Histol Histopathol (2002) 17: 707-714 Histology and http://www.hh.um.es Histopathology Cellular and Molecular Biology Influence of chronic treatment with the growth hormone secretagogue Ipamorelin, in young female rats: somatotroph response in vitro L. Jiménez-Reina1, R. Cañete2, M.J. de la Torre2 and G. Bernal1 1Department of Morphological Sciences, Section of Human Anatomy, School of Medicine, Córdoba, Spain and 2Department of Pediatrics, Pediatric Endocrinology Unit, School of Medicine, Córdoba, Spain Summary. Growth hormone (GH) is secreted in the Key words: GH secretagogues, Ipamorelin, anterior pituitary gland by the somatotroph cells. Somatotroph cells Secretion is regulated by growth hormone releasing hormone (GHRH) and somatostatin. Morever, GH secretagogues (GHS) can exert a considerable effect on Introduction GH secretion. In order to determine the effects of chronic treatment with the GHS Ipamorelin on the The biosynthesis and secretion of growth hormone composition of the somatotroph cell population and on (GH) in the anterior pituitary gland is under complex somatotroph GH content, an in vitro analysis was hormone regulation. It is regulated by two hypothalamic performed of the percentage of somatotroph cells (% of hormones, somatostatin and growth hormone-releasing total), the ratio of different GH cell types hormone (GHRH), which oppose one another and act (strongly/weakly-staining) and individual GH content, in through distinct membrane receptors. Somatostatin pituitary cell cultures obtained from young female rats inhibits GH release via a family of GTP-binding protein- receiving Ipamorelin over 21 days (Ipamorelin group) coupled membrane receptors (Jakobs et al., 1983; and the effects were compared with those of GHRH Yamada et al., 1992; Buscail et al., 1994) and GHRH (GHRH group) or saline (saline group). The activates GH release through a stimulatory G-protein- ultrastructure of somatotroph cells did not change, but coupled receptor (Dhanasekaran et al., 1995). GH the volume density of secretion granules was increased regulates its own release through negative feedback (P<0.05) by previous in vivo Ipamorelin or GHRH loops acting at the level of the hypothalamus, via GH treatment. In 3-day basal pituitary cell monolayer receptors (Burton et al., 1992; Bennet et al., 1995), and cultures, the percentage of somatotroph cells showed no at pituitary level (Jiménez-Reina et al., 2000; Peng et al., modifications between groups, nor was there any change 2001). in the ratio of strongly/weakly immunostaining GH cells. In addition to GHRH and somatostatin, other In the Ipamorelin group alone, in vitro treatment with neuropeptides can exert important modulatory effects on Ipamorelin (10-8 M), or GHRP 6 (10-8 M), or GHRH GH secretion, by acting either directly on the pituitary (10-8 M) for 4 hours, increased the percentage of gland and/or at hypothalamic level. Major neuropeptides somatotroph cells, without modifying the ratio of that affect GH release are the synthetic molecules termed strongly/weakly immunostained GH cells. Basal GH secretagogues (GHS) (Giustina and Veldhuis, 1998). intracellular GH content in somatotroph cells over 4 GHS act through membrane receptors that increase hours was lower in the Ipamorelin group and the GHRH intracellular concentrations of inositol triphosphate, and group than in the saline group. Only in the Ipamorelin enhance the activity of protein kinase C (Cheng et al., group did Ipamorelin (10-8 M), GHRP 6 (10-8 M) and 1991; Herrington and Hille, 1994; Lei et al., 1995). GHRH (10-8 M) prompt increased intracellular GH Recently, an endogenous peptide ligand for the GHS content. These data suggest that, at least in the young receptor, ghrelin, was purified from the rat stomach and female rat, the GHS Ipamorelin is able to exert a subsequently cloned (Kojima et al., 1999) dynamic control effect on the somatotroph population Synthesis and release of GH in the pituitary takes and on GH hormone content. place in somatotroph cells, a cell population traditionally reported as heterogeneous on the basis of both physical Offprint requests to: L. Jiménez-Reina, Departamento de Ciencias cell-separation methods (Snyder et al., 1977; Hall et al., Morfológicas, Facultad de Medicina, Avda. Menéndez Pidal s/n, 14004 1982; Lindstrom and Savendahl, 1996), and Córdoba, Spain. Fax : 957 218246. e-mail: [email protected] ultrastructural features (Kurosumi, 1986; Takahasi, 708 Somatotroph responses to Ipamorelin 1991). Changes take place both in cell morphology and diced into small pieces for cell dispersion or for in the relative proportions of the different types of ultrastructural study. somatotrophs under different physiological and experimental conditions, such as hormone treatment Ultrastructural and stereological analysis (González-Parra et al., 2000), chronic IGF-1 administration (Pellizas et al., 2000), GH autoregulation The small pieces were fixed with fixative solution (Asa et al., 2000; Jiménez-Reina et al., 2000), pituitary (1% glutaraldehyde and 2% formaldehyde in 0.2 M hyperplasia (Vidal et al., 2001), and in genetic and cacodylate buffer) for 2 hours, and postfixed with 1% transgenic models with perturbations of individual osmium tetroxide for 1 hour. Following dehydration with components of the GH regulatory system (Frohman et ethanol they were embedded in Durcupan‚ ACM. al., 2000). Sections of approximately 300 nm were collected on Since GHRH plays an essential part in regulating the 300-mesh nickel grids. GH cells were identified on functional and proliferative activity of somatotroph cells ultrathin sections by immunogold (Roth, 1983). After (Giustina and Veldhuis, 1998; Mayo et al., 2000, etching with a saturated aqueous solution of sodium Frohman et al., 2000), thus affecting the morphology metaperiodate, rabbit serum anti-rat GH (UCB and composition of the somatotroph cell population Bioproducts, diluted 1:1000, 20h at 4 °C) as the first (Dobado-Berrios, 1996a), it may be hypothesized that antiserum, and goat anti-rabbit IgG conjugated with GHS also play a major role in the dynamic control of the colloidal gold (15 nm in diameter) (Janssen Life Sci, somatotroph cell population. Glen, Belgium; diluted 1:10) were used. For washing The present study aimed to ascertain, in vitro, the and dilution of sera, tris buffer (0.05 M, pH 7.4) (TRIS) influence of chronic administration of a recently- and 1% albumin bovine serum (BSA) in TRIS were synthesized GHS, Ipamorelin (Raun et al., 1998), on the used, respectively. After immunostaining, grids were composition of the somatotroph cell population and on stained with uranyl acetate and lead citrate. Preparations the individual GH content of these cells in young female were examined using a Phillips CM 10 electron rats. microscope and 40 micrographs were taken per animal (20 at x1950 and 20 at x6400). The immunoreaction Materials and methods specificity for rat GH was analysed by omission of the specific antiserum, replacing the antiserum with normal Animals rabbit serum, and adsorption of the specific antiserum with its homologous (rat GH) or heterologous (rat Sixty-day-old female Wistar rats were used. Animals prolactin) hormone. were given free access to rat chow (IPM R-20, Letica The volume density of nucleus (VvNu), cytoplasm S.A., Hospitalet, Barcelona, Spain) and tap water. They (VvCy) and secretion granules (VvGr; % cytoplasm), were housed individually and kept under conventional calculated by the formula of Weibel and Bolender conditions (temperature: 22±2 °C 12:12 h light/dark (1973), were selected as stereological parameters. cycle with lights on at 06.30) in the laboratory animal center of the School of Medicine in Córdoba. The rats Cell dispersion and cell culture were cared for, and used, in accordance with European Council directive 86/609/ EEC (24/11/1987). Pituitary cell cultures were prepared according to Jiménez-Reina et al. (2000) Briefly, pituitaries were Experiment design diced into small pieces and dispersed in Erlenmeyer flasks with a mixture of 0.02 g/l collagenase (Type V, The duration of the experiment was 21 days, and rats Sigma Chemical Co.) and 0.01 g/l trypsin 1:250 (Sigma were weighed daily. They were divided into three groups Chemical Co.) in Dulbecco Modified Eagle's Medium (n=6): the Ipamorelin-pretreated group (Ipamorelin (DMEM) (Sigma Chemical Co.). The Erlenmeyer flask group); the GHRH-pretreated group (GHRH group); and was placed in a humidified atmosphere of 5% CO2 in air, the saline-pretreated group (saline group). In the at 37 °C, for 60 minutes. The cell suspension obtained Ipamorelin-pretreated group, animals were injected was gently pipetted for ten minutes, washed in DMEM, subcutaneously (sc) daily at 9 am with 100 µg/kg body and centrifuged (100 g for 10 min) twice. The cellular weight of diacetate salt of Ipamorelin (Novo-Nordisk, pellet was washed again, centrifuged, and resuspended Denmark). In the GHRH-pretreated group, animals were in 10% fetal bovine serum (FBS)-DMEM. Cell yield and injected sc daily at 9 am with 10 µg/kg body weight of viability were checked by the trypan blue exclusion 6 GHRH1-29 (Serono Labs., Spain). The Saline-pretreated method: cell yields were 1.64±0.12x10 /pituitary, and group received the same treatment as the above groups, cell viability was over 90% in all cell dispersions. The but received saline serum instead of Ipamorelin or cellular suspension was dispensed into tissue culture GHRH. consisting of 96 wells (2x104 cells/well/200 µl 10% After the last injection, animals were decapitated at FBS-DMEM) and incubated in a humidified atmosphere 1 pm and pituitaries were removed. The posterior of 5% CO2 in air, at 37 °C, for 3 days. Monolayer pituitaries were discarded, and the anterior pituitaries cultures were washed twice with DMEM and fresh 709 Somatotroph responses to Ipamorelin serum-free-DMEM was added. After 4 hours’ incubation removing the antiserum, replacing the antiserum with at 37 °C and 5% CO2, with DMEM, or with Ipamorelin normal rabbit serum, and preadsorbing the antiserum (10-8 M), or with GHRH (10-8 M), or with GHRP-6 (10-8 with rat GH (Biogenesis Ltd.) at 4 °C for 24 hours.