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 and characterization of new functional truncated variants of somatostatin receptor subtype 5 in rodents.

Somatostatin and cortistatin exert multiple biological actions through five receptors (sst1-5); however, not all their effects can be explained by activation of sst1-5. Indeed, we recently identified novel truncated but functional human sst5-variants, present in normal and tumoral tissues. In this study, we identified and characterized three novel truncated sst5 variants in mice and one in rats displaying different numbers of transmembrane-domains [TMD; sst5TMD4, sst5TMD2, sst5TMD1 (mouse-variants) and sst5TMD1 (rat-variant)]. These sst5 variants: (1) are functional to mediate ligand-selective-induced variations in [Ca(2+)]i and cAMP despite being truncated; (2) display preferential intracellular distribution; (3) mostly share full-length sst5 tissue distribution, but exhibit unique differences; (4) are differentially regulated by changes in hormonal/metabolic environment in a tissue- (e.g., central vs. systemic) and ligand-dependent manner. Altogether, our results demonstrate the existence of new truncated sst5-variants with unique ligand-selective signaling properties, which could contribute to further understanding the complex, distinct pathophysiological roles of somatostatin and cortistatin.

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.

Identification and characterization of two novel truncated but functional isoforms of the somatostatin receptor subtype 5 differentially present in pituitary tumors.

CONTEXT: Somatostatin and its related peptide cortistatin exert multiple actions on normal and tumoral tissue targets through a family of receptors termed somatostatin receptor (sst)1-5. Despite the considerable advances in the knowledge on these receptors and their (patho)physiological roles, there is still evidence that additional receptors for these peptides should exist to fully explain their actions. OBJECTIVE: The growing number of spliced variants found in similar receptor families, often present in tumors, and results from our group obtained on sst5 from other species (pig) led us to explore the existence of new human sst5 isoforms. DESIGN AND RESULTS: A rapid amplification of cDNA ends PCR approach on samples from a human pituitary tumor and a cell line enabled identification of two novel alternatively spliced sst5 receptor variants. The sequences obtained encode putative proteins that correspond to truncated isoforms of five and four transmembrane domains (TMDs), accordingly named sst5TMD5 and sst5TMD4, respectively. Both novel receptors show a differential expression pattern in normal tissues and are also present in pituitary tumors of diverse etiology including nonfunctioning adenomas, corticotropinomas, somatotropinomas, and a prolactinoma. In contrast to the predominant plasma membrane localization of full-length sst5, both sst5TMD5 and sst5TMD4 show a preferentially intracellular localization. Despite their truncated nature, both receptors are functional, as shown by their ability to mediate selective, ligand-induced rises in free cytosolic calcium concentration. Specifically, whereas sst5TMD5 is selectivity activated by somatostatin compared with cortistatin, cells transfected with sst5TMD4 almost exclusively respond to cortistatin and not to somatostatin. CONCLUSIONS: Our results demonstrate the existence of two previously unidentified sst5 spliced variants with distinct distribution in normal tissues and pituitary tumors, unique ligand-selective signaling properties, and subcellular distribution, which could contribute to somatostatin and cortistatin signaling in normal and tumoral cells.

Dimerization of G protein-coupled receptors: new avenues for somatostatin receptor signalling, control and functioning.

Somatostatin acts through binding and activation of five G protein-coupled receptors (GPCRs) termed somatostatin receptors or ssts (sst1-sst5). These receptors, as many other GPCRs are not just monomers but display a differential tendency to homodimerize, which varies depending on the sst subtype. Moreover, there is evidence that pairs of distinct receptors such as ssst2-sst3 and sst1-sst5 crosstalk by establishing a physical interaction, which results in altered pharmacological or/and functional properties. In addition, ssts can also heterodimerize with other families of GPCRs, as opioid and dopamine receptors, originating heterodimers which properties are different to those of their separated receptors. The present review summarizes the current knowledge on ssts homodimerization, heterodimerization, and interaction with other GPCRs, as well as how interactions affect different aspects of the normal functioning of these receptors.

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.

RT-PCR analysis of the expression of POMC and its processing enzyme PC1 in amphibian melanotropes.

The frog intermediate lobe comprises two functionally distinct cell subtypes, referred to as secretory and storage melanotropes, which differ in their ultrastructure, secretory, and synthetic rates, and display dissimilar responses to hypothalamic regulatory factors. All these differences make melanotrope subtypes an excellent model to analyze the expression and regulation of genes involved in the control and maintenance of the secretory state of endocrine cells. However, quantification of the expression levels of genes involved in the secretory process requires the characterization of a gene whose expression remains constant irrespective of the secretory state of the cells. In this study, we have cloned the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from frog pituitary and have evaluated its suitability as internal standard in gene expression studies in melanotropes. A semiquantitative RT-PCR system developed to this end revealed that secretory melanotropes and storage melanotropes possess similar expression levels of GAPDH, whereas, as expected, secretory melanotropes showed higher levels of POMC transcripts than storage cells. Furthermore, we found that the expression of the convertase PC1, an intracellular protease involved in POMC processing, parallels that of POMC, thus suggesting that the higher secretory rate of the POMC-derived peptide alpha-MSH exhibited by secretory melanotropes is supported by their higher PC1 expression levels. In addition, we have shown that both POMC and PC1 mRNAs are up-regulated by the hypothalamic factor TRH in melanotrope cell cultures. In contrast, the inhibitory factor NPY reduced the expression level of the convertase but did not modify that of POMC. Taken together, these results demonstrate that PC1 expression is regulated in melanotropes by both stimulatory (TRH) and inhibitory (NPY) hypothalamic signals, in a manner which essentially parallels that observed for the precursor POMC.

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.

Differential contribution of nitric oxide and cGMP to the stimulatory effects of growth hormone-releasing hormone and low-concentration somatostatin on growth hormone release from somatotrophs.

There is increasing evidence that nitric oxide (NO) produced by NO synthase (NOS), and their signalling partners, guanylyl cyclase and cGMP, play a relevant role in growth hormone (GH) secretion from somatotrophs. We previously demonstrated that both GH-releasing hormone (GHRH; 10(-8) M) and low concentrations of somatostatin (10(-15) M) stimulate pig GH release in vitro, whereas a high somatostatin concentration (10(-7) M) inhibits GHRH-induced GH secretion. To ascertain the possible contribution of the NOS-NO and guanylyl cyclase-cGMP routes to these responses, cultures of pituitary cells from prepubertal female pigs were treated (30 min) with GHRH (10(-8) M) or somatostatin (10(-7) or 10(-15) M) in the absence or presence of activators or blockers of key steps of these signalling cascades, and GH release was measured. Two distinct activators of NO route, SNAP (5x10(-4) M) or L-AME (10(-3) M), similarly stimulated GH release when applied alone (with this effect being blocked by 10(-7) M somatostatin), but did not alter the stimulatory effect of GHRH or 10(-15) M somatostatin. Conversely, two NO pathway inhibitors, NAME (10(-5) M) or haemoglobin (20 microg/ml) similarly blocked GHRH- or 10(-15) M somatostatin-stimulated GH release. 8-Br-cGMP (10(-8) to 10(-4) M) strongly stimulated GH release, suggesting that cGMP may function as a subsequent step in the NO pathway in this system. Interestingly, 10(-7) M somatostatin did not inhibit the stimulatory effect of 8-Br-cGMP. Moreover, although 8-Br-cGMP did not modify the effect of GHRH, it enhanced GH release stimulated by 10(-15) M somatostatin. Accordingly, a specific guanylyl cyclase inhibitor, LY-83, 583 (10(-5) M) did not alter 10(-15) M somatostatin-induced GH release, whereas it blocked GHRH-induced GH secretion. These results demonstrate for the first time that the NOS/NO signalling pathway contributes critically to the stimulatory effects of both GHRH and low-concentration somatostatin on GH release, and that, conversely, the subsequent guanylyl cyclase/cGMP step only mediates GHRH- and not low-concentration somatostatin-induced GH secretion from somatotrophs.

New insights in the mechanism by which SRIF influences GH secretion.

Once thought to act only as a somatotropin release-inhibiting factor (SRIF), SRIF is currently viewed as a pleiotropic neuroendocrine factor controlling secretion, gene expression, apoptosis and signalling in many different targets. Actually, despite the numerous studies that have characterized SRIF action on somatotropes, new facets are continuously being discovered which help enlightening the biology of this cell type. As an example, ten years ago we demonstrated that SRIF exerts a dual, inhibitory/stimulatory effect on GH release from cultured pig somatotropes, which depends on the concentration of the peptide and on a divergent responsiveness of the two main cell subsets comprising the somatotrope population. Specifically, very low, picomolar doses of SRIF were found to stimulate GH release in vitro from intact cultures of dispersed pig pituitary cells and from purified somatotrope subpopulations. Conversely, higher (10(-7)M) SRIF concentrations inhibited, as expected, GHRH-induced GH release from intact pituitary cells and from one of the somatotrope subtypes; yet, at this same dose, it stimulated GH release from the other somatotrope subset. Analysis of second messenger pathways revealed that cAMP is the main signal conveying the stimulatory effects of low-dose SRIF. This peptide also exerts a distinct, dose-dependent regulation of the expression of three of its receptor subtypes (sst1, sst2 and sst5) at the pituitary. Indeed, acute in vitro treatment with a high SRIF dose increased mRNA levels of all three subtypes, whereas a low SRIF concentration only increased that of sst5. Interestingly, short term treatment with GHRH or ghrelin reduced the expression of sst5, and not that of sst1 and sst2. Hopefully, ongoing studies on cloning and individual characterization of porcine sst will help to unravel the complex and exciting response of somatotropes to SRIF.

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.

Melanotrope cells as a model to understand the (patho)physiological regulation of hormone secretion.

Regulation of hormone secretion is a complex process that comprises the sequential participation of numerous subcellular mechanisms. Hormone secretion is dictated by extracellular stimuli that are transduced intracellularly into activation/deactivation of different mechanisms, such as hormone expression, processing and exocytosis, which will ultimately determine the precise availability of hormone to be secreted. Malfunction in any of these steps may result in deficient or excessive hormone release and the subsequent appearance of endocrine disorders. Given the complexity of this system, it is difficult to find appropriate cellular models wherein to investigate the multiple components of the secretory process in a physiologically relevant, experimentally manipulable setting. In this review, we present recent evidence on the use of the intermediate lobe (IL) of the pituitary as a powerful tool to understand different aspects of the regulated secretory pathway. IL is composed of a single endocrine cell type, alpha-melanocyte stimulating hormone (alpha-MSH)-producing melanotropes, a fact that greatly facilitates its study. Furthermore, melanotropes can be separated using classic cell separation techniques into two cell subtypes showing opposite morphophysiological phenotypes of hypo- and hypersecretory cells. Comparison of their gene expression fingerprints has unveiled the existence of certain genes preferentially expressed in each melanotrope subtype. Because of their direct participation in the secretory pathway, we postulate that characterization of these gene products in an endocrine cell type may represent novel and useful markers for reliably determining the general secretory status in an endocrine gland, as well as a valuable new tool to further investigate this complex process.

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.

Secretory plasticity of pituitary cells: a mechanism of hormonal regulation.

Pituitary somatotropes and melanotropes have enabled us to investigate the molecular basis and functional dynamics underlying secretory plasticity, an ability of endocrine cells to adapt their activity to the changing physiologic requirements, which generates discrete cell subpopulations within each cell hormonal type. Porcine somatotropes comprise two morphologically distinct subpopulations of low- (LD) and high-density (HD) cells, separable by Percoll gradient, that respond differently to hypothalamic regulators. In LD somatotropes, somatostatin (SRIF) inhibits growth hormone (GH)-releasing hormone (GHRH)-induced GH secretion. Conversely, SRIF alone stimulates GH release from HD somatotropes. These disparate SRIF actions entail a molecular signaling heterogeneity, in that SRIF increases cAMP levels in HD but not in LD cells as a requisite to stimulate GH release. GHRH-stimulated GH release also involves differential signaling in LD and HD cells: although it acts primarily through the cAMP/extracellular Ca2+ route in both somatotrope subsets, full response of LD somatotropes also requires the inositol phosphate/intracellular Ca2+ pathway. Amphibian melanotropes, which regulate skin adaptation to background color by secreting POMC-derived alpha-melanocyte-stimulating hormone (alphaMSH), also comprise two subpopulations with divergent secretory phenotypes. LD melanotropes show high biosynthetic and secretory activities and high responsiveness to multiple hypothalamic factors. Conversely, HD melanotropes constitute a hormone-storage subset poorly responsive to regulatory inputs. Interestingly, in black-adapted animals most melanotropes acquire the highly-secretory LD phenotype, whereas white-background adaptation, which requires less alphaMSH, converts melanotropes to the storage HD phenotype. These same interconversions can be reproduced in vitro using appropriate hypothalamic factors, thus revealing the pivotal role of the hypothalamus in regulating the functional dynamics of the secretory plasticity. Furthermore, this regulation likely involves a precise control of the secretory pathway, as suggested by the differential distribution in LD and HD melanotropes of key components of the intracellular transport, processing, and storage of secretory proteins. Hence, molecular signaling heterogeneity and unique secretory pathway components seem to relevantly contribute to the control of secretory plasticity, thereby enabling endocrine cells to finely adjust their dynamic response to the specific hormonal requirements.

Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone.

The triakontatetraneuropeptide TTN increases [CA2+]i in rat astrocytes through activation of peripheral-type benzodiazepine receptors.

Astrocytes synthesize a series of regulatory peptides called endozepines, which act as endogenous ligands of benzodiazepine receptors. We have recently shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present study was to determine the mechanism of action of TTN on cultured rat astrocytes. Binding of the peripheral-type benzodiazepine receptor ligand [3H]Ro5-4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fragment (TTN[17-34], the octadecaneuropeptide ODN) did not compete for [3H]Ro5-4864 binding. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) with the fluorescent probe indo-1 showed that TTN (10(-10) to 10(-6) M) provokes a concentration-dependent increase in [Ca2+]i in cultured astrocytes. Simultaneous administration of TTN (10(-8) M) and Ro5-4864 (10(-5) M) induced an increase in [Ca2+]i similar to that obtained with Ro5-4864 alone. In contrast, the effects of TTN (10(-8) M) and ODN (10(-8) M) on [Ca2+]i were strictly additive. Chelation of extracellular Ca2+ by EGTA (6 mM) or blockage of Ca2+ channels with Ni2+ (2 mM) abrogated the stimulatory effect of TTN. The calcium influx evoked by TTN (10(-7) M) or by Ro5-4864 (10(-5) M) was not affected by the N- and T-type calcium channel blockers omega-conotoxin (10(-6) M) and mibefradil (10(-6) M), but was significantly reduced by the L-type calcium channel blocker nifedipine (10(-7) M). Patch-clamp studies showed that, at negative potentials, TTN (10(-7) M) induced a sustained depolarization. Reduction of the chloride concentration in the extracellular solution shifted the reversal potential from 0 mV to a positive potential. These data show that TTN, acting through peripheral-type benzodiazepine receptors, provokes chloride efflux, which in turn induces calcium influx via L-type calcium channels in rat astrocytes.

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.

Amphibian melanotrope subpopulations respond differentially to hypothalamic secreto-inhibitors.

The melanotrope population of the frog intermediate lobe consists of two subtypes of cells, referred to as high-(HD) and low-density (LD) melanotrope cells, which differ markedly in their basal morphofunctional features as well as their in vitro response to hypothalamic factors, such as the stimulator thyrotropin-releasing hormone (TRH) and the inhibitor dopamine. In this study, we have investigated whether other major hypothalamic regulators of the release of alpha-melanocyte-stimulating hormone (alpha-MSH), such as gamma-aminobutyric acid (GABA) and neuropeptide Y (NPY), also differentially regulate frog melanotrope subpopulations. Our results show that in LD cells, both factors markedly inhibited proopiomelanocortin (POMC) mRNA accumulation and alpha-MSH secretion. In contrast, the secretory and biosynthetic activity of HD cells was not modified by GABA. NPY inhibited POMC transcript accumulation and tended to reduce alpha-MSH secretion in HD cells, yet these effects were less pronounced than those evoked in LD cells. In addition, GABA and NPY inhibited the KCl-induced rise in cytosolic free calcium levels in both subpopulations. Taken together, these results further indicate that frog melanotrope subpopulations are differentially regulated by the hypothalamus and strongly suggest that the intensity of such regulation is directly related to the activity of the cell subset. Thus, the LD subpopulation represents a highly responsive cell subset which is regulated by multiple neuroendocrine factors (TRH, dopamine, GABA and NPY), whereas the hormone storage HD subpopulation shows a moderate response to single stimulatory (TRH) and inhibitory (NPY) inputs.