Characterization of voltage-dependent Ca2+ channels in beta-cell line.
Although there is compelling pharmacological evidence based on Ca2+-channel antagonist studies suggesting that the voltage-dependent Ca2+ channels regulate insulin release, no direct comparison with Ca2+ currents exists. This is particularly important because of the recent demonstration in other cell types of one and possibly two Ca2+ channels that are insensitive to Ca2+-channel antagonists, the dihydropyridines and the phenylalkylamines. Using an SV40-transformed pancreatic beta-cell line (HIT cells), we determined how voltage-dependent Ca2+ channels are involved in stimulus-secretion coupling. Ca2+ currents were measured with the tight-seal technique for whole-cell recording. The cytosolic free-Ca2+ concentration ([Ca2+]i) was followed with the fluorescent probe Fura 2, and the measurements were compared with insulin secretion stimulated by depolarizing the cells with K+. The Ca2+ current contained two components: a rapidly decaying current activated at -50 to -40 mV that decayed with a time constant of 25 ms and a very slowly decaying component activated at -40 mV. Both components were sensitive to the Ca2+-channel antagonist nimodipine. There is excellent agreement in the concentration of nimodipine that inhibited Ca2+ and the increase in [Ca2+]i in response to K+ depolarization (IC50 of 15 and 6 nM, respectively). Nimodipine inhibited insulin release over a similar dose-response range with an IC50 of 1.5 x 10(-9) M. These studies indicate that the increase in [Ca2+]i in response to beta-cell depolarization can be accounted for by the influx of this ion through a single class of dihydropyridine-sensitive Ca2+ channels in the cell membrane.
Keahey
H H
HH
Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Rajan
A S
AS
Boyd
A E
AE
Kunze
D L
DL
D000818:Animals; D001464:Barium / Q000378:metabolism; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D016257:Fura-2; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D009553:Nimodipine / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology
D001572:Benzofurans; D015220:Calcium Channels; D007328:Insulin; D001464:Barium; D009553:Nimodipine; D011188:Potassium; D002118:Calcium; D016257:Fura-2