Mouse hypothalamic somatostatin release: roles of calcium and calmodulin.

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.

Inhibition of rat hypothalamic somatostatin release by somatostatin: evidence for somatostatin ultrashort loop feedback.

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.

Inhibition of hypothalamic somatostatin release by beta-adrenergic antagonists.

A number of in vivo studies suggest that hypothalamic somatostatin (SRIF) tone is stimulated by the beta-adrenergic system. Employing dispersed adult male rat hypothalamic cells, we studied the effects of beta-adrenergic antagonists on the release of hypothalamic SRIF. Propranolol, at concentrations of 1-100 microM, had no detectable effect on basal SRIF release, but caused dose-dependent inhibition of SRIF release stimulated by ouabain. Two other beta-adrenergic antagonists, labetolol and metoprolol, also caused inhibition of ouabain-stimulated SRIF release. The alpha 2-agonist clonidine was without effect on SRIF release under basal or stimulated conditions. GH secretion from monolayers of dispersed rat anterior pituitary cells was also examined. Propranolol (1-100 microM) had no significant effect on basal GH secretion or GH secretion stimulated by rat GRF. In conclusion, 1) beta-adrenergic antagonists caused inhibition of stimulated SRIF release; 2) clonidine had no detectable effect on SRIF release; and 3) propranolol did not affect GH secretion in vitro. These findings support the hypothesis that beta-adrenergic antagonists augment GH responsivity by inhibiting hypothalamic SRIF release.

Effects of vasopressin on insulin secretion and inositol phosphate production in a hamster beta cell line (HIT).

The recent isolation of vasopressin (VP) from the rat and human pancreas led us to investigate the effects of VP on insulin secretion. In the SV 40-transformed hamster beta cell line (HIT), 0.1-1.0 nM VP caused rapid stimulation of insulin secretion. Slight but significant inhibition of insulin secretion was observed in the presence of 10 pM VP. These effects of VP on insulin secretion were paralleled by dose-dependent changes in inositol phosphate (IP) production, indicating mediation by V1-type VP receptors. VP stimulated IP3 production at 30 sec and production of IP1 by 60 sec. VP (0.1 nM to 1 microM) failed to stimulate the release or cellular content of cAMP, whereas forskolin was an effective stimulus. Forskolin and VP together caused at least additive stimulation of insulin secretion. Taken together, these observations indicate that VP is not acting via V2-mediated pathways. However, VP-induced stimulation of insulin and IP production were only slightly inhibited by a V1a pressor antagonist in 100- or 1,000-fold excess, indicating that VP effects are not mediated by V1a receptors. The V1 receptor involved may represent a V1b or a novel type of VP receptor. These observations suggest a potential physiological role of VP in regulating insulin secretion.

CSF somatostatin in Alzheimer's disease.

Studies have previously demonstrated low somatostatin levels in autopsy cortical tissue from Alzheimer's disease (AD) patients and low somatostatin levels in CSF obtained from subjects with dementia. We evaluated levels of this peptide in 21 non-depressed subjects, 10 with AD, 2 with Parkinson's disease (PD), and 9 with other neurological conditions. The AD patients had significantly lower mean CSF somatostatin than the "other" neurological patients (14.6 +/- 1.5 S.E.M. versus 26.7 +/- 3.2 pg/ml, p less than 0.005). A demented PD subject had a level in the range of the AD group, while the non-demented PD patient had a value above this range. Thus, all 11 patients with AD or PD dementia, analogous disorders, had levels below 21.8 mg/ml, while 7 of the 10 remaining patients had values above 21.8 pg/ml. Age did not explain this finding.

Pre-exposure of rat anterior pituitary cells to somatostatin enhances subsequent growth hormone secretion.

The precise roles of GH-releasing factor (GRF) and somatostatin (SRIF) in the orchestration of pulsatile GH secretion have not yet been fully determined. We examined the interactions of rat GRF and SRIF in the concentration ranges present in rat hypophysial-portal blood, on the secretion of GH from dispersed male rat anterior pituitary cells in monolayer culture. The effects of exposing cells to GRF and/or SRIF (0.01-1.nmol/l) for 1 h were compared with the effects of preincubation of cells with SRIF before experimental incubations. As anticipated, the stimulatory effects of 0.1-1 nmol GRF/1 were abolished by concurrent incubation with SRIF at an equimolar concentration, although SRIF, at these concentrations, did not significantly inhibit basal GH secretion. Conversely, pre-exposure to 0.1 nmol SRIF/1 for 30 or 60 min, resulted in an increase in GH secretion during a subsequent 60-min incubation period, both in the absence or in the presence of GRF (0.01-1 nmol/l). Pretreatment with GRF caused increased responsivity to GRF rather than significant sensitization of the GH response to GRF. These observations demonstrate actions of SRIF, at low and probably physiological concentrations, which are more complex than those of a pure inhibitor of GH secretion. Pre-exposure of the pituitary to SRIF enhances subsequent GH secretion, suggesting that SRIF may play an additional physiological role in amplifying the GRF signal.

Timing of exposure to somatostatin relative to growth hormone-releasing factor dictates the rat anterior pituitary cell growth hormone response.

The temporal interactions of rat GH-releasing factor (GRF) and somatostatin (SRIF) on the secretion of GH from perifused rat anterior pituitary cells have been studied. SRIF and GRF were employed at concentrations in a range close to levels reported in the hypophysial circulation of the rat. GH secretion was inhibited by pulses of 1 nmol SRIF/l (6 min) or 0.3 nmol SRIF/l (6 or 20 min). No rebound GH stimulation was observed. Exposure of cells to 6-min pulses of 0.3 nmol rat GRF/l repeated three times resulted in rapid stimulatory responses which became attenuated. Concomitant exposure to 0.3 nmol SRIF/l during a GRF pulse resulted in transient inhibition followed by a delayed and enhanced GH response, measured as the area under the curve but not peak height, whereas continuation of SRIF after a GRF pulse abolished the GH response. Exposure to 0.3 nmol SRIF/l prior to but not during a GRF pulse did not delay or inhibit the GH response. The area under the curve was increased under these conditions because of the lowered baseline in cells perifused with SRIF. SRIF alters somatotroph responsivity to equimolar concentrations of rat GRF and under different temporal conditions and can inhibit, enhance or delay GH secretion.

Anticonvulsants inhibit rat neuronal somatostatin release.

Somatostatin (SRIF), a peptide widely distributed in the central nervous system, has been implicated in the genesis of seizure activity in a number of animal models of epilepsy. We examined the effects of the anticonvulsants, phenytoin, carbamazepine and diazepam, on the release of SRIF from dispersed adult rat neuronal cells in short-term culture. Each of these agents caused dose-dependent inhibition of ouabain-stimulated SRIF release in a well-characterized hypothalamic dispersed cell system. We also examined the effects of phenytoin on SRIF release from dispersed rat cortical cells and inhibition of stimulated SRIF secretion was again observed. These findings support the hypothesis that the inhibition of neuronal SRIF release may represent a pharmacological mechanism of action of anticonvulsants.

GHRF causes biphasic stimulation of SRIF secretion from rat hypothalamic cells.

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.

Evidence that diminished pituitary responsivity to GHRF is secondary to intracellular GH pool depletion.

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.

Somatostatin release from dispersed hypothalamic cells: effects of diabetes.

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.