Sulfonylureas enhance exocytosis from pancreatic beta-cells by a mechanism that does not involve direct activation of protein kinase C.
Hypoglycemic sulfonylureas stimulate insulin release by binding to a regulatory subunit of plasma membrane ATP-sensitive K+ (K(ATP)) channels. The consequent closure of K(ATP) channels leads to depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx, and a rise in intracellular [Ca2+]. Recently, however, it has been suggested that sulfonylureas may have an additional action on secretion, independent of changes in intracellular [Ca2+] but dependent on the activity of protein kinase C (PKC). We have investigated the mechanisms involved in the PKC-dependent effect of sulfonylureas on the secretion machinery in beta-cells. In MIN6 beta-cells permeabilized by streptolysin O, insulin release was stimulated by elevation of [Ca2+] from 10(-8) to 10(-5) mol/l. At a [Ca2+] of 10(-8) mol/l, insulin release from permeabilized beta-cells was stimulated by addition of GTP-gamma-S, or by addition of a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA, but not GTP-gamma-S, also increased insulin release when [Ca2+] was 10(-5) mol/l. Insulin release from permeabilized beta-cells was stimulated by tolbutamide (0.1-1 mmol/l) at 10(-8) but not at 10(-5) mol/l Ca2+. The effect of tolbutamide was blocked either by inhibition of PKC or when phorbol ester-sensitive PKC isoforms were maximally stimulated by TPA. Meglitinide and glibenclamide also stimulated insulin release from permeabilized beta-cells. To assess the possibility that direct activation of PKC mediates the exocytotic response to sulfonylureas, we studied the effect of tolbutamide and glibenclamide on PKC activity. Purified brain PKC was not activated by tolbutamide or glibenclamide, whether tested in the absence or presence of phosphatidylserine or TPA, or at low or high [Ca2+]; nor was the total PKC activity in extracts of MIN6 beta-cells affected by tolbutamide. Neither tolbutamide nor glibenclamide elicited translocation of any isoform of PKC in intact or permeabilized beta-cells under conditions in which TPA evoked a marked redistribution of PKC alpha- and epsilon-isoforms. We conclude that although the plasma membrane K(ATP) channel-independent stimulation of exocytosis by sulfonylureas may require functional PKC, the mechanism does not involve a direct activation of the enzyme.
Tian
Y A
YA
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, UK.
Johnson
G
G
Ashcroft
S J
SJ
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects*; D005905:Glyburide / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007340:Insulinoma; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D010190:Pancreatic Neoplasms; D010718:Phosphatidylserines / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D013453:Sulfonylurea Compounds / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014044:Tolbutamide / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007004:Hypoglycemic Agents; D007527:Isoenzymes; D010718:Phosphatidylserines; D015221:Potassium Channels; D013453:Sulfonylurea Compounds; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014044:Tolbutamide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D005905:Glyburide; D002118:Calcium