EFFECTS OF CAFFEINE ON CYTOPLASMIC FREE CA2+ CONCENTRATION IN PANCREATIC D-CELLS ARE MEDIATED BY INTERACTION WITH ATP-SENSITIVE K+ CHANNELS AND L-TYPE VOLTAGE-GATED CA2+ CHANNELS BUT NOT THE RYANODINE RECEPTOR

In the pancreatic beta-cell, an increase in the cytoplasmic free Ca2+ concentration ([Ca2+](i)) by caffeine is believed to indicate mobilization of Ca2+ from intracellular stores, through activation of a ryanodine receptor-like channel. It is not known whether other mechanisms, as well, underlie caffeine-induced changes in [Ca2+](i). We studied the effects of caffeine on [Ca2+](i) by using dual-wavelength excitation microfluorimetry in fura-2-loaded beta-cells. In the presence of a non-stimulatory concentration of glucose, caffeine (10-50 mM) consistently increased [Ca2+](i). The effect was completely blocked by omission of extracellular Ca2+ and by blockers of the L-type voltage-gated Ca2+ channel, such as D-600 or nifedipine. Depletion of agonist-sensitive intracellular Ca2+ pools by thapsigargin did not inhibit the stimulatory effect of caffeine on [Ca2+](i). Moreover, this effect of caffeine was not due to an increase in cyclic AMP, since forskolin and 3-isobutyl-1-methylxanthine (IBMX) failed to raise [Ca2+](i) in unstimulated beta-cells. In beta-cells, glucose and sulphonylureas increase [Ca2+](i) by causing closure of ATP-sensitive K+ channels (K-ATP channels). Caffeine also caused inhibition of K-ATP channel activity, as measured in excised inside-out patches. Accordingly, caffeine (> 10 mM) induced insulin release from beta-cells in the presence of a non-stimulatory concentration of glucose (3 mM). Hence, membrane depolarization and opening of voltage-gated L-type Ca2+ channels were the underlying mechanisms whereby the xanthine drug increased [Ca2+](i) and induced insulin release. Paradoxically, in glucose-stimulated beta-cells, caffeine(> 10 mM) lowered [Ca2+](i). This effect was due to the fact that caffeine reduced depolarization-induced whole-cell Ca2+ current through the L-type voltage-gated Ca2+ channel in a dose-dependent manner. Lower concentrations of caffeine (2.5-5.0 mM), when added after glucose-stimulated increase in [Ca2+](i), induced fast oscillations in [Ca2+](i). The latter effect was likely to be attributable to the cyclic AMP-elevating action of caffeine, leading to phosphorylation of voltage-gated Ca2+ channels. Hence, in beta-cells, caffeine-induced changes in [Ca2+](i) are not due to any interaction with intracellular Ca2+ pools. In these cells, a direct interference with K-ATP channel- and L-type voltage-gated Ca2+-channel activity is the underlying mechanism by which caffeine increases or decreases [Ca2+](i).