sq-23377 and Insulinoma

Noradrenaline effects on voltage-operated calcium channels and exocytosis were studied, for the first time, in single patch-clamped RINm5F insulin-secreting cells. Noradrenaline, despite small and variable inhibition of calcium currents, strongly inhibited the increase in membrane capacitance (a measure of exocytosis) stimulated by both step depolarizations and the calcium ionophore, ionomycin. Noradrenaline similarly inhibited KCl- and ionomycin-induced [3H]serotonin release from RINm5F cell populations. Noradrenaline effects were mediated by PTX-sensitive G proteins. Noradrenaline inhibitory effects on secretion are, therefore, mainly exerted downstream from Ca2+ influx.

Topics: Animals; Calcium; Calcium Channels; Electrophysiology; Exocytosis; Insulinoma; Ionomycin; Ionophores; Norepinephrine; Patch-Clamp Techniques; Potassium Chloride; Rats; Serotonin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM-dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP-dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I-like protein.

Topics: Animals; Autoradiography; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Insulin; Insulin Secretion; Insulinoma; Ionomycin; Mice; Mice, Transgenic; Pancreatic Neoplasms; Phosphorylation; Potassium Chloride; Substrate Specificity; Synapsins; Time Factors; Tolbutamide; Tumor Cells, Cultured

In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.

Topics: Adenoma, Islet Cell; Animals; Benzofurans; Calcium; Carbachol; Cell Line; Cell Membrane; Cytosol; Ethers; Fluorescent Dyes; Fura-2; Insulinoma; Ionomycin; Kinetics; Membrane Potentials; Pancreatic Neoplasms; Potassium Chloride; Rats; Tetradecanoylphorbol Acetate