Neuroscience Letters, 135 (1992) 117-120 117 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 08345

HS-142-1, a novel non-peptide ANP antagonist, blocks the cyclic GMP production elicited by natriuretic peptides in PC12 and NG108-15 cells

Shinichiro Toki, Yoshikazu Morishita, Tomoyuki Sano" and Yuzuru Matsuda

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, Tokyo (Japan)

(Received 28 August 1991; Revised version received 16 October 1991; Accepted 18 October 1991)

Key words. Natriuretic peptide; Cyclic GMP formation; HS-142-1; PCI2; NG108-15; Particulate guanylyl cyclase

HS-142-1 is a novel non-peptide antagonist for atrial natriuretic peptide (ANP) receptor. The effect of HS-142-1 on the cyclic GMP production elicited by natriuretic peptides in neuronal cell lines, PC12 and NGI08-15 was examined. Natriuretic peptides such as ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) enhanced cyclic GMP production in a dose-dependent manner. HS-142-1 inhibited cyclic GMP accumulation elicited by natriuretic peptides in a dose-dependent fashion in both cells.The results suggest that HS-142-1 will be an important tool for identification and understanding of the mechanisms by which natriuretic peptides act in nervous systems.

The family of natriuretic peptides (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) [21] and C-type natriuretic peptide (CNP) [22]) are circulating hormones that regulate body fluid volume and blood pressure [11]. The existence of natriuretic peptides and their receptors have been described in widespread area in the brain [13, 15, 19, 25]. The distribution of the peptides in the brain suggests important roles of these peptides as neuromodulators of blood pressure [6], cerebrospinal fluid production [20] and hormone secretion [16].

The existence of at least two functionally and structur- ally distinct ANP receptor types has been reported; ANP functional receptor contains an intracellular guanylyl cyclase domain in its molecule and mainly responsible for the various physiological activities of ANP [2, 23], whereas the ANP-C receptor is apparently not coupled to the guanylyl cyclase and is considered to mediate the metabolic clearance and degradation of ANP [4, 7, 12]. The cloning and expression of cDNA encoding a mam- malian plasma membrane form of guanylyl cyclase has recently revealed the existence of two different subtypes of guanylyl cyclase-containing natriuretic peptide recep- tor [1, 18]. Due to the lack of specific antagonist for the functional receptor, the true functions of the hormones

* Present address: Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Nagaizumicho, Sunto-gun, Shizuoka 411, Japan.

Correspondence." Y. Matsuda, Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida-shi, Tokyo 194, Japan.

are still unclear; the development of such an antagonist has been awaited [11].

Recently, HS-142-1, a novel and non-peptide ANP an- tagonist, was isolated from the culture broth of Aureo- basidium sp. [8]. HS-142-1 competitively and selectively inhibits [125I]ANP binding to its guanylyl cyclase con- taining receptor and effectively blocks the actions of ANP through acting specifically on the guanylyl cyclase- containing ANP receptor [9, 10]. In order to clarify whether HS-142-1 blocks the actions of natriuretic pep- tides on nervous systems, in this report, we examined the effects of HS-142-1 on the cyclic GMP accumulation in- duced by natriuretic peptides in two neurally derived cell lines, the PC12 rat pheochromocytoma and the NG108- 15 mouse neuroblastoma-rat glioma hybridoma.

PC12 cells were grown on 150 cm 2 flask in Dulbecco's modified Eagle's media (DMEM) supplemented with 7.5% fetal calf serum, 7.5% horse serum, 100 U/ml peni- cillin G sodium, and 100 ~tg/ml streptomycin sulfate at 37°C in a humidified atmosphere of 95% air and 5% CO2 as described [3]. NG108-15 cells were cultured on 150 cm 2 flask in DMEM containing 10% horse serum, 100 /~M hypoxanthine, 16/~M deoxythymidine, and 1 ~M aminopterin at 37°C in a humidified atmosphere of 90% air and 10% CO2 as described [24]. Cyclic GMP contents were separately determined intracellularly and extracel- lularly in PC 12 cells but not separately in NG 108-15 cells as reported [3, 24]. In either case, all assays were in tripli- cate and cyclic GMP formed was measured using a radioimmunoassay kit (Yamasa, Japan). Rat ANP (rANP = ANP (1-28), porcine BNP (pBNP -- BNP (1-

118

32)), and porcine CNP (pCNP = CNP (l 22)) were ob- tained from Peptide Institute, Inc., Osaka, Japan.

When PC12 cells were incubated with rANE both ex- tracellular and intracellular levels of cyclic GMP in- creased 10- and 6-fold, respectively, above the basal val- ues seen in the absence of the peptides (Fig. 1A). pBNP stimulated the cyclic GMP accumulation as did rANP with almost equal potency, whereas pCNP was about

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10-fold less effective than rANP and pBNR HS-142-1 inhibited both the extracellular and the intracellular in- creases in cyclic GMP stimulated by 10 7 M of rANP with ICs0 values of 13.5 and 3.4/tg/ml, respectively (Fig. 2A). HS-142-1 inhibited the intracellular and extracellu- lar cyclic GMP productions induced by 10 7 M of pBNP in a concentration-dependent manner as did rANP; ICs0 values were calculated to be 2.8 and 2.4/~g/ml, respec-

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Iog[peptide ( M ) ] rig. 2. The effects of HS-142-1 on the elevation of cyclic ~ P content Fig. 1. Concentration-dependent cyclic GMP accumulation induced by natriuretic peptides in PC12 (A) and NG108-15 (B) cells. Cells were incubated in the presence of rANP ((~), pBNP (~) and pCNP (,",,) for 20 min (PC12) or 10 rain (NGI08-15). A: open symbols indicate the ex- tracellular level of cyclic GMP and closed symbols show cytosolic level. B: all symbols show the total level of both intracellular and extracel- lular cyclic GMP produced. Data are the means _+ S.E.M. (error bars) from an experiment performed in triplicate. Basal value seen in the

absence of natriuretic peptides is also indicated.

induced by natriuretic peptides in PC12 cells, Cyclic GMP production elicited by 10 7 M rANP (A), 10 7 M pBNP (B) or 10 v M pCNP (C) was measured in the presence of varying concentrations of HS- 142-1. Cyclic GMP content was determined at 20 min after the addition of natriuretic peptides. Open bars mean the intracellular levels of cyclic GMP and solid bars indicate the extracellular levels of cyclic GMP. Data are the means _+ S.E.M. (error bars) from an experiment performed in tripli- cate. Basal value seen in the absence of natriuretic peptides is also

indicated.

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tively (Fig. 2B). HS-142-1 also inhibited pCNP-induced intracellular and extracellular cyclic GMP accumula- tions at similar concentrations as did rANP and pBNP (Fig. 2C); ICs0 values were approximately 1.4 and 2.0 /lg/ml, respectively. At a concentration of 100/~g/ml, HS- 142-1 completely abolished all the cases of natriuretic peptide-induced responses (Fig. 2A - C).

In NG108-15 cells, rANP caused a dose-dependent cy- clic GMP accumulation as did in PC12 cells (Fig. 1B). However, it is worthy to note that pBNP is several-fold less potent in stimulating cyclic GMP formation than rANE A significant change of cyclic GMP levels by pCNP was not seen at concentrations up to 10 6 M. Again, HS-142-1 attenuated the increase of cyclic GMP level induced by 10 -7 M rANP or 10 7 M pBNP with almost equal potency; ICs0 values were calculated to be approximately 30 #g/ml in either case (Fig. 3). The inhi- bitory efficacy of HS-142-1 in NG108-15 cells was weaker than that observed in PC12 cells; the cyclic GMP level elevated by rANP of pBNP did not revert to the basal level in the presence of 100/~g/ml of HS-142-1, which was clearly distinct from the results obtained in PC12 cells. The cyclic GMP formation by PC12 of NG108-15 cells stimulated by sodium nitroprusside, which is an activator of soluble guanylyl cyclase, was not inhibited by HS- 142-1 at a concentration up to 100/lg/ml (data not shown). HS-142-1 alone at concentrations up to 100 pg/ml had no effect on the intracellular and ex- tracellular accumulations of cyclic GMP in PC12 and NG108-15 cells. These results indicate that HS-142-1 selectively inhibits natriuretic peptide-induced accumula- tion of cyclic GMP in the two neuronal cell lines.

In order to examine whether HS-142-1 blocks the ac- tion of natriuretic peptides on nervous systems, we used in the present study neurally derived cell lines, PC12 and NG108-15, which are well-characterized and widely used as models of neuronal cells [3, 14, 24]. Natriuretic pep- tides stimulated a dose-dependent accumulation of cyclic GMP in these cells but to different extents, suggesting that receptor subtypes are present in the two cells in dif- ferent portions. Recently, Koller et al. [5] revealed a lig- and specificity of the two different subtypes of ANP functional receptor, ANP A-type (ANPR-A) and B-type (ANPR-B) receptors. ANP and, to a lesser extent, BNP are efficient activators of ANPR-A. The ANPR-B is acti- vated most efficiently by CNP. BNP is also effective at stimulating this cyclase, but is less potent. If so, PC12 cell has both ANPR-A and ANPR-B, but the amount of ANPR-A is much higher than that of ANPR-B, and NG108-15 cell has very little amount of ANPR-B. Fur- ther studies are needed to clarify a ligand/receptor rela- tionship in the two neuronal cell lines. HS-142-1 prevent- ed, with almost equal potency, all cases of cyclic GMP

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Fig. 3. The effects of HS-142-1 on the elevation of cyclic G M P content

induced by natriuretic peptides in N G 108-15 cells. Cyclic G M P produc- tion elicited by 10 -7 M rANP (A) or 10 7 M pBNP (B) was measured in

the presence of varying concentrations of HS-142-1. Cyclic G M P con-

tent was determined at 10 rain after the stimulation with natriuretic

peptides. Each bar means the sum of both intracellular and extracellu-

lar levels of cyclic GMP. Data are the means + S.E.M. (error bars) from

an experiment carried out in triplicate. Basal value seen in the absence

of natriuretic peptides is also indicated.

productions in PC12 cells stimulated by rANE pBNP, and pCNP, suggesting that HS-142-1 equally recognizes the ANPR-A and ANPR-B in PC12 cells. Similar results were obtained by using cultured vascular smooth and endothelial cells in combination with the three natriuretic peptides and HS-142-1 (unpublished observations). However, in NG108-15 cells, HS-142-1 rather weakly in- hibited cyclic GMP response induced by rANP or pBNP

120

and d id n o t abo l i sh c o m p l e t e l y the e l e v a t i o n i nduced by

p B N P to the basa l level at a c o n c e n t r a t i o n o f 100/~g/ml.

A l t h o u g h it is n o t c lear w h y HS-142-1 w e a k l y inh ib i t s the

cycl ic G M P p r o d u c t i o n in N G 1 0 8 - 1 5 cells, the p re sen t

resul ts suggest the poss ib le ex is tence o f a nove l sub type

o f A N P func t iona l recep tor , wh ich is insens i t ive to H S -

142-1, in N G 108-15 cells. To def in i te ly d e t e r m i n e the re-

c e p t o r sub type specif ici ty o f HS-142-1 , m o r e de ta i l ed ex-

p e r i m e n t s wi th cells tha t specif ical ly express each sub-

type h a v e to be u n d e r t a k e n . In any case, t ha t HS-142-1

b locks the ac t ions o f na t r i u r e t i c pep t i de s on n e u r o n a l cell

l ines indica tes the usefulness o f HS-142-1 to cha r ac t e r i z e

and u n d e r s t a n d the m e c h a n i s m s by which na t r i u r e t i c

pep t ides act in n e r v o u s systems. In o u r p r e v i o u s p a p e r

[17], we also s h o w e d tha t HS-142-1 a n t a g o n i z e s ex-

o g e n o u s A N P - i n d u c e d renal r e sponses such as d iures is

and na t r iu res i s in v ivo system. A c c o r d i n g l y , HS-142-1

p r o v i d e s a p o w e r f u l p h a r m a c o l o g i c a l too l fo r e luc ida t ing

the phys io log ica l roles o f na t r iu re t i c pep t ides in n e r v o u s

sys tems which poss ib ly c o n t r i b u t e to the n e u r o m o d u l a -

t ions o f b l o o d pressure and h o r m o n e secre t ion .

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