Muscarinic modulation of voltage-dependent Ca2+ channels in insulin-secreting HIT-T15 cells.
Potentiation of insulin secretion from pancreatic beta-cells by acetylcholine requires ongoing cyclic electrical activity initiated by other depolarizing secretagogues. Patch-clamp recordings in glucose-free solutions were made from the clonal beta-cell line HIT-T15 to determine whether the muscarinic agonist bethanechol (BCh) modulated voltage-dependent Ca2+ channels independent of effects on membrane potential. Only high-threshold, dihydropyridine-sensitive (L-type) Ca2+ channels with a mean conductance of 26 pS were observed in cell-attached patches. BCh (100 microM) caused a two- to threefold increase in both fractional open time and mean current of single Ca2+ channels. These changes resulted from a 44% decrease in the longer of two apparent mean closed times and a 25% increase in the mean open time. Similar BCh-stimulated increases in macroscopic Ca2+ currents were recorded in whole cell, perforated-patch recordings. The role of protein kinase C (PKC) in the muscarinic activation of Ca2+ channels was tested using a variety of PKC activators and inhibitors. Acute application of either the active phorbol ester phorbol 12-myristate 13-acetate (PMA) or the membrane-permeable diacylglycerol analog 1,2-didecanoyl-rac-glycerol mimicked the effects of BCh, whereas an inactive phorbol (4 alpha) had no effect. Depletion of PKC activity by chronic exposure to PMA or acute application of the PKC inhibitor staurosporine greatly reduced or abolished muscarinic activation of Ca2+ channels. These results are consistent with muscarinic activation of L-type, voltage-dependent Ca2+ channels mediated in large part by PKC.
Love
J A
JA
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson 39216, USA.
Richards
N W
NW
Owyang
C
C
Dawson
D C
DC
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000109:Acetylcholine / Q000378:metabolism*; D018723:Bethanechol / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D015640:Ion Channel Gating; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D007328:Insulin; D018721:Muscarinic Agonists; D018723:Bethanechol; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D011493:Protein Kinase C; D009543:Nifedipine; D000109:Acetylcholine