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Readers are referred to a recent symposium (1) and reviews (2–4) for a comprehensive and broad introduction to somatostatin (SS) and its actions. This chapter will be restricted to the mechanisms (primarily ionic) by which SS inhibits growth hormone (GH) release. The abundant literature on the actions of SS on other cell types will not be discussed unless directly relevant to ionic mechanisms in somatotrophs. We will begin by summarizing the ionic mechanisms by which growth hormone releasing factor (GRF) stimulates the acute release of GH via cyclic AMP (cAMP) and cytosolic free Ca++ ([Ca++]i) as second messengers (Fig. 2.1). This is discussed in more detail elsewhere in this volume.

Acid extracts of a) acutely dispersed rat pars intermedia (PI) cells, b) media after incubation of PI cells, c) whole nervosa-intermedia, and d) whole pars distalis, were chromatographed on Sephadex G-50 Fine in 1% acetic acid. Three peaks of ACTH biological activity were resolved in all four extracts. Peak I eluted in the void volume of the column, peak III co-eluted with synthetic ACTH1–39, and peak II eluted in an intermediate position. The predominant ACTH activity derived from the PI tissue was peak I, amounting to over 70% of the total ACTH activity present in that lobe. The positions of PI peaks I and II remained unaltered after rechromatography as well as after treatment with and chromatography in 8 M urea. However, peak I of PI ACTH was further resolved into two separate peaks by chromatography on Sephadex G-100 SF. Thus pars intermedia ACTH activity appears to be composed of four separate entities, with the predominant forms being larger than ACTH1–39.

Studies were carried out to further define the sites of interaction of Ca++, cAMP, and somatostatin (SRIF) in the mechanisms governing GH release from acutely dispersed purified somatotrophs obtained from rat adenohypophyses. Both high [K+] and the Ca++ ionophore A23187 stimulated the release of GH. The release induced by high [K+] was accompanied by a small but significant transient increase in intracellular cAMP, while A23187 did not alter basal cAMP levels. The augmented release of GH induced by both secretagogues was blocked by SRIF (1 ng/ml) and by removing Ca++ from the incubation medium (85 microM). The transient increase in cAMP induced by high [K+] was not blocked by SRIF or by low Ca++ incubation. These results are consistent with a model whereby an increase in free cytosol Ca++ will result in GH release by exocytosis. This increase in free cytosol Ca++ can come from either intracellular bound Ca++ or from the extracellular compartment. Secretagogues which act via cAMP increase intracellular cAMP levels. The increase in cAMP would then stimulate the translocation of Ca++ from a bound to a free cytosol form. Secretagogues may also stimulate GH release by bypassing cAMP to increase free cytosol Ca++ by directly increasing the influx of Ca++ into the cells or by increasing the intracellular movement of Ca++ to the free cytosol compartment. SRIF would block GH release by acting at the level of the plasma membrane to block Ca++ influx and by inhibiting a step(s) subsequent to the increase in cytosol Ca++.

The exposure of rat adenohypophysial tissue to iced media for periods of 30–60 min causes accumulation of Ca2+ by the tissue and an increased "basal" release of growth hormone into the media. The Ca2+ permeability following the chill, estimated from the uptake of 45Ca2+ by the tissue at 37 °C, is unchanged by the exposure to iced media. This suggests that the accumulation of Ca2+, which occurs during the chill, may be due simply to decreased efflux of Ca2+. The insensitivity of 45Ca2+ influx to the increased cellular content is readily explained if Ca2+ is rapidly sequestered after it has entered the cell. Our previous investigations snowed an increase in 45Ca2+ uptake (permeability) associated with hormone release which had been induced by elevated K+ or by partially purified releasing factor. We used tissue that had been chilled during collection. Our studies here indicate that our previous observations of increased uptake are qualitatively correct despite the cellular accumulation of Ca2+ that must have occurred before exposure to the secretagogues. The release of hormone seems to be related to the absolute cytoplasmic concentration of Ca2+ and release will occur in any circumstance that increases this concentration. The source of Ca2+ is not critical.

The presence of immunoreactive α melanocyte stimulating hormone (IαMSH) was investigated in both mucosal and muscular layers of the various areas of the gastrointestinal tract. IαMSH was present in both layers in all areas of the gastrointestinal tract but the esophageal mucosa and muscularis in saline extracts. The highest concentrations were found in the duodenum. Hypophysectomized males tended to have higher content than intact males. There was no difference between intact estrogen-primed females and hypophysectomized females up to 1 month post hypophysectomy in any area. The tract of 3 month hypophysectomized females showed lower levels than the intact estrogen-primed females in 5 areas; however, in similar groups of 3 month hypophysectomized females which were estrogen primed, 8 of the 10 areas contained more IαMSH than the intact estrogen-primed females. Acid extracts from female rats during the estrous cycle showed no cycle-dependent differences. Comparison of acid and saline extracts showed an absence of IαMSH in gastric tissues and a decrease in the duodenal muscularis in acid extracts but no consistent differences were found in other areas. These results suggest that the IαMSH found in the gastrointestinal tract is not of pituitary origin but may be produced in the gastrointestinal tract. The induction of increased content by estrogen priming in hypophysectomized rats suggests that estrogen priming may induce production. The absence of IαMSH in acid extracts of the stomach suggests that a difference in distribution of pro-opio-cortin products may exist in the gastrointestinal tract.

cAMP is thought to be an essential intracellular mediator in the release of GH from somatotrophs. Ca++ is required in the incubation medium to elicit GH release iin vitro. We have carried out studies using a purified preparation of rat somatotrophs to see whether the Ca++ requirement precedes or follows the accumulation of cAMP induced by prostaglandin E2 (which increases adenylate cyclase activity) and 3-isobutyl-lmethylxanthine (a phosphodiesterase inhibitor). Incubation of somatotrophs in low Ca++ medium (

In 28 6-h experiments on 10 conscious resting trained male dogs, plasma growth hormone (GH) was determined at 5-min intervals by radioimmunoassay. For all experiments, the basal GH concentration in plasma was 0.80 ± 0.06 ng mL−1. In each experiment, 1–3 secretory bursts of GH occurred, raising plasma GH 2.4 to 15.3 times basal concentrations (for all 43 bursts, 6.6 ± 0.4 times the basal value). Metabolic clearance rates (MCR) and apparent distribution volumes (V) were determined, using stepwise infusions of canine GH. The MCR (3.99 ± 0.30 mL kg−1 min−1) and V (57.9 ± 5.5 mL kg−1) were used to transform the GH concentration versus time data into GH secretion rates, using a single compartment approach. Basal GH secretion rates for all 28 experiments were 3.12 ± 0.24 ng kg−1 min−1. The secretory bursts yield peak GH secretion rates of 9.4 ± 0.8 times basal secretion and these steep-sloped bursts last 25.1 ± 1.2 min. Six-hour infusions of 0.15 μg kg−1 min−1 of somatostatin (SRIF) abolished all secretory bursts but did not lower basal secretion rates. In five of seven SRIF infusion experiments in which samples were taken after the infusion ceased a secretory burst was seen in the hour following cessation of infusion (in four cases within 10 min). These secretory bursts lasted 23.0 ± 2.9 min and were similar to those seen in control experiments. Infusions of SRIF at 0.05 μg kg−1 min−1 had no effect. These results imply that during basal GH secretion, a surfeit of SRIF impinges on the somatotrophs, as extra SRIF does not further lower basal secretion. However, during secretory bursts, very little SRIF must be present, as exogenous SRIF blocks these bursts. The bursts are similar in duration to overshoots provoked in perifused dispersed rat somatotrophs by removal of an SRIF signal. It seems likely that their cause in vivo is similar. (All values are means ± SEM.)

Sheep hypothalamic extracts, chromatographed on Sephadex G-25, revealed 3 peaks with CRF biological activity. The first eluting peak co-chromatographed with ACTH. After separation from ACTH by CMC, this CRF peak contained vasopressin immunoactivity and pressor activity. The second eluting CRF peak also contained vasopressin immunoactivity and pressor activity and was found to represent the presumed authentic vasopressin. The third eluting CRF peak contained vasopressin immunoactivity but no pressor activity. Further processing of this putative CRF on Sephadex G-10 resulted in a 500-fold purification. The material is inactivated by proteolytic enzymes. We tentatively conclude that this CRF is a peptide with a molecular weight of 700–1000 and that it shares common antigenic determinants with vasopressin.

The problems inherent in the study of the control of adenohypophyseal hormone secretion in vitro using intact tissue or tissue fragments are fourfold: variability of response, lack of viability of tissue in the gland core, relative lack of sensitivity, and the heterogeneity of cell types within the gland, which precludes interpretation of intracellular metabolic events within a specific cell type. Several investigators have used acutely dispersed or cultured adenohypophyseal cells to examine the effects of hypothalamic regulatory hormones as described in recent reviews (Labrie et al., 1976b; Vale et al., 1976). These preparations overcome the problems of variability, viability, and sensitivity associated with the classic whole or hemipituitary studies. However, the difficulties arising from the heterogeneity of the cell type remain. It is not possible, using presently available preparations, save for the use of autoradiographic techniques, to localize alterations of intracellular metabolite involved in the release of one hormone to a specific adenohypophyseal cell type. In light of much evidence showing a lack of specificity of several of the hypothalamic hypophysiotropic hormones (Labrie et al., 1976a,b; Vale et al., 1976), it has become imperative to study a uniform cell population. Cloned tumor cell lines have been used in an attempt to overcome this problem (Tashjian et al., 1968; Hertelendy and Keay, 1974; Dannies et al., 1976), but there is no assurance that intracellular events are not grossly altered in tumor cells.

The parenchymal cells of the pars intermedia (PI) and corticotrophs of the pars distalis (PD) synthesize pro-opiomelanocortin (POMC), which, through posttranslational processing, gives rise to a group of structurally related peptides, including MSHs, ACTH, CLIP, LPHs and endorphins. We investigated the control of release of these peptides using an in vitro system. We perifused either intact neurointermediate lobes (NI) or PD halves obtained from rats. Perifusion medium and tissue extracts were subjected to a battery of bioassays (BA) and radioimmunoassays (RIA) (including MSH-BA, alpha-MSH-RIA, ACTH-BA, ACTH-RIA, LPH-RIA) and a receptor-binding assay for morphine-like activity (MLA). The relative amounts of released peptide activities were examined under basal conditions and after challenging with synthetic ovine corticotrophin-releasing factor (CRF) and somatostatin. CRF stimulated the release of all assayed peptides from both the PD and PI in a dose-related manner. Stimulated release was immediate (within 3 min), constant, reversible and repeatable. Somatostatin (up to 100 ng/ml) did not alter basal release from either PD or PI. Somatostatin did block CRF-induced release from the PI but not from the PD. These observations support an action of both CRF and somatostatin in the control of secretion of POMC-derived peptides from the PI.

In order to define the roles of intracellular Ca++ and cAMP in the mechanisms governing GH release, we have carried out perifusion studies using a purified preparation of acutely dispersed somatotrophs obtained from rat adenohypophyses. Two groups of secretagogues were used: those that act by increasing intracellular cAMP [(Bu)2cAMP], the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, and prostaglandin E2; and those thought to act primarily by increasing Ca++ influx into the cells (high extracellular K+ and the Ca++ ionophore A23187). The release of GH induced by the three secretagogues that increase cAMP levels is instantaneous and maintained during perifusion, whereas the release induced by high K+ and A23187, unlike the cAMP-induced release, reaches a peak within 2 min and then falls rapidly back to baseline levels, even though the secretagogues are still present. After GH release has returned to baseline levels but while high K+ is maintained, the somatotrophs respond briskly to (Bu)2cAMP, 3...

The purpose of this study was to ascertain whether the pars intermedia of the rat adenohypophysis, isolated from direct innervation via the infundibular stem, could maintain adrenal cortical weight and plasma corticosterone levels. We compared the adrenal cortical response of rats 40 days after either complete hypophysectomy, hypophysectomy with reinsertion of only the pars distalis, or hypophysectomy with reinsertion of only the nervosa-intermedia. Adrenal weight and plasma corticosterone levels were partially maintained in the group with reinserted pars distalis. These parameters were not different from the complete hypophysectomy group in the animals with reinserted nervosa-intermedia. Thus, the pars intermedia, with its nerve supply disrupted, cannot maintain adrenal cortical function.

Studies were carried out to simultaneously measure cAMP and cGMP accumulation and GH release from acutely dispersed purified somatotrophs obtained from rat adenohypophyses. cAMP accumulation was dramatically increased by both prostaglandin E2 (10(-6) M) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor, 0.5 mM) within 1 min of their addition, while there was a delay of 8--16 min before a significant increase in GH release was seen. SRIF (100, 10, or 1 ng/ml) completely blocked the stimulated release of GH. SRIF also consistently decreased the elevation of cAMP induced by the two secretagogues, but this decrease was small and not always significant. cGMP was unmeasurable (less than 0.02 fmol/1000 cells) in all of our experiments, while basal cAMP levels were about 1 fmol/1000 cells. We conclude that cAMP plays a role in the intracellular mechanisms governing GH release and that SRIF primarily acts subsequent to cAMP elevation, with a possible secondard or minor action on cAMP formation.

An acutely dispersed and purified preparation of somatotrophs obtained from rat adenohypophyses was used to study the mechanism of action of GH-releasing factor (GRF). Synthetic GRF [human pancreatic, hpGRF-(1–40)-OH] stimulated the immediate (within 4 min) release of GH in a dose-related manner, with a preceding or concurrent increase in cAMP in the somatotrophs. Somatostatin, at concentrations as low as 1.0 ng/ml, completely blocked the GRF-induced increase i n GH release, with only a partial reduction in the GRF-induced accumulation of cAMP in the somatotrophs. 3-Isobutyl-l-meth-ylxanthine, a phosphodiesterase inhibitor, potentiated the action of GRF in increasing cAMP in the somatotrophs, with subsequent GH release. These results along with those of previous studies suggest that cAMP is an intracellular mediator in the action of GRF and that somatostatin has a major effect on blocking GH release at a step subsequent to cAMP accumulation. (Endocrinology 117: 2364–2370, 1985)