C-type natriuretic peptide stimulates secretion of growth hormone from rat-pituitary-derived GH3 cells via a cyclic-gmp-mediated pathway

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1 Eur. J. Biochem. 222, (1994) 0 FEBS 1994 C-type natriuretic peptide stimulates secretion of growth hormone from rat-pituitary-derived GH3 cells via a cyclic-gmp-mediated pathway Yoshiyuki SHIMEKAKE, Shigeki OHTA and Kiyoshi NAGATA Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan (Received January 18, 1994) - EJB /1 Although C-type natriuretic peptide (CNP) has been shown to exist at the highest concentration in the anterior pituitary in rat tissues, its physiological role(s) there is (are) not clear. In this study, we report a novel function of CNP examined with anterior pituitary-derived cell lines, GH3 and AtT20/D16v-F2. Both CNP and atrial natriuretic peptide (ANP) increased cellular cgmp levels in both cell lines in dose-dependent manners. CNP, but not ANP, stimulated growth hormone (GH) release from GH3 cells. In contrast, neither ANP nor CNP had any significant effect on the corticotropin release from AtT20/D16v-F2 cells. An activator for cgmp-dependent protein kinase (cgk), dibutyryl cgmp, mimicked the stimulation of GH release from GH3 cells by CNP. Constitutive GH release from GH3 cells was greatly diminished in the presence of inhibitors for CAMP-dependent protein kinase, while stimulative GH release by CNP was not affected. However, inhibitors which can block cgk almost completely diminished the stimulative effect of CNP. An inhibitor for protein kinase C did not show any effect on either constitutive or CNP-stimulative GH release. Our observations indicate that the stimulation of GH release from GH3 cells by CNP is mediated mainly by the cgk signal-transduction pathway, not by CAMP-dependent protein kinase or protein kinase C, through a CNP-specific receptor (possibly ANP-B receptor). Thus, CNP may act as a local modulator in the anterior pituitary. C-type natriuretic peptide (CNP) is the third member of the mammalian natriuretic peptide family discovered following atrial natriuretic peptide (ANP) [l] and brain natriuretic peptide (BNP) [2]. CNP was first isolated from porcine brain [3, 41, then identified in rat and human brains [5, 61. CNP possesses sequence similarity to ANP and BNP. According to the cloned cdna and genes for CNP from various species [5-91, its structure is highly conserved among species and, actually, rat, porcine and human CNP-22, the smaller molecular form with 22 amino acid residues, are identical. CNP has been shown to exert pharmacological actions similar to ANP and BNP, for example, natriuretic, diuretic and vasorelaxant effects [lo, ll], albeit with a lesser Correspondence to K. Nagata, Shionogi Research Laboratories, Shionogi & Co., Ltd., Sagisu, Fukushima-ku, Osaka, Japan 553 Abbreviations. CNP, C-type natriuretic peptide ; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GH, growth hormone ; ACTH, corticotropin ; PRL, prolactin ; cgk, cgmp-dependent protein kinase; cak, CAMP-dependent protein kinase ; PKC, protein kinase C ; db-camp, N6,2 -O-dibutyryladenosine 3 :5 -cyclic monophosphate (sodium salt) ; db-cgmp, iv,2 -O-dibutyrylguanosine 3 :5 -cyclic monophosphate (sodium salt); H-85, N-[2-(N-formyl-p-chlorocinnamylamino)ethyl]-5-isoquinolinesulfona~de; H- 88, N-(2-cinnamylaminoethyl)-5-isoquinolinesulfonamide ; H-89, N- [2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; KT- 5720, (SR, 9S, 11S)-( -)-9-hydroxy-9-hexyloxycarbonyl-8-methy1-2,3,9,10- tetrahydro- 8,11 -epoxy- lh$h,llh -2,7b,11 a- triazadibenzo(a,g)cycloocta(c,d,e)trinden-1 -one; KT5823, (8R,9S,11 S)-(-)- 9-methoxy -9-rnethoxycarbonyl- 8 -methyl-2,3,9,10- tetrahydro- 8,llepoxy - lh,sh,llh - 2,7b,ll a - triazadibenzo(a,g) cycloocta(c,d,e)- trinden-1 -one. potency. While ANP and BNP are mainly cardiac hormones [12], CNP is localized in the brain and little is detected in other peripheral tissues including the heart [5, 6, 131. A receptor specific for CNP, ANP-B (or GC-B) [14], has been shown to be located in the brain and the pituitary gland [15, 161. Therefore, CNP may function in a manner distinct from ANP and BNP as a neuromodulator; it is actually reported to have some effect on the rat central nervous system [ In rat, CNP has been found in the anterior pituitary at the highest level of all the tissues tested [5]. Although the origin of CNP in the pituitary is unknown, we showed in a previous report that somatomammotrophs or somatotrophs could produce CNP [20]. These observations suggest that CNP may also play an important role in the anterior pituitary. To assess the physiological function(s) of CNP in the pituitary gland, we examined the effect of CNP on anterior pituitary-derived established cell lines which have been used in many endocrinology studies. In this paper, we report that CNP, but not ANP, stimulates growth hormone (GH) release from GH3 cells by a cgmp-mediated pathway, not by a CAMP-mediated pathway which has been reported to be the main regulatory mechanism of GH secretion [21, 221. MATERIALS AND METHODS Materials Rat ANP-(1-28), rat CNP-22 were from Peptide Institute Inc. N6,2 -O-Dibutyryladenosine 3 : 5 -cyclic monophosphate (sodium salt) (db-camp), NZ,2 -O-dibutyrylguanosine 3 : 5 -cyclic monophosphate (sodium salt) (db-cgmp) and

2 646 staurosporine were from Sigma Chemical Co. N-[2-(N-Formy1 -p- chlorocinnamy1amino)ethylj isoquinoline - sulfon - amide (H-85), N-(2-cinnamylaminoethy1)-5-isoquinolinesulfonamide (H-88) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) were from Seikagaku Co. (8R,9S, 11 S)-( -)-9-hydroxy-9-hexyloxycarbonyl-8-methyl-2,3,9,10-tetrahydroro-8,11 -epoxy- lh,8h,llh-2,7b,ll a-triazadibenzo(a,g)cycloocta(c,d,e)trinden- 1 -one(kt5720) and (8R,9S,llS)-(- )-9-methoxy -9-methoxycarbonyl- 8 -methyl- 2,3,9,10 - tetrahydro - 8,ll- epoxy - lh,8h,llh - 2,7b,11a- triazadibenzo(a,g)cycloocta(c,d,e)trinden-1-one (KT5823) were from Kyowa Medex Co., Ltd. Cell cultures Established cell lines used in this study were from the American Type Culture Collection. Rat-pituitary-derived GH3 cells were cultured in Ham's F10 medium (Flow Laboratories) supplemented with 15 % horse serum and 2.5 % fetal calf serum. Mouse-pituitary-derived AtT20/D16v-F2 cells were cultured in Dulbecco's modified minimum essential medium (DMEM; Gibco Laboratories) plus 10% fetal calf serum. All cultures were maintained at 37 "C under a 5 % CO, atmosphere log(ipeptldev4 ** I Measurement of cellular cgmp and camp levels Cellular cgmp and CAMP levels were measured as reported elsewhere [lo]. Cells were seeded on 12-well plates (2X105 cells/well) and cultured at 37 C for two days. After washing twice with the incubation medium (serum-free medium, Ham's F10 or DMEM, containing 20mM Hepes, ph 7.4) plus 0.1% bovine serum albumin and 0.5 mm 3-isobutyl-1-methylxanthine (Sigma), cells were incubated in the presence or absence of peptides in the same medium (1 ml/ well) at 37 C for an appropriate period. The cells were immediately disrupted by the addition of ice-cold 6% trichloroacetic acid and then the cellular cgmp and camp levels were measured using RIA kits from Yamasa. Hormone determinations After seeding and washing as described above, cells were incubated with peptides or cyclic nucleotide analogs in the serum-free incubation media at 37 C. After an appropriate period of incubation, cultured supernatants were collected in microcentrifuge tubes, centrifuged at 1000 rpm for 10 min, and the supernatants were subjected to hormone assays. Rat GH and corticotropin (ACTH) concentrations were determined with RIA kits from Amersham and Peninsula Laboratories Inc., respectively. In the studies with inhibitors, cells were incubated with each compound in the serum-free medium for 30 min before addition of CNP. Data analysis Data are presented throughout as means 2SEM (n = 4-6). Statistical analysis was performed by the Student's t test or Dunnett multiple comparison. RESULTS Response of pituitary-derived cells to CNP and ANP Immunoreactive CNP and CNP-specific ANP-B receptor as well as ANP-specific ANP-A receptor has been detected log([peptideym) Fig. 1. Cellular response of pituitary-derived cell lines to CNP and ANP in terms of cgmp generation. Cellular cgmp was measured after 10-min incubation with 0-1 pm CNP (closed circles) or ANP (open circles). (A) GH3 cells; (B) AtT20/D16v-F2 cells. Significantly different (*, P<O.O5 or **, P<O.Ol) from basal (without peptides) levels by Dunnett multiple comparison. in anterior pituitary glands [5, 15, 161. To investigate whether CNP has any biological effect(s) on pituitary-derived cell lines, we first examined the response of those cells to CNP by monitoring the intracellular cgmp level which should increase if guanylate-cyclase-coupled ANP receptor exists on the cell surface. Rat GH3 cells which secrete GH and prolactin (PRL) constitutively, therefore, with somatomammotroph or somatotroph-origin, and mouse AtT20/D16v-F2 cells which secrete ACTH, therefore, with corticotroph-origin, were treated with rat ANP-(1-28) or CNP-22 for 10 min (Fig. 1). Both ANP and CNP increased intracellular cgmp levels in both cell lines. ANP was more effective than CNP in the generation of cgmp in GH3 cells, while CNP was more effective in AtT20/D16v-F2 cells. Taking the receptor selectivity of natriuretic peptides into account [ 14, 231, these results suggest the existence of both ANP-A and ANP-B receptors in these pituitary-derived cells. Effects of CNP and ANP on hormone release from pituitary-derived cells The anterior pituitary is an important tissue for endocrine control and secretes several hormones. Therefore, we next examined the effects of CNP and ANP on hormone release from pituitary-derived cell lines. ACTH release from AtT20I

3 time(h) B T I log([peptidelw Fig. 2. Cellular response of AtT20/D16v-F2 cells to CNP and ANP in terms of ACTH release. (A) Time course of ACTH release. ACTH release was measured after an appropriate period of incubation with 0.1 pm CNP (closed circles), ANF (open circles) or without peptides (open squares). (B) Dose-dependent response of ACTH release to CNP and ANP. ACTH release was measured after 3-h incubation with 0-1 pm CNP (closed symbols) or ANP (open symbols) in the presence (triangles) or absence (circles) of 1 pm corticotropin-releasing factor. D16v-F2 cells was almost linear at least up to 5 h of incubation, and the presence of 0.1 pm CNP or ANP in the media did not produce any significant difference in secreted ACTH levels (Fig. 2A). Neither CNP nor ANP up to 1 pm had any statistically significant effect on ACTH release from these cells 3 h after the addition of peptide in the presence or absence of rat corticotropin-releasing factor (Fig. 2B). GH release from GH3 cells was almost linear at least up to 6 h of incubation, and CNP at 1 pm significantly stimulated GH release even 30 min after its addition (Fig. 3A). CNP stimulated GH secretion in a dose-dependent manner, and exhibited significant stimulation at concentrations higher than 0.1 nm (Fig. 3B). In contrast, ANP up to 1 pm did not show any significant stimulative effect on GH release (Fig. 3B). Possible involvement of cgmp-dependent protein kinase in the stimulation of GH release from GH3 cells by CNP CNP at 1 pm significantly increased intracellular cgmp levels in rat GH3 cells within 10 min and the increased cgmp level lasted for at least 6 h (Fig. 4), being correlated with the stimulation of GH release from GH3 cells by CNP 6 - In P I time (h) 180j ** 80 I % log([paptideym) Fig. 3. Cellular response of GH3 cells to CNP and ANP in terms of GH release. (A) Time course of GH release. GH release was measured after an appropriate period of incubation with 1 pm CNP (closed circles) or without CNP (open circles). ## Significantly dif- ferent (P<O.Ol) from basal (without CNP) levels by Student s t test. (B) Dose-dependent response of GH release to CNP and ANP. GH release was measured after 3-h incubation with 0-1 pm CNP (closed circles) or ANP (open circles). Significantly different (*, P < 0.05 or **, P < 0.01) from basal (without peptides) levels by Dunnett multiple comparison. 300 ~ ## - ## ## I time (h) Fig. 4. Time course of cgmp generation in GH3 cells in response to CNP. Cellular cgmp was measured after an appropriate period of incubation in the presence (closed circles) or absence (open circles) of 1 pm CNP. ## Significantly different (P < 0.01) from basal (without CNP) levels by Student s t test.

4 time (h) "" I ** I C (mm) Fig.5. Db-cGMP is effective on GH release from GH3 cells at concentration lower than those of db-camp. (A) GH release was measured after an appropriate period of incubation with 5 mm dbcamp (triangles), 5 mm db-cgmp (squares) or without (circles). (B) GH release was measured after 1-h incubation with 0-5 mh4 db-camp (triangles) or db-cgmp (squares). ** Significantly different (P < 0.01) from basal level (0 mm) by Dunnett multiple comparison. which lasted up to at least 6 h after its addition (Fig. 3A). The camp level in GH3 cells was not significantly altered by CNP treatment up to 6 h (data not shown), suggesting the improbability of direct camp generation by CNP. Addition of db-camp [an activator for CAMP-dependent protein kinase (cak)] at 5 mm, which has been reported to cause the maximal response of GH release from GH3 cells [24], led to release stimulation up to 6 h after addition (Fig. 5A). The same concentration of db-cgmp [an activator for cgmp-dependent protein kinase (cgk)] also stimulated GH release. A high concentration of db-cgmp may cross-activate cak [25]. As shown in Fig. 5B, db-cgmp stimulated GH release at the lower concentration than db-camp (db-cgmp was effective at 0.2 mmj. In addition, the cellular camp level in GH3 cells was about 30-times higher than the cgmp level, even after stimulation by CNP. These results show that the stimulation of GH release by CNP through cross-activation of cak with cgmp is quite unlikely. The mechanism of stimulation by CNP was further examined using protein kinase inhibitors (Fig. 6). An inhibitor selective to cak, H-89 [26] at 10 pm, caused almost complete loss of constitutive GH release, while the net stimulation by CNP (amount of GH released in the presence of CNP was subtracted from that in the absence of CNP) was not diminished. The analogous compound H-85, which has a much lower affinity and selectivity for protein kinases, did not show any significant loss of both constitutive and CNP-stimulated GH release from GH3 cells at concentrations up to 30 pm. Another analog H- 88, which inhibits both cak and cgk, greatly diminished constitutive as well as CNP-stimulated GH release at 30 pm. A newly developed protein kinase inhibitor with higher selectivity for cak, KT5720 [27], showed a similar result to H-89, causing about 80% loss of constitutive GH release from GH3 cells without any loss of the net stimulated release by CNP at 10 pm, the concentration which has been shown to be effective for fibroblast [28]. The analogous compound, KT5823, which was developed as a cgk-selective inhibitor [27, 281, diminished CNP-stimulated GH release from GH3 cells almost completely without affecting the constitutive GH release. An inhibitor for protein kinase C (PKC), staurosporine, affected neither constitutive nor CNP-stimulated GH release at 0.1 pm, the concentration reported to inhibit PKC effectively in GH3 cells [29]. Thus, PKC should not be involved in constitutive or CNP-stimulated GH release. These results strongly suggest that the constitutive GH release from GH3 cells is mediated by cak pathway and that the stimulation of GH release by CNP is mediated by cgk pathway. General protein and RNA syntheses in GH3 cells were not affected by CNP because neither [3Hluridine nor [?3]methionine incorporation was altered in the presence of 1 pm CNP up to 3 h (data not shown). Northern blot analysis of total RNA from GH3 cells did not exhibit any significant increase of rat GH mrna during the CNP treatment up to 6 h (data not shown). Immunoprecipitation experiments revealed that 14? 2% (n = 4) of newly synthesized GH molecules in GH3 cells was secreted into the cultured medium over 3 h. No significant change in the amount of immunoprecipitable cellular GH was observed up to 3 h of incubation with CNP. Therefore, CNP seemed not to augment specific synthesis of GH at either the RNA or protein level, but to stimulate the release of the hormone stored in secretory granules. DISCUSSION The anterior pituitary contains the highest concentration of CNP in rats [5], which led us to examine the effect of CNP on anterior pituitary cells. ANP has been shown to inhibit ACTH release as a hypothalamic neurohormone [30]. Studies with primary cultures of rat anterior pituitary cells have shown the inhibitory effect of ANP on ACTH and GH release [31, 321. The mechanism, however, was not examined, but was suggested to result from the decrease of the cellular camp level [31]. CNP has been shown to inhibit leuteinizing hormone secretion and to stimulate PRL secretion in vivo through hypothalamic action [ No action of CNP on primary culture cells has been reported. Therefore, the observations in this study are the first to show that CNP may have direct effect(s) on the anterior pituitary. Hormone secretion from the anterior pituitary is regulated mainly by a cak pathway [21, 22, 33-35] which increases the intracellular Ca" by opening voltage-dependent Ca2+ channels [36, 371. In the case of GH, camp stimulates transcription of the GH gene through transcription factor Pit- 1/GHF1 [38]. In contrast, thyrotropin-releasing hormone action is mediated primarily by a PKC pathway [ To date, there has been no direct evidence for the involvement of the cgk pathway in the regulation of hormone secretion.

5 649 H-85 H-88 H-88 H-89 H-89 KT5720 KT5720 KT5823 ST 30pM 1WM 30pM 5pM 1OpM 1pM 10pM lopm O.lpM Fig. 6. Effects of protein kinase inhibitors on GH release from GH3 cells. Cells were incubated for 30 min without (control) or with inhibitors at indicated concentrations, then incubated with 1 pm CNP (hatched bars) or without CNP (open bars). After 3 h, secreted GH was measured. ST, staurosporine. ** Significantly different (P < 0.01) from the basal level (the open bar of the control) by Dunnett multiple comparison. ## Significantly different (P < 0.01) from levels without CNP (open bars in respective sets) by Student s t test. Natriuretic peptides have been shown to exert their biological functions through their guanylate-cyclase-coupled receptors [ Recently, functions mediated by other signal transduction pathways, such as adenyl cyclaseicamp system, through ANP-C (clearance) receptor have also been shown [46]. Stimulation of GH release from GH3 cells by CNP was correlated to cellular cgmp increase (Figs 3 and 4), while CNP did not alter the camp level in GH3 cells. The camp level in the non-treated GH3 cells (5.5 t 0.3 pmol/105 cells in the presence of 0.5 mm 3-isobutyl-1-methylxanthine) was about 10-times higher than that in primary anterior pituitary cells stimulated by 100 nm growth hormone releasing hormone [47], suggesting that permanent activation of a certain type of adenylate cyclase occurs in these tumor cells to cause the constitutive production and secretion of GH at a higher level than normal GH-producing cells. Db-cGMP stimulated GH release from GH3 cells at a concentration lower than dbcamp (Fig. 4). Inhibitors for cak and PKC (H-89, KT5720 and staurosporine) did not affect the net stimulation of GH release by CNP, while inhibitors for cak and cgk (H-88) and for cgk (KT5823) greatly diminished the stimulation (Fig. 6). These results revealed that this stimulation was mainly via the cgk pathway and not the cak or PKC pathway, and the possibility of cross-activation of cak by cgmp in this system was quite unlikely. In addition, no significant augmentation of RNA and protein syntheses (non-specific and specific to GH) by CNP was observed. We have observed similar stimulation of PlU release from GH3 cells by CNP (Shimekake, Y., unpublished results). These observations suggest that the stimulation occurs on the activation of specific protein(s) which promote the secretion pathway (formation of secretory granules, transport to cell membrane, opening of Ca channels, release by exocytosis, etc.) and not on hormone synthesis, because many secretory granules in somatomammotrophs contain both GH and PRL and the release of both hormones is regulated in a parallel manner [48, 491. Surprisingly, ANP increased the cgmp level in GH3 cells more effectively than CNP but had very little effect on GH release (Figs 1 and 3). A possible explanation is that the stimulation of GH release mediated by the cgk pathway is specifically through CNP-specific ANP-B receptor linking to some unkown factor(s) which is involved in the hormonerelease pathway. In other words, the guanylate cyclase do- main of a biological ANP receptor and a certain subtype of cgk may be closely located to each other, and phosphorylate specific substrate(s) to confer receptor-subtype-specific functions. Db-cGMP permeating from the extracellular medium probably stimulates the ANP-B receptor-linked cgk as well as other types of cgks and, therefore, mimicks the effects of CNP on GH release. The stimulation pathway of hormone release by CNP also seems specific to somatomammotrophs or somatotrophs, because CNP could not affect ACTH release from AtT20/D16v-F2 cells, although it effectively stimulated cellular cgmp level in those cells (Figs 1 and 2). The possibility that rat peptides were not effective in mouse cells is unlikely because of the very high conservation in ANP and CNP peptide sequences among species and of the high dosage of up to 1 pm used in this study. Several reports showed the existence of both ANP-A and ANP-B receptors in pituitary glands [16, 171. Observations here suggest that ANP-A and ANP-B receptors may have distinct roles in pituitary glands, as we previously reported the possibility of different roles of ANP receptors in the kidney, that ANP-A may stimulate while ANP-B may repress natriuresis and diuresis [lo]. The concentration of CNP in rat anterior pituitary is about 20 nm [5], and CNP at this concentration can stimulate GH release from GH3 cells by about 50% (Fig. 3B). We do not know whether or not the increase of GH release by this level has a physiological significance, but CNP may play a physiological role in the regulation of GH release because, first, anterior cells may respond to CNP more efficiently than GH3 cells as the response to growth-hormone-releasing hormone has been shown to be much lower in GH3 cells (36% increase at 1 nm in 3 h) than in primary cells (sixfold increase) [50] and, second, the CNP level itself may be stimulated by some other factors in vivo as shown in the cases of bovine aortic endothelial cells [51], chromaffin cells [52] and human THP-1 cells [53]. Actually, GH release from primary anterior pituitary cells was stimulated about fivefold by 0.1 pm CNP (Shimekake, Y., unpublished results), supporting the first possibility. In conclusion, CNP stimulates the GH release from GH3 cells by a cgk pathway in a manner specific to that of the ANP-B receptor and somatomammotrophs. The control of GH release from GH3 cells by CNP is the first system revealing direct involvement of the cgk pathway in the regulation

6 of hormone secretion. Our observations suggest that CNP may be a local modulator within the anterior pituitary in autocrine or paracrine action. REFERENCES 1. Flynn, T. G., De Bold, M. & De Bold, A. J. (1983) Biochem. Biophys. Res. Commun. 117, Sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (1988) Nature 332, Minamino, N., Kangawa, K. & Matsuo, H. (1990) Biochem. Biophys. Res. Commun. 170, Sudoh, T., Minamino, N., Kangawa, K. & Matsuo, H. (1990) Biochem. Biophys. Res. Commun. 168, Komatsu, Y., Nakao, K., Suga, S., Ogawa, Y., Mukoyama, M., Arai, H., Shirakami, G., Hosoda, K., Nakagawa, O., Hama, N., Kishimoto, I. & Imura, H. (1991) Endocrinology 129, Ogawa, Y., Nakao, K., Nakagawa, O., Komatsu, Y., Hosoda, K., Suga, S., Arai, H., Nagata, K., Yoshida, N. & Imura, H. (1992) Hypertension 19, Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (1990) FEBS Lett. 276, Tawaragi, Y., Fuchimura, K., Nakazato, H., Tanaka, S., Minamino, N., Kangawa, K. & Matsuo, H. (1990) Biochem. Biophys. Res. Commun. 172, Tawaragi, Y., Fuchiyama, K., Tanaka, S., Minamino, N., Kangawa, K. & Matsuo, H. (1991) Biochem. Biophys. Res. Comrnun. 175, Shimekake, Y., Kawabata, T., Nakamura, M. & Nagata, K. (1992) FEBS Lett. 309, Furuya, M., Takehisa, M., Minamitake, Y., Kitajima, Y., Hayashi, Y., Ohnuma, N., Ishihara, T., Minamino, N., Kangawa, K. & Matsuo, H. (1990) Biochem. Biophys. Res. Commun. 170, Ogawa, Y., Nakao, K., Mukoyama, M., Hosoda, K., Shirakami, G., Arai, H., Saito, Y., Suga, S., Jougasaki, M. & Imura, H. (1991) Circ. Res. 69, Ueda, S., Minamino, N., Aburaya, M., Kangawa, K., Matsukura, S. & Matsuo, H. (1991) Biochem. Biophys. Rex Comrnun. 175, Koller, K., Lowe, D. G., Bennett, G. L., Minamino, N., Kangawa, K., Matsuo, H. & Goeddel, D. V. (1991) Science 252, Konrad, E. M., Thibault, G. & Schiffrin, E. L. (1992) Regul. Pep. 39, Wilcox, J. N., Augustine, A,, Goeddel, D. V. & Lowe, D. G. (1991) Mol. Cell. Biol. 11, Huang, F.-L. S., Skala, K. D. & Samson, W. K. (1992) J. Neuroendocrinol. 4, Huang, F.-L. S., Skala, K. D. & Samson, W. K. (1992) J. Neuroendocrinol. 4, Samson, W. K., Skala, K. D. & Huang, F.-L. S. (1991) Brain Res. 568, Ohta, S., Shimekake, Y. & Nagata, K. (1993) Mol. Cell. Biol. 13, Bilezikjian, L. M. & Vale, W. (1983) Endocrinology 113, Law, G. J., Ray, K. P. & Wallis, M. (1985) FEBS Lett. 179, Suga, S., Nakao, K., Hosoda, K., Mukoyama, M., Ogawa, Y., Shirakami, G., Arai, H., Saito, Y., Kambayashi, Y., Inouye, K. & Imura, H. (1992) Endocrinology 130, Sletholt, K., Haug, E. & Gautvik, K. M. (1987) Biosci. Rep. 7, Schumacher, H., Miiller, D. & Mukhopadhyay, A. K. (1992) Mol. Cell. Endocrinol. 90, Geilen, C. G., Wieprecht, M., Wieder, T. & Reutter, W. (1992) FEBS Lett. 309, Kase, H., Iwahashi, K., Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A. & Kaneko, M. (1987) Biochem. Biophys. Res. Commun. 142, Gadbois, D. M., Crissman, H. A., Tobey, R. A. & Bradbury, E. M. (1992) Proc. Natl Acad. Sci. USA 89, MacEwan, D. J., Mitchell, R., Thomson, F. J. &Johnson, M. S. (1991) Biochim. Biophys. Acta 1094, Fink, G., Dow, R. C., Casley, D., Johnston, C. I., Lim, A. T., Copolov, D., L., Bennie, J., Carroll, S. & Dick, H. (1991) J. Endocrinol. 131, R9-Rl Shibasaki, T., Naruse, M., Yamauchi, N., Masuda, A., Imaki, T., Naruse, K., Demura, H., Ling, N., Inagami, T. & Shizume, K. (1986) Biochem. Biophys. Res. Commun. 135, Dayanithi, G. & Antoni, F. A. (1990) J. Endocrinol. 125, Rotsztejn, W. H., Dussaillant, M., Nobou, F. & Rosselin, G. (1981) Proc. Natl Acad. Sci. USA 78, Frohman, L. A. & Jansson, J.-0. (1986) Endocrine Reviews 7, Narayanan, Nm, Lussier, B. T., French, M., Moor, B. & Kraicer, J. (1989) Endocrinology 124, Lussier, B. T., Frebch, M. B., Moor, B. C. & Kraicer, J. (1991) Endocrinology 128, Lussier, B. T., Frebch, M. B., Moor, B. C. & Kraicer, J. (1991) Endocrinology 128, McCormick, A., Brady, H., Theill, L. E. & Karin, M. (1990) Nature 345, Sutton, C. A. & Martin, T. F. J. (1982) Endocrinology 110, Rebecchi, M. J. & Gershengorn, M. C. (1983) Biochem. J. 216, Rebecchi, M., Kolesnick, R. M. & Gershengorn, M. C. (1983) J. Biol. Chem. 216, Lowe, D. G., Chang, M.-S., Hellmiss, R., Chen, E., Singh, S., Garbers, D. L. & Goeddel, D. V. (1989) EMBO J. 8, Chinkers, M., Garbers, D. L., Chang, M.-S., Lowe, D. G., Chin, H., Goeddel, D. V. & Schulz, S. (1989) Nature 341, Chang, M.-S., Lowe, D. G., Lewis, M., Hellmiss, R., Chen, E. & Goeddel, D. V. (1989) Nature 341, Schulz, S., Singh, S., Bellet, R. A., Singh, G., Tubb, D. J., Chin, H. & Garbers, D. L. (1989) Cell 58, Anand-Srivastava, M. B. & Trachte, G. J. (1993) Pharmacol. Rev. 45, Puttagunta, A. L., Chik, C. L., Girard, M., O Brien, L. & Ho, A. K. (1992) J. Endocrinol. 135, Nikitovitch-Winer, M. B., Atkins, J. & Maley, B. E. (1987) Endocrinology 121, Kashio, Y., Chomczynski, P., Downs, T. R. & Frohman, L. A. (1990) Endocrinology 127, Gabriel, S. M., Milbury, C. M., Alexander, S. M., Nathanson, J. A. & Martin, J. B. (1989) Neuroendocrinology 50, Suga, S., Nakao, K., Itoh, H., Komatsu, Y., Ogawa, Y., Hama, N. & Imura, H. (1992) J. Clin. Invest. 90, Babinski, K., Fethiere, J., Roy, M., De Lean, A. & Ong, H. (1992) FEBS Lett. 313, Ishizaka, Y., Kangawa, K., Minamino, N., Ishii, K., Takano, S., Eto, T. & Matsuo, H. (1992) Biochem. Biophys. Rex Comrnun. 189,