) and small for gestational age (2). Allpatients gave their informed consent and the project wasapproved by the Ethical Committee of Uppsala University.

Progesterone, verapamil, anisomycin, EGTA [ethylene-glycolbis-(-aminoethylether)-N,N,n0,n0-tetraacetic acid] andsalts were purchased from Sigma Chemical Co. (St Louis,MO, USA), and oxytocin (Syntocinon) from Sandoz Ltd (Basel,Switzerland). [3H]-leucine (145 Ci/mmol) was obtained fromNew England Nuclear (Boston, MA, USA).

The biopsies were taken from the upper edge of the uterineincision before the administration of any oxytocics. Thebiopsies were immediately immersed in ice-cold and oxy-genated Hepes buffer containing (mmol/L): Naþ 122.0, Kþ 4.7,

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60

0

Inte

nsit

y o

f co

ntr

acti

on

s (

mN

)

20 min

(c)

60

0

(b)

60

0

(a)

Fig. 1 (a) Spontaneous contractile activityand effects of (b) progesterone (10mM) aloneand (c) progesterone (10mM) plusanisomycin (100mM) on term humanmyometrial strips (n ¼ 8).

Ca2þ 2.5, Mg2þ 2.5, Cl¹ 136.7, PO¹4 1.2, D-glucose 5.0 and

Hepes 11.0 (pH adjusted to 7.35–7.40 by 2M NaOH). Insome cases this buffer was also supplemented with 10mmol/lprogesterone (dissolved in ethanol; final concentration<0.1%)or with progesterone plus 100mmol/l anisomycin or with100mmol/l anisomycin alone. The experiments were conductedin 5 groups. Biopsies used in 1 group were not re-used in anyother group.

Group I was used for testing the effect of progesteroneand progesterone plus anisomycin on myometrial contractileactivity (n ¼ 8). Myometrial biopsy from each patient wasdivided into 3 muscle strips (1.5 × 0.3 × 0.3 cm). They werepreincubated in Hepes buffer, progesterone 10mM andprogesterone 10mM plus anisomycin 100mmol/l, respectively,for 4 hours at 378C. After preincubation, each muscle stripwas mounted in a 4 ml tissue chamber. The initial tensionwas set to 5–6 mN. Each muscle strip was continuouslyperfused (1 ml/min) with the same composition of the mediumas they preincubated in, at 378C for more than 3 hours. Therecording techniques has been reported previously (Fuet al.,1993).

Group II was used for testing the effects of anisomycin onspontaneous contractile activity (n ¼ 5). Myometrial biopsyfrom each patient was divided into 5 muscle strips(1.5 × 0.3 × 0.3 cm) and immersed in Hepes buffer or supple-mented with 2, 20, 50 and 100mmol/l anisomycin, respectively.They were preincubated in those media for 4 hours at 378Cbefore measuring the contractile activity.

In group III, the protein synthesis was measured as [3H]-leucine incorporation (n ¼ 4). Eighteen myometrial strips(64 6 6 mg) were prepared from each patient. Six strips wereincubated with Hepes buffer, 6 with 10mmol/l progesterone,and 6 with 10mmol/l progesteroneþ 100mmol/l anisomycin at378C. Uptake of [3H]-leucine was measured during the first 6hours of incubation at one hour interval in 1 ml mediumsupplemented with 0.02mCi/ml [3H]-leucine. After incubation,muscle strips were briefly washed 3 times with nonradioactiveHepes buffer. Each strip was then homogenized in 1 ml 5% (byvol) trichloroacetic acid, the pellet was spun down and thesupernatant discharged. The pellet was dissolved in 1 ml 0.5M

NaOH and the radioactivity determined in a liquid scintillationcounter after addition of 10 ml Ready Safe scintillation fluid(Beckman, USA).

Group IV was used for testing the effects of verapamil onspontaneous activity and on progesterone- or oxytocin-inducedcontractions (n ¼ 8). Three muscle strips (1.5 × 0.3 × 0.3 cm)were obtained from each patient. They were mounted andsuperfused with Hepes buffer, 10 mU/ml oxytocin and10mmol/l progesterone, respectively. After stable contractionswere established, 2.5mmol/l verapamil was applied to thedifferent tissue chambers.

Group V for testing the influence of Ca2þ-free media onspontaneous activity and progesterone- or oxytocin-inducedcontractions (n ¼ 5). Three muscle strips (1.5 × 0.3 × 0.3 cm)were obtained from each patient. They were mounted and

Progesterone, calcium channels and myometrial contractions 351

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Time after concentrations began (min)

120

8

220

To

nu

s (

mN

) 6

4

60 80 10040

(c)

120

5000

020

Acti

vit

y a

rea (

mN

sec)

4000

2000

60 80 10040

(b)

120

12

020

Co

ntr

acti

on

s in

terv

al (m

in)

6

4

60 80 10040

(a)

1000

3000

8

10

2

Fig. 2 Effects of progesterone (10mM) and progesterone plusanisomycin (100mM) on contractile intervals, activity-area and tonusof term human myometrial strips. The average contractile interval,activity-area and tonus during 120 min were significantly changed byprogesterone (P < 0.01), but anisomycin had no additional effects.Each point represents the mean of 8 experiments6 SD. Control (A),progesterone (B) and progesterone plus anisomycin (X).

superfused with Hepes buffer, 10 mU/ml oxytocin and10mmol/l progesterone, respectively, in the presence ofextracellular calcium. After stable contractions were estab-lished the extracellular Ca2þ was replaced with 1 mM EGTAwhich prevents calcium influx. Subsequently the extracellularCa2þ was added and EGTA removed from the solutions.

A contraction was defined as an increase in tension of atleast 0.5mN followed by a decline. The contraction interval wasmeasured as the time between the start of 2 consecutivecontractions. The activity-area was defined as the integratedforce from the start of a contraction until 95% recovery of

the peak force, and tonus as the lowest tension betweencontractions.

Mean values6 standard deviation (SD) are presented.Statistical significance of the difference between mean valueswas analysed by repeated measures of analysis of variance,followed by the least-significant-difference test.P < 0.05 wasconsidered significant.

Results

After having been mounted in the tissue chamber, the myo-metrial strips resumed spontaneous contractile activity withinthe 736 13.1 min range. The contraction interval was7 6 0.48 min and increased slightly during 120 minutes. Theactivity-area was 37316 257.4 mNsec and the tonus remainedclose to the initially adjusted level (5 mN) (Figs 1A and 2,n ¼ 8). When the strips were exposed continuously to10mmol/l progesterone after biopsy, the average frequency ofthe myometrial contractile activity during a 120-minute periodincreased 5-fold. Progesterone also enhanced the tonus by 26%and decreased the activity area of the contractions by 84%(Figs 1B and 2, n ¼ 8). The protein synthesis inhibitoranisomycin (100mmol/l) had no effect either on myometrialcontractile activity induced by progesterone (Figs 1C and 2,n ¼ 8) or on the spontaneous contractile activity (data notshown,n ¼ 5).

Under control conditions, the protein synthesis of themyometrial strips measured as [3H]-leucine incorporationincreased with time reaching an apparent saturation after 3hours (Fig. 3,n ¼ 4). The [3H]-leucine incorporation was notaffected by progesterone alone, but it was markedly reduced in

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6

1000

01

Time (hour)

[3H

]-le

ucin

e in

co

rpo

rati

on

(CP

M/m

g w

et

weig

ht)

5

800

600

400

200

2 3 4

Fig. 3 Effects of progesterone (10mM) and progesterone plusanisomycin (100mM) on [3H]-leucine incorporation into myometrialstrips. Myometrial strips were incubated with [3H]-leucine (0.02mCi/ml) for 1 to 6 hours. The results are presented as the mean of 4experiments6 SD. Control (A), progesterone (B) and progesteroneplus anisomycin (X).

40

0

Inte

nsit

y (

mN

)

15 min

20

0.1verapamil 0.25 0.5 1.0 1.5 2.0 2.5

Fig. 4 Effects of increasing concentrations of verapamil on the spontaneous contractile activity of a myometrial strip (n ¼ 3).

strips exposed to the combination of progesterone andanisomycin.

When the myometrial strips were exposed to increasingconcentrations of verapamil the spontaneous contractileactivity was gradually inhibited and completely blocked at2.5mmol/l (Figs 4 and 5A). In 5 of 8 strips exposed toprogesterone the contractile activity was blocked immediatelyby 2.5mmol/l verapamil whereas 5 minutes was required in theremaining 3 strips (Fig. 5). However, the contractile activityobserved in the presence of 10 mU/ml oxytocin was much lesssensitive to 2.5mmol/l verapamil, which gradually reduced theactivity-area but did not entirely eliminate the contractions(Fig. 5C).

The spontaneous contractile activity was rapidly blockedby omission of Ca2þ and simultaneous addition of 1 mM

EGTA (Fig. 6A). The increase in tonus by progesterone wasalso rapidly reversed by Ca2þ omission (Fig. 6B), but thecontractile activity observed in the presence of oxytocin didnot disappear until after 20–30 minutes (Fig. 6C). Uponreintroduction of Ca2þ all these effects were rapidly reversed(Fig. 6).

Discussion

Progesterone has previously been found to have complex effectson term human myometrial contractile activity characterized by

Progesterone, calcium channels and myometrial contractions 353

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60

0

Inte

nsit

y o

f co

ntr

acti

on

s (

mN

)

(c)

60

0

(b)

60

0

(a)

15 min

Verapamil

Verapamil

Verapamil

Fig. 5 Effects of 2.5mM verapamil on thespontaneous, progesterone- and oxytocin-induced contractile activity of term humanmyometrial strips (n ¼ 8). a. control, b.progesterone 10mM, c. 10 mU/ml oxcytocin.

60

0

Inte

nsit

y o

f co

ntr

acti

on

s (

mN

)

15 min

(c)

60

0

(b)

60

0

(a)

–Ca2+

+Ca2+

–Ca2+

+Ca2+

–Ca2+

+Ca2+

Fig. 6 Effect of Ca2þ omission on spontaneous progesterone- andoxytocin-induced contractile activity of term human myometrialstrips (n ¼ 5). The omission of Ca2þ was combined with the additionof 1 mM EGTA. a. control, b. 10mM progesterone, c. 10 mU/mloxytocin.

increased contractile frequency, elevated myometrial tonus andreduced activity-area of contractions (Fuet al., 1993). It isgenerally accepted that progesterone exerts most of its effects ontarget cells by interacting with cytosolic receptors which promotethe translation process by binding in the nucleus (Jensenet al.,1979). Under the present experimental conditions, the proteinsynthesis of myometrial strips, expressed as [3H]-leucineincorporation, reached an apparent maximum after 3 hours ofincubation. This synthesis was sustained by endogenous sourcesof amino acids since the medium only contained trace amounts[3H]-leucine. Progesterone had no effect on synthesis, but theprotein synthesis inhibitor anisomycin caused a pronouncedreduction of [3H]-leucine incorporation. The lack of effect ofprogesterone may be due to downregulation of the cytosolichormone receptors at term pregnancy (Illingworthet al., 1975;Giannopouloset al., 1979).

Despite the lack of effect of progesterone on proteinsynthesis the hormone had pronounced effects on thecontractile activity of the myometrial strips. Moreover, theseeffects persisted even after inhibition of protein synthesis byanisomycin. Also, progesterone effect was eliminated asextracellular Ca2þ was removed, and rapidly reversed afterCa2þ was readded. Consequently it seems that a nongenomicmechanism is involved in the action of progesterone.Progesterone has been found to stimulate Ca2þ influx in frogoocytes and human sperm by a nongenomic mechanismafter binding to cell surface receptors (Wassermanet al., 1980;Blackmoreet al., 1990). The hormone has also been found toincrease the Ca2þ current in nonpregnant rat myometrial cells,although it was not clarified whether that was due to a genomicor nongenomic action (Rendtet al., 1992). Whereas nifedipinehad little effect on Ca2þ currents in rat myometrial cells, inthis study verapamil was found to block the spontaneous as wellas the progesterone-induced contractile activities completely,indicating that they rely on Ca2þ entry through L-type channels.The observation that verapamil had a much less pronouncedeffect on the oxytocin-induced contractions is consistent withthe fact that this hormone promotes contraction both bymobilizing intracellular Ca2þ and by activating influx (Izumietal., 1990; Tasakaet al., 1991).

In conclusion, the present results indicate that non-genomic action of progesterone plays a role in regulation ofthe contractile activity in term human myometrium. Apartfrom a direct effect on the voltage-dependent Ca2þ channels itis possible that the hormone promotes Ca2þ entry bydepolarizing the cells. In human sperm it has thus beenproposed that progesterone induces a depolarising Naþ current(Foresta et al., 1995). An alternative explanation of theeffects of progesterone is that the hormone inhibits Ca2þ

extrusion by the plasma membrane Ca2þ-ATPase or Naþ/Ca2þ

countertransport.

Acknowledgements

This study was supported by the Swedish Medical ResearchCouncil, no. 174-3495.

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