Faculty Bibliography

The complex interactions of the hypothalamic releasing peptides somatostatin (SRIF) and growth hormone (GH)-releasing hormone (GHRF), which regulate GH secretion, are still incompletely understood. To further scrutinize these interactions, we have studied the effects of GHRF on SRIF secretion from dispersed adult rat hypothalamic cells. Rat GHRF caused calcium- and dose-dependent stimulation of SRIF release in static 1-h incubations. SRIF release was stimulated by GHRF in a concentration range of 1-100 nM. However, the extended dose-response curve was biphasic in nature, with a significantly lower SRIF response in the presence of 1 microM GHRF vs. 100 nM GHRF. SRIF release, stimulated by another secretagogue (10 microM veratridine), was not affected by the presence or absence of 1 microM GHRF, suggesting the lack of toxic impairment of hypothalamic cell function by GHRF at this concentration. In conclusion, a biphasic stimulatory pattern of SRIF secretion in response to GHRF was observed in experiments employing dispersed rat hypothalamic cells. The biphasic SRIF response pattern to GHRF may be relevant in the physiological regulation of GH secretion

The tumor promoter, tetradecanoylphorbolacetate (TPA), causes a significant increase in both insulin secretion and the incorporation of 32Pi into phosphatidylcholine (PC) in RIN insulinoma cells. The peptide hormone, arginine vasopressin (AVP), also stimulates these functions, although to a lesser degree. When added together, the effects on secretion and PC metabolism are synergistic. At the same time, TPA inhibits the AVP-stimulated rise in phosphoinositide (PI) metabolism. Neither phloretin nor tamoxifen, reported to be inhibitors of protein kinase C activity, are able to block the effects of TPA on secretion, although both influence PC metabolism

In a perifused dispersed rat anterior pituitary cell system, growth hormone (GH) secretion became attenuated in response to repeated pulsatile or prolonged exposure to submaximal stimulatory concentrations of rat growth hormone-releasing factor (GHRF). However, persistent intracellular GH stores could be released upon subsequent challenge with the membrane depolarizing agent KCl, forskolin, or the phorbol ester, tetradecanoylphorbol acetate (TPA). The GH secretory response to repeated pulsatile administration of either KCl or forskolin also became attenuated. In these experiments, persistent intracellular GH stores could be released upon subsequent GHRF stimulation. Repeated challenge with pulses of TPA failed to elicit any GH release after the initial stimulatory response, although a subsequent GHRF pulse was stimulatory, indicating persistence of intracellular GH stores. These data are compatible with the hypothesis that the decreased GH secretory responsivity to GHRF, which was observed in the course of these experiments, is caused by the functional depletion of specific secretagogue-sensitive pools of intracellular GH, rather than by receptor-mediated desensitization

We examined the release of growth hormone-release inhibiting factor (somatostatin) from dispersed hypothalamic cells obtained from mature diabetic rodents and normal age-matched controls, in an attempt to demonstrate a possible hypothalamic defect which might underlie some of the reported abnormalities in somatotrophic function in diabetes mellitus. Insulinopoenic diabetes was induced by either streptozotocin or alloxan. Somatostatin release from cells from diabetic rats was diminished both basally and after stimulation by membrane depolarisation. Stimulated release was calcium dependent in cells from both normal and diabetic animals. The defect was present in both streptozotocin and alloxan induced diabetes. We also compared hypothalamic somatostatin release from cells obtained from obese hyperinsulinaemic C57 BL/Ks db/db diabetic mice and non diabetic lean litter mates (db/-). Despite longstanding marked hyperglycaemia, no significant alteration in somatostatin release was apparent. Likewise, starvation of rats for 5 days did not result in significant diminution of somatostatin release. These observations document a defect in hypothalamic somatostatin release in experimentally induced insulinopoenic diabetes, which is not apparent in the db/db mouse, suggesting that glucose per se is not responsible. Rather than the anticipated increase in hypothalamic somatostatin release in insulinopoenic diabetes, a reduction in release was observed. These observations are compatible with the hypothesis that increased hypothalamic somatostatin release is not responsible for abnormal growth hormone secretion in this model

To determine whether hypothalamic somatostatin (SRIF) release might be modulated by a negative feedback loop, we examined the effects of exogenous SRIF on the release of SRIF immunoreactivity from dispersed adult rat hypothalamic cells. Added SRIF-(1-14) caused dose-dependent inhibition of endogenous SRIF release in this system (IC50, 140 pM). SRIF-(1-28) and Tyr1-SRIF also inhibited SRIF release, whereas other peptides present in the hypothalamus were without demonstrable effects when tested at a concentration of 100 pM. Ala9-SRIF, a SRIF analog with reduced bio- and immunoreactivities, had no effect on SRIF release at concentrations as high as 1 nM. SRIF release from hypothalamic cells increased with lengthening periods of incubation from 1-3 h, but the increase was not linear in nature. Likewise, when hypothalamic cell density in the incubation medium was increased, less than the expected increment in SRIF release was observed. Conversely, when the incubation volume was increased and cell density was reduced, an increment in SRIF release was observed. These data demonstrate that SRIF when added at physiologically relevant concentrations inhibited endogenous SRIF release, confirming the presence of an ultrashort loop feedback effect at a hypothalamic level. Our observations also suggest that endogenous SRIF may modulate and inhibit its own release. These findings may be of physiological relevance in the control of the hypothalamic-somatotropic axis

We employed a short term system of dispersed adult mouse hypothalamic cells, as previously described in the rat, to examine the roles of calcium and calmodulin in SRIF release. Incubation studies were performed 24 h after hypothalamic cell dispersion. SRIF release, as determined by RIA was stimulated in a dose-dependent manner by the membrane-depolarizing agents KCl, ouabain, and veratridine as well as by the calcium ionophore A23187. The stimulation of SRIF release induced by depolarizing agents was abolished or diminished by 1) omission of extracellular calcium, 2) chelation of extracellular calcium by EGTA, and 3) the calcium channel blocker verapamil, indicating calcium dependence of this process. Three chemically distinct groups of calmodulin inhibitors were employed to study the role of calmodulin in stimulus-secretion coupling of hypothalamic SRIF. The neuroleptic calmodulin inhibitors trifluperazine (1 microM), chlorpromazine (10 microM), and promethazine (10 microM) as well as the naphthalene sulfonamide calmodulin inhibitor W7 (1 microM) and compound 48/80 (50 micrograms/ml) were all demonstrated to have an inhibitory effect on the stimulation of SRIF release induced by membrane depolarization. No inhibitory effect of the less potent naphthalene sulfonamide agent W5 was observed at 1 microM, although an inhibitory effect was seen at 10 microM. The stimulation of SRIF release induced by A23187 was not inhibited by omission of extracellular calcium or by verapamil, but was inhibited by EGTA and trifluperazine. These data demonstrate the role of calcium in membrane depolarization-induced stimulus-secretion coupling of mouse hypothalamic SRIF. Inhibition of the stimulatory response by low concentrations of three distinct groups of calmodulin inhibitors, i.e. neuroleptics, naphthalenesulfonamide agents and compound 48/80, suggests a role for calmodulin in this process