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.

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.

Direct effects of growth hormone (GH)-releasing hexapeptide (GHRP-6) and GH-releasing factor (GRF) on GH secretion from cultured porcine somatotropes.

Growth hormone (GH)-releasing hexapeptide (GHRP-6) belongs to the expanding family of synthetic GH secretagogues (GHSs). Previous studies have shown that non-peptidyl GHRP-6 analogues stimulate GH release in vivo in pigs, and interact synergistically with GH-releasing factor (GRF), but its direct effects on porcine somatotropes have not been addressed hitherto. In the present study, we have evaluated the response of cultured porcine pituitary cells to GHRP-6, and its interaction with GRF and somatostatin (SRIF). Secretory response of somatotropes was assessed by using two distinct techniques. GH released by monolayer cell cultures was evaluated by enzyme immunoassay, whereas that secreted by individual somatotropes was measured by immunodensitometry using a cell blotting assay. Our results demonstrate that both GHRP-6 and GRF stimulated GH release from monolayer cultures at doses equal to or above 10(-9) M. Use of cell immunoblot assay demonstrated that, like GRF, the hexapeptide acts directly upon porcine somatotropes to exert its action. Moreover, regardless of the technique applied, combined administration of GHRP-6 (10(-6) or 10(-9) M) and GRF (10(-8) M) resulted in an additive, but not synergistic, stimulatory GH response. Finally, SRIF (10(-7) M) inhibited the stimulatory effect of GHRP-6 alone or in combination with GRF. These results indicate that GHRP-6 directly and effectively stimulates GH secretion from porcine somatotropes in vitro, and acts additively when coadministered with GRF. Therefore, the synergistic stimulatory effect of GHSs and GRF reported in vivo in this species might require additional factors that are lacking in the in vitro situation.

Somatostatin plays a dual, stimulatory/inhibitory role in the control of growth hormone secretion by two somatotrope subpopulations from porcine pituitary.

Previous results from our laboratory demonstrated the existence of two subpopulations of porcine somatotropes of low- (LD) and high density (HD) that exhibit differences in ultrastructure and respond in an opposite manner to somatostatin (SRIF) in vitro. In LD cells, SRIF did not affect basal growth hormone (GH) release but partially blocked the stimulatory effect induced by GH-releasing factor (GRF). Conversely, SRIF paradoxically stimulated the secretory activity of HD somatotropes. Here, we have analysed in detail the basic parameters that characterize this differential response. To this end, the time- and dose-dependent effects of SRIF-14 were evaluated on separate monolayer cultures of both subpopulations. Likewise, the direct effect of the peptide on individual somatotropes from each subset was assessed by cell immunoblot assay. Finally, we compared the effects of SRIF-14 and SRIF-28 on cultures of LD and HD cells. SRIF-14 (10(-7) M) induced a rapid (30 min) and sustained (4 h) 2-fold increase in GH release from HD cells, whereas it did not affect GH secretion from LD somatotropes. Surprisingly, a low dose of SRIF (10(-15) M) stimulated GH release from both LD (154.1 +/- 8.2% of basal, P < 0.05) and HD (337.2 +/- 55.5% of basal, P < 0.05) subpopulations, even more effectively than higher doses of the peptide. Results from cell blotting showed that SRIF stimulatory effects were exerted directly upon individual somatotropes. Finally, SRIF-28 elicited similar responses to those observed for SRIF-14 in both somatotrope subpopulations, yet 10(-15) M SRIF-28 was less potent than the same dose of SRIF-14 in stimulating GH release from HD cells. Our present findings demonstrate that SRIF can function as a true GH-releasing factor in cultures of porcine pituitary cells by acting specifically and directly upon somatotropes. Furthermore, together with previous observations, these results strongly suggest that SRIF is not merely an inhibitor of GH release in pigs, but might play a dual modulatory role. Heterogeneity of the somatotrope population contributes greatly to this divergent effect of SRIF.

Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells.

Previous results demonstrate that porcine somatotropes can be separated by density gradient centrifugation into low density (LD) and high density (HD) subpopulations. In rat, two analog somatotrope subpopulations differ morphologically and functionally. In an attempt to determine whether morphological differences were also present within LD and HD porcine somatotropes, we undertook a quantitative electron microscope study of the subcellular organelles of immunoidentified LD and HD somatotropes. In addition, to test for the existence of functional differences, cultures of separated HD and LD subpopulations were treated for 4 h with or without 10 microM GRF-(1-29) and/or 100 microM somatostatin (SRIF), and porcine GH release and intracellular content were evaluated using a homologous enzyme immunoassay. Morphometric results demonstrate that LD somatotropes are smaller in size (P < 0.05) and contain fewer secretory granules (P < 0.05) and more rough endoplasmic reticulum (P < 0.05) than HD somatotropes. In terms of secretion, LD somatotropes showed a classical response; GRF increased GH release 1.7-fold (n = 6; P < 0.05) over the control value, whereas treatment with SRIF alone did not affect basal GH release in this subpopulation, but partially blocked GRF-induced GH release. HD somatotropes responded to GRF with a similar 1.7-fold increase in GH release. However, SRIF administered alone or in combination with GRF exerted a paradoxical stimulatory effect on HD somatotropes (2.15- and 2.12-fold over control value, respectively; n = 6; P < 0.05). These results demonstrate that the porcine somatotrope population is composed of two major subpopulations that display a distinctive pattern of ultrastructural organization and a markedly divergent secretory response to in vitro SRIF treatment.