Effects of calcium channel inhibitors on insulin-induced increase in calcium influx: To determine the pharmacological profile of the calcium influx in HTC cells, several inhibitors known to interfere with calcium entry pathways were administered using the manganese quench protocol. Inhibitors were introduced with a calcium-free standard buffer at the same time as 50 pM manganese chloride. First, the divalent cations zinc, cobalt and nickel were tested. Administration of zinc blocked the basal rate of manganese influx, whereas cobalt and nickel ions by themselves were found to quench FURA-2 fluorescence at the monitored calcium-insensitive wavelength of 357 nm (data not shown). Consequently, the data using these divalent cations could not be interpreted. Second, 100 pM verapamil was used, which is a known blocker of voltage-operated calcium channels.
In the presence of verapamil, insulin accelerated basal manganese influx rate by 1.42±0.1-fold (n=10), which was not statistically different from the effect of insulin alone (not significant by unpaired ANOVA). Then, gadolinium was used, an ion of the lanthanide series known to block calcium entry pathways triggered by the depletion of intracellular calcium pools in cultured hepatocytes and to inhibit non- selective cation channels in hepatoma cells. At a concentration of 100 pM, gadolinium prevented insulin from increasing the basal manganese influx rate (1.05±0.09-fold increase, n=25, P<0.05 compared with insulin alone by unpaired ANOVA). Finally, 10 pM SKF96365, a compound known to interfere with receptor-operated calcium inflow in rat hepatocytes, also abolished the effect of insulin on basal manganese influx rate (0.65±0.08-fold increase, n=12, P<0.05 compared with insulin alone by unpaired ANOVA). Neither gadolinium, SKF96365, nor verapamil had a significant effect on the steady-state baseline manganese-induced quench rate (not significant by unpaired ANOVA).
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