title: Binding of the dihydropyridine calcium channel blocker (+)-[3H] isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-3-pyridinecarboxylate (PN200-110) to RINm5F membranes and cells: characterization and functional significance.
abstract: This report provides direct evidence for a dihydropyridine receptor/calcium channel in the insulin-secreting beta-cell line RINm5F. The receptor/channel can modulate the intracellular Ca++ concentration and the resultant insulin secretion by regulating the influx of extracellular Ca++ through dihydropyridine-sensitive voltage-dependent L-type Ca++ channels. Elevated extracellular K+ or the dihydropyridine Ca++ channel agonist, BAY k 8644 [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl- phenyl)pyridine-5-carboxylate], stimulated the uptake of 45Ca++, raised [Ca++]i, and increased insulin secretion in a concentration-dependent manner. These actions were inhibited by L-type Ca++ channel blockers including nitrendipine, verapamil and diltiazem. (+)-[3H]PN200-110 bound specifically with high affinity to RINm5F cell membranes (Kd approximately 200 pM). Specific binding was inhibited competitively by dihydropyridines whereas phenylalkylamines inhibited incompletely (+)-[3H]PN200-110 binding, consistent with an allosteric interaction. The benzothiazepine diltiazem had no effect on (+)-[3H]PN200-110 binding in the presence of Ca++, but increased binding allosterically in the absence of Ca++ (in the presence of EGTA). Maximal (+)-[3H]PN200-110 binding required divalent cations, with Mg++, Mn++ and Ba++ essentially as effective as Ca++ in reversing the effects of EGTA, whereas binding was not supported by Cd++ or La . Specific high affinity (+)-[3H]PN200-110 binding was also demonstrated in intact RINm5F cells and shown to be modulated by membrane potential. Depolarization of the cells by raising extracellular K+ from 5 to 80 mM increased the affinity of (+)-[3H]PN200-110 4- to 5-fold (decreased Kd) with no significant effect on the maximum number of binding sites.
authors.affiliation: Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca.
authors.forename: G C
authors.initials: GC
authors.lastname: Yaney
authors.forename: G A
authors.initials: GA
authors.lastname: Stafford
authors.forename: J D
authors.initials: JD
authors.lastname: Henstenberg
authors.forename: G W
authors.initials: GW
authors.lastname: Sharp
authors.forename: G A
authors.initials: GA
authors.lastname: Weiland
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D017275:Isradipine; D008564:Membrane Potentials / Q000187:drug effects; D010069:Oxadiazoles / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D007328:Insulin; D010069:Oxadiazoles; D011189:Potassium Chloride; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D002118:Calcium; D017275:Isradipine