Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations

Store-operated Ca2+ channels in liver cells have been shown previously to exhibit a high selectivity for Ca2+ and to have properties indistinguishable from those of Ca2+-release-activated Ca2+ (CRAC) channels in mast cells and lymphocytes [Rychkov, Brereton, Harland and Barritt (2001) Hepatology 33, 938-947]. The role of CRAC channels in the maintenance of hormone-induced oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+](cyt)) in isolated rat hepatocytes was investigated using several inhibitors of CRAC channels. 2-Aminoethyl diphenylborate (2-APB; 75 muM), Gd3+ (1 muM) and 1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF 96365; 50 muM) each inhibited vasopressin- and adrenaline (epinephrine)-induced Ca2+ oscillations (measured using fura-2). The characteristics of this inhibition were similar to those of inhibition caused by decreasing the extracellular Ca2+ concentration to zero by addition of EGTA. The effect of 2-APB was reversible. In contrast, LOE-908 {(R,S)-(3,4-dihydro-6,7-dimetlioxy-isochinolin-1-yl)-2-phenyl-N,N-di[2- (2,3,4-trimethoxyphenyl)ethyl]acetamide mesylatel (30 muM), used commonly to block Ca2+ inflow through intracellular-messenger-activated, non-selective cation channels, did not inhibit the Ca2+ oscillations. In the absence of added extracellular Ca2+, 2-APB, Gd3+ and SK&F 96365 did not alter the kinetics of the increase in [Ca induced by a concentration of adrenaline or vasopressin that induces continuous Ca2+ oscillations at the physiological extracellular Ca2+ concentration. Ca2+ inflow through non-selective cation channels activated by maitotoxin could not restore Ca2+ oscillations in cells treated with 2-APB to block Ca2+ inflow through CRAC channels. Evidence for the specificity of the pharmacological agents for inhibition of CRAC channels under the conditions of the present experiments with hepatocytes is discussed. It is concluded that Ca2+ inflow through CRAC channels is required for the maintenance of hormone-induced Ca2+ oscillations in isolated hepatocytes.