Abstract
We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.
Highlights
Somatostatin Inhibits InsulinSecretion by a G-Protein-mediated Decrease in Ca2+ Entry through Voltage-dependent Ca2+ Channinels the Beta Cell*
Somatostatin (SRIF)' is a neuropeptide originally isolated inhibits insulin secretion from an SV40 transformed from the hypothalamus that inhibitsgrowth hormone secrehamster beta cell line (HIT cells) by an effect on the tion
Measurement of [Ca2+]i andMembraneP~tential--[Ca*+]~ was We first established that two secretagogues, Bay K 8644 (a measured in fura-2-loaded HIT cell suspensions in modified KrebsRinger bicarbonate medium buffer containing[20] HEPES, 1.5 CaC12, 4 glucose, and 0.1% bovine serum albumin as previously described (22)
Summary
Fura-2AM andbisoxonol were obtained from Molecular Probes (Eugene, OR) and RPMI 1640 medium and fetal bovine serum from GIBCO (Grand Island, NY). The studies were initiated by removing the medium, washing, and incubating thceells with Krebs-Ringer bicarbonatemedium (1.67 mM glucose and minus &RbCl) containing the test agentsover a 40min time course at20 "C. The cells were grown in RPMI 1640 medium with Cells were scrapedin 1%sodium dodecyl sulfate,and radioactive. All experiments were performed with diazoxidewasused as apositive control to demonstrate K' efflux cells from passage 64-76. Insulin Secretion-HIT cells were plated into12-wellCostar plates centage of"Rb' radioactive content at time 0 which is inversely at 5 X lo[5] cells/25-mm well and grown for 3 days. Insulinsecretionfrom HIT cell Statistical Analyses-Results were analyzed by Student's t test for monolayers incubated in Krebs-Ringer bicarbonatemedium contain- paired and unpairedvalues. Insulin in the supernatants was measured by radioimmunoassay as previously described (21)
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