Resistin regulates pituitary lipid metabolism and inflammation in vivo and in vitro.

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function and also regulates growth hormone (GH) secretion in rat adenopituitary cells cultures with the adipokine. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues, including the pituitary. The aim of this study is to investigate the possible action of resistin on the lipid metabolism in the pituitary gland in vivo (rats in two different nutritional status, fed and fast, treated with resistin on acute and a chronic way) and in vitro (adenopituitary cell cultures treated with the adipokine). Here, by a combination of in vivo and in vitro experimental models, we demonstrated that central acute and chronic administration of resistin enhance mRNA levels of the lipid metabolic enzymes which participated on lipolysis and moreover inhibiting mRNA levels of the lipid metabolic enzymes involved in lipogenesis. Taken together, our results demonstrate for the first time that resistin has a regulatory role on lipid metabolism in the pituitary gland providing a novel insight in relation to the mechanism by which this adipokine can participate in the integrated control of lipid metabolism.

The ghrelin O-acyltransferase-ghrelin system reduces TNF-α-induced apoptosis and autophagy in human visceral adipocytes.

AIMS/HYPOTHESIS: Proinflammatory and proapoptotic cytokines such as TNF-α are upregulated in human obesity. We evaluated the association between ghrelin isoforms (acylated and desacyl ghrelin) and TNF-α in obesity and obesity-associated type 2 diabetes, as well as the potential role of ghrelin in the control of apoptosis and autophagy in human adipocytes. METHODS: Plasma concentrations of the ghrelin isoforms and TNF-α were measured in 194 participants. Ghrelin and ghrelin O-acyltransferase (GOAT) levels were analysed by western-blot, immunohistochemistry and real-time PCR in 53 biopsies of human omental adipose tissue. We also determined the effect of acylated and desacyl ghrelin (10 to 1,000 pmol/l) on TNF-α-induced apoptosis and autophagy-related molecules in omental adipocytes. RESULTS: Circulating concentrations of acylated ghrelin and TNF-α were increased, whereas desacyl ghrelin levels were decreased in obesity-associated type 2 diabetes. Ghrelin and GOAT were produced in omental and subcutaneous adipose tissue. Visceral adipose tissue from obese patients with type 2 diabetes showed higher levels of GOAT, increased adipocyte apoptosis and increased expression of the autophagy-related genes ATG5, BECN1 and ATG7. In differentiating human omental adipocytes, incubation with acylated and desacyl ghrelin reduced TNF-α-induced activation of caspase-8 and caspase-3, and cell death. In addition, acylated ghrelin reduced the basal expression of the autophagy-related genes ATG5 and ATG7, while desacyl ghrelin inhibited the TNF-α-induced increase of ATG5, BECN1 and ATG7 expression. CONCLUSIONS/INTERPRETATION: Apoptosis and autophagy are upregulated in human visceral adipose tissue of patients with type 2 diabetes. Acylated and desacyl ghrelin reduce TNF-α-induced apoptosis and autophagy in human visceral adipocytes.

The new truncated somatostatin receptor variant sst5TMD4 is associated to poor prognosis in breast cancer and increases malignancy in MCF-7 cells.

Somatostatin receptors (sst1-5) are present in different types of tumors, where they inhibit key cellular processes such as proliferation and invasion. Although ssts are densely expressed in breast cancer, especially sst2, their role and therapeutic potential remain uncertain. Recently, we identified a new truncated sst5 variant, sst5TMD4, which is related to the abnormal response of certain pituitary tumors to treatment with somatostatin analogs. Here, we investigated the possible role of sst5TMD4 in breast cancer. This study revealed that sst5TMD4 is absent in normal mammary gland, but is abundant in a subset of poorly differentiated human breast tumors, where its expression correlated to that of sst2. Moreover, in the MCF-7 breast cancer model cell, sst5TMD4 expression increased malignancy features such as invasion and proliferation abilities (both in cell cultures and nude mice). This was likely mediated by sst5TMD4-induced increase in phosphorylated extracellular signal-regulated kinases 1 and 2 and p-Akt levels, and cyclin D3 and Arp2/3 complex expression, which also led to mesenchymal-like phenotype. Interestingly, sst5TMD4 interacts physically with sst2 and thereby alters its signaling, enabling disruption of sst2 inhibitory feedback and providing a plausible basis for our findings. These results suggest that sst5TMD4 could be involved in the pathophysiology of certain types of breast tumors.

Identification of novel genes involved in the plasticity of pituitary melanotropes in amphibians.

Melanotrope cells from the amphibian intermediate lobe are composed of two subpopulations that exhibit opposite secretory behavior: hypersecretory and hormone-storage hyposecretory melanotropes. Isolation of these subpopulations allowed a comparison of their gene expression profiles by differential display, leading to the identification of a number of genes differentially expressed in hypersecretory or hyposecretory melanotropes. Among them, we chose two (preferentially expressed in hyposecretory cells) of unknown function but structurally related to proteins involved in the secretory process: Rab18 and KIAA0555. We demonstrate that, upon activation of the regulated secretory pathway, Rab18 associates with secretory granules, inhibits their mobilization, and, consequently, reduces the secretory capacity of neuroendocrine cells. The other gene, KIAA0555, was predicted by in silico analysis to encode a protein with a long coiled-coil domain, a structural feature also shared by different proteins related to intracellular membrane traffic (i.e., golgins), and a hydrophobic C-terminal domain that could function as a transmembrane domain. A database search unveiled the existence of a KIAA0555 paralogue, KIAA4091, displaying a long coiled-coil region highly similar to that of KIAA0555 and an identical C-terminal transmembrane domain. Both KIAA0555 and KIAA4091 were found to be predominantly expressed in tissues containing cells with regulated secretory pathway, that is, endocrine and neural tissues. Moreover, when exogenously expressed in HEK293 cells, both proteins showed a yuxtanuclear distribution, which partially overlaps with that of a Golgi complex marker, thus suggesting a possible role of these two proteins in the control of the secretory process.

Ghrelin induces growth hormone secretion via a nitric oxide/cGMP signalling pathway.

The presence of ghrelin and its receptor, growth hormone (GH) secretagogue receptor, in the hypothalamus and pituitary, and its ability to stimulate GH release in vivo and in vitro, strongly support a significant role for this peptide in the control of somatotroph function. We previously demonstrated that ghrelin elicits GH secretion directly in somatotrophs by activating two major signalling cascades, which involve inositol phosphate and cAMP. In as much as nitric oxide (NO) and its mediator cGMP have been recently shown to contribute substantially to the response of somatotrophs to key regulatory hormones, including GH-releasing hormone, somatostatin and leptin, we investigated the possible role of this signalling pathway in ghrelin-induced GH release in vitro. Accordingly, cultures of pituitary cells from prepuberal female pigs were challenged with ghrelin (10(-8) m, 30 min) in the absence or presence of activators or blockers of key steps of the NO synthase (NOS)/NO/guanylate cyclase (GC)/cGMP route and GH secretion was measured. Two distinct activators of the NO route, S-nitroso-N-acetylpenicillamine (SNAP) (5 x 10(-4) m) and L-arginine methyl ester hydrochloride (L-AME) (10(-3) m), comparably stimulated GH secretion when applied alone. The presence of L-AME enhanced ghrelin-stimulated GH secretion, whereas SNAP did not alter its effect. Conversely, two different NOS/NO pathway inhibitors, N(w)-nitro-L-arginine methyl ester hydrochloride (10(-5) m) or haemoglobin (20 microg/ml), similarly blocked ghrelin-induced (but not basal) GH release, thus indicating that NO contributes critically to ghrelin action in somatotrophs. Moreover, incubation with a permeable cGMP analogue, 8-Br-cGMP (10(-8) m) stimulated GH secretion, but did not modify the stimulatory action of ghrelin, suggesting that cGMP could mediate the action of NO. Indeed, inhibition of GC by 10 microm LY-53,583 did not alter basal GH secretion but abolished the GH-releasing action of ghrelin. Taken together, our results provide novel evidence indicating that ghrelin requires activation of the NOS/NO route, and its subsequent GC/cGMP signal transduction pathway, as necessary steps to induce GH secretion from somatotrophs.

Identification and characterization of two novel (neuro)endocrine long coiled-coil proteins.

We have identified a novel vertebrate-specific gene by applying a Differential Display method on two distinct subtypes of pituitary melanotropes showing divergent secretory phenotypes of hypo- and hypersecretion. A paralogue of this gene was also identified. The existence of a long coiled-coil domain and a C-terminal transmembrane domain in the sequences, together with the Golgi distribution of the proteins in transfected cells, suggest that they can be considered as new members of the golgin family of proteins. Both genes were primarily expressed in (neuro)endocrine tissues in vertebrates thus supporting a role for these proteins in the regulated secretory pathway.

Direct pituitary effects of kisspeptin: activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion.

Recent, compelling evidence indicates that kisspeptins, the products of KiSS-1 gene, and their receptor GPR54, represent key elements in the neuroendocrine control of reproduction, and that they act primarily by regulating gonadotrophin-releasing hormone (GnRH) secretion at the hypothalamus. Conversely, and despite earlier reports showing GPR54 expression in the pituitary, the potential physiological roles of kisspeptins at this gland have remained elusive. To clarify this issue, cultures of rat pituitary cells were used to evaluate expression of KiSS-1 and GPR54, and to monitor the ability of kisspeptin-10 to stimulate Ca(2+) responses in gonadotrophs and to elicit luteinising hormone (LH) secretion in vitro. The results obtained show that both GPR54 and KiSS-1 are expressed in the pituitary of peripubertal male and female rats. Moreover, kisspeptin-10 induced a rise in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) in approximately 10% of male rat pituitary cells. Intriguingly, kisspeptin-responsive cells included not only gonadotrophs, in which a 62.8 +/- 16.0%[Ca(2+)](i) rise was observed, but also somatotrophs, wherein kisspeptin induced a 60.3 +/- 5.5%[Ca(2+)](i) increase. Accordingly, challenge of dispersed pituitary cells with increasing kisspeptin-10 concentrations induced dose-related LH and growth hormone (GH) secretory responses, which were nevertheless of lower magnitude than those evoked by the primary regulators GnRH and GH-releasing hormone, respectively. In particular, 10(-8) M kisspeptin caused maximal increases in LH release (218.7 +/- 23.6% and 180.4 +/- 7.2% in male and female rat pituitary cells, respectively), and also stimulated maximally GH secretion (181.9 +/- 14.9% and 260.2 +/- 15.9% in male and female rat pituitary cells, respectively). Additionally, moderate summation of kisspeptin- and GnRH-induced LH responses was observed after short-term incubation of male rat pituitary cells. In conclusion, our results provide unequivocal evidence that kisspeptins exert direct pituitary effects in peripubertal male and female rats and suggest a possible autocrine/paracrine mode of action. The precise relevance and underlying mechanisms of this potential new actions of kisspeptins (i.e. the direct modulation of gonadotrophic and somatotrophic axis at the pituitary) deserve further analysis.

Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat.

Kisspeptins have recently emerged as essential regulators of gonadotropin secretion and puberty onset. These functions are primarily conducted by stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion. However, relevant aspects of KiSS-1 physiology, including the ontogeny and major signaling systems of its stimulatory action, remain to be fully elucidated. To cover these issues, the effects of kisspeptin-10 on GnRH and LH secretion were monitored at early stages of postnatal maturation, and potential changes in the sensitivity to kisspeptin were assessed along the pubertal transition in the rat. In addition, the signaling cascades involved in kisspeptin-induced GnRH secretion were explored by means of pharmacological blockade using rat hypothalamic explants. Despite sexual immaturity, kisspeptin-10 potently elicited GnRH release ex vivo and LH secretion in vivo at early stages (neonatal to juvenile) of postnatal development. Yet, LH responsiveness to low doses of kisspeptin was enhanced in peri-pubertal animals. Concerning GnRH secretion, the stimulatory action of kisspeptin-10 required activation of phospholipase-C, mobilization of intracellular Ca2+ and recruitment of ERK1/2 and p38 kinases, but was preserved after blockade of type 2 cyclo-oxygenase and prostaglandin synthesis. In summary, our present data document the ontogeny, sensitivity and intracellular signals for the stimulatory action of kisspeptin on the GnRH/LH axis in the rat. Although LH responses to low doses of kisspeptin appeared to be enhanced at puberty, kisspeptin was able to readily activate the GnRH system at early stages of postnatal maturation. These observations further stress the essential role of kisspeptin in normal, and eventually pathological, timing of puberty.

Cortistatin mimics somatostatin by inducing a dual, dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotropes.

Cortistatin is a recently discovered neuropeptide that is structurally related to somatostatin, the classic inhibitor of growth hormone (GH) release. Cortistatin binds with high affinity to all five somatostatin receptors (sst1-5), and, like somatostatin, cortistatin inhibits in vivo GH release in man and rats. In this report, we compared the in vitro actions of cortistatin and somatostatin using primary pig pituitary cell cultures. In this species, we have previously reported that somatostatin not only inhibits GH-releasing hormone (GHRH)-stimulated GH release at high doses, but also stimulates basal GH release at low (pM) doses, a dual response that is markedly dependent on the subpopulation of pituitary somatotropes examined. Results reported herein demonstrate that cortistatin closely mimics the dose-dependent inhibitory and stimulatory effects of somatostatin on GH secretion. As cortistatin, unlike somatostatin, binds to the human receptor for ghrelin/GH secretagogs (GHS-R), we also investigated whether cortistatin stimulates GH release through this receptor by using a synthetic, short form of cortistatin, cortistatin-8 (CST8), which lacks the sst-binding capacity of full-length cortistatin but retains its GHS-R-binding capacity. Interestingly, CST8 stimulated GH release only at low doses (10(-15) M), and did not reduce GH secretion stimulated by GHRH, ghrelin, or low-dose, full-length cortistatin, yet it counteracted that induced by a nonpeptidyl GHS, L-163 255. Taken together, our results indicate that the dual, inhibitory and stimulatory effects of cortistatin on GH release closely parallel those of somatostatin and are probably mediated by the same receptor(s) and signaling pathway(s) for both peptides. Furthermore, they suggest that the pathway(s) activated by cortistatin (and somatostatin) to stimulate GH release are not initiated by GHS-R activation.

Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat.

Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element in the neuroendocrine system controlling development and function of the reproductive axis; the HP unit being highly sensitive to the organizing effects of endogenous and exogenous sex steroids. To gain knowledge on the molecular mode of action and potential biomarkers of exposure to estrogenic compounds at the HP unit, we screened for differentially expressed genes at the pituitary and hypothalamus of rats after neonatal exposure to estradiol benzoate. Our analyses identified persistent up-regulation of alpha- and beta-globin mRNAs at the pituitary following neonatal estrogenization. This finding was confirmed by combination of RT-PCR analyses and in situ hybridization. Induction of alpha- and beta-globin mRNA expression at the pituitary by neonatal exposure to estrogen was demonstrated as dose-dependent and it was persistently detected up to puberty. In contrast, durable up-regulation of alpha- and beta-globin genes was not detected at the hypothalamus, cortex, cerebellum, liver and testis. Finally, enhanced levels of alpha- and beta-globin mRNAs at the pituitary were also demonstrated after neonatal administration of the anti-androgen flutamide. In summary, alpha- and beta-globin genes may prove as sensitive, pituitary-specific biomarkers of exposure to estrogenic (and/or anti-androgenic) compounds at critical periods of sex development, whose potential in the assessment of endocrine disrupting events at the HP unit merits further investigation.

Gonadotropin-secreting cells in ovariectomized rats treated with different oestrogen receptor ligands: a modulatory role for ERbeta in the gonadotrope?

In the rat, oestrogen is a key regulator of gonadotrophin synthesis and release through activation of oestrogen receptors (ERs). Gonadotropes express alpha and beta isoforms of ER and both can activate transcription in response to oestrogen. These experiments were aimed at evaluating the relative contribution of ERalpha and ERbeta on gonadotrope morphology, progesterone receptor (PR) expression and LH secretion. Ovariectomized rats were daily injected over 3 days with 25 microg oestradiol benzoate, 0.3 or 1.5 mg of the selective ERalpha agonist propylpyrazole triol (PPT) with or without 1.5, 3.0 or 4.5 mg of the selective ERbeta agonist diarylpropionitrile (DPN), DPN alone, and 0.3 or 3 mg of tamoxifen. Controls were given 0.2 ml oil. Serum concentration and pituitary content of LH, gonadotrope PR expression, pituitary PR content, and gonadotrope morphology were analyzed by RIA, immunohistochemistry, Western blotting and light and electron microscopy, respectively. Results showed that PPT reversed all consequences of ovariectomy, DPN mimicked the effects of PPT except for its LH-releasing action and tamoxifen had ERalpha-like responses. When combined with PPT, DPN attenuated ERalpha effects without interfering with its LH-releasing activity. Oestradiol benzoate had similar effects to those of combined PPT and DPN. It is suggested that (i) the structural reorganization of the cytoplasmic organelles provided by oestrogen, and the shrinkage of the ovariectomy-induced hypertrophy of gonadotropes, which precedes the expression of PR, are evoked by ERalpha and modulated, in a ying-yang fashion, by ERbeta; and (ii) the oestrogen-dependent exocytosis of LH, the final step in the secretory process, is dependent on ERalpha exclusively.

Differential expression and processing of chromogranin A and secretogranin II in relation to the secretory status of endocrine cells.

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Analysis of Rab18 and a new golgin in the secretory pathway.

Two new amphibian genes have been isolated and characterized from frog melanotropes, and the level of expression of these genes is related to the secretory status of the cells. Both genes, Rab18 and a novel member of the golgin family of proteins, are ubiquitously expressed in endocrine and nonendocrine tissues, and their corresponding proteins appear to show intracellular distributions associated with discrete vesicular and tubular structures, respectively, suggesting that they may play relevant roles in the regulation of the secretory pathway.

Ghrelin induces growth hormone (GH) secretion via nitric oxide (NO)/cGMP signaling.

Ghrelin, a recently discovered 28-aa peptide, stimulates GH release through a mechanism involving PLC- and cAMP-related signaling pathways. Recently, nitric oxide (NO) and its mediator, cGMP, have been shown to be required for the response of somatotropes to various regulators (GHRH, somatostatin, leptin). Here, we explore the possible role of the NO synthase (NOS)/NO/guanylate cyclase (GC)/cGMP signaling pathway in ghrelin-induced GH release from cultured pig somatotropes using blockers or activators of this route.

Homologous and heterologous in vitro regulation of pig pituitary somatostatin receptor subtypes, sst1, sst2 and sst5 mRNA.

Somatostatin (SRIF) is commonly regarded as an inhibitor of GH release in rodents and humans. However, in pigs, SRIF can stimulate the release of GH at low (picomolar) doses, while inhibiting GHRH-stimulated GH release at high (nanomolar) doses in primary pituitary cell cultures. One possible mechanism by which pig cells respond differently to the actions of SRIF is by differential expression and regulation of SRIF receptor subtypes. As no information is available on the homologous regulation of SRIF receptors in pigs, we examined the acute (4 h) in vitro effects of SRIF on mRNA levels of SRIF receptors sst1, sst2 and sst5 by multiplex RT-PCR. These particular sst subtypes were selected because all three have been implicated in the inhibitory effects of SRIF on GH release in both rodents and humans. At a high dose (10(-7) M), SRIF stimulated the expression of sst1, sst2 and sst5 in pig pituitary cell cultures. At a low dose (10(-13) M), SRIF markedly increased sst1, without affecting sst2 or sst5. Given that our laboratory has shown SRIF at high and low doses stimulates cAMP production in a subpopulation of pig somatotropes, we sought to determine if this signaling pathway may be responsible for the stimulatory effect of SRIF on its own receptor expression. The receptor-independent cAMP activator forskolin elevated sst1 and sst2 mRNA levels but did not affect sst5 expression, suggesting the stimulatory actions of high- and low-dose SRIF on sst1 and high-dose SRIF on sst2 mRNA levels can be mediated by activation of cAMP, whereas the stimulatory effect of high-dose SRIF on sst5 mRNA is elicited by a cAMP-independent pathway. Interestingly, both GHRH (10(-8) M) and ghrelin (10(-6) M), which release GH in pig pituitary cell cultures via cAMP-dependent mechanisms, decreased sst5 without altering sst1 or sst2 mRNA levels. Since the actions of GHRH and ghrelin on sst expression markedly contrasted with that observed for SRIF and forskolin these results clearly indicate GHRH and ghrelin are regulating sst5 mRNA levels by a cAMP-independent signaling pathway. Taken together, our results demonstrate that expression of pig SRIF receptors is under a complex, receptor subtype-selective regulation, wherein the concerted actions of key regulators of somatotrope function would play divergent and dose-dependent effects.

Research progress in the stimulatory inputs regulating growth hormone (GH) secretion.

A review is presented on progress in the research of stimulatory inputs that regulate growth hormone secretion, including recent results on the action of the hypothalamic peptides growth-hormone releasing factor (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP), as well as that of both peptidic (growth hormone-releasing hexapeptide; GHRP-6) and non-peptidyl (L-163,255) synthetic GHSs on somatotrope cell function.

Melanotrope cell plasticity: a key mechanism for the physiological adaptation to background color changes.

The intermediate lobe of the pituitary secretes the melanotropic hormone alpha-MSH, which in amphibians plays a crucial role in skin color adaptation. It has been previously demonstrated that, in the frog Rana ridibunda, the intermediate lobe is composed of two distinct subpopulations of melanotrope cells that can be separated in vitro by using Percoll density gradients. These two melanotrope cell subsets, referred to as high-density (HD) and low-density (LD) cells, differ in their ultrastructural characteristics as well as in their biosynthetic and secretory activity. However, the specific, physiological role of the heterogeneity displayed by melanotrope cells remains elusive. In the present study, we investigated the effects of background color adaptation on melanotrope cell subpopulations. We found that adaptation of frogs to dark or white environment did not modify either the overall number of cells per intermediate lobe or the apoptotic and proliferation rates of melanotrope cells. On the other hand, adaptation of the animals to a white background significantly increased the proportion of hormone-storage HD cells and caused a concomitant decrease in that of LD cells (which exhibit higher levels of alpha-MSH release and POMC messenger RNA than HD cells). Conversely, after black-background adaptation the proportion of LD cells was markedly increased, suggesting that interconversion of HD cells to LD cells occurs during physiological activation of the intermediate lobe. In addition, black-background adaptation also enhanced alpha-MSH release by both cell subpopulations and increased inositol phosphate production in LD cells. These data indicate that, in frog, the proportions of the two melanotrope cell subsets undergo marked modifications during skin color adaptation, likely reflecting the occurrence of a secretory cell cycle whose dynamics are highly correlated to the hormonal demand imposed by the environment.

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 activate common and distinct intracellular signaling pathways to stimulate growth hormone secretion from porcine somatotropes.

We have recently shown that the two bioactive forms of pituitary adenylate cyclase-activating polypeptide, PACAP38 and PACAP27, stimulate GH release and GH messenger RNA (mRNA) accumulation in cultured porcine pituitary cells. However, dose- and time-related differences in the response to both peptides suggested that the signaling mechanisms activated by PACAP38 and PACAP27 in this cell type could differ. To test this hypothesis, we have evaluated hormone release and GH mRNA content after PACAP treatment in combination with selective activators and inhibitors of the adenylate cyclase/cAMP/protein kinase A and the phospholipase C/inositol phosphate (IP)/protein kinase C pathways, and with blockers of intra- and extracellular Ca2+. Our results show that activation of the adenylate cyclase/cAMP/protein kinase A system, and extracellular Ca2+ entry through L-type Ca2+-channels are prevailing and requisite signals for the transduction of the stimulatory effects of both PACAP38 and PACAP27 on GH release and transcription in porcine somatotropes. However, phospholipase C and intracellular Ca2+ also contribute, although partially, to PACAP38-induced, but not to PACAP27-induced increase in porcine GH secretion and mRNA levels. These findings demonstrate that in normal somatotropes, PACAP38 can activate multiple transduction pathways that differ from those employed by PACAP27. Moreover, these differences could account for the previously described divergences in the actions of either peptide in porcine somatotropes.