Phorbol ester-stimulated insulin secretion by RINm5F insulinoma cells is linked with membrane depolarization and an increase in cytosolic free Ca2+ concentration.
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.
Yada
T
T
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401.
Russo
L L
LL
Sharp
G W
GW
D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004987:Ethers / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D007340:Insulinoma / Q000378:metabolism*; D015759:Ionomycin; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D001572:Benzofurans; D004987:Ethers; D005456:Fluorescent Dyes; D015759:Ionomycin; D011189:Potassium Chloride; D002217:Carbachol; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2