Direct effects of tolbutamide on mitochondrial function, intracellular Ca2+ and exocytosis in pancreatic β-cells

Using the whole-cell voltage-clamp method to measure ATP-sensitive K+(K-ATP) currents, changes in cell capacitance to measure secretion and microfluorimetry to monitor intracellular Ca2+ and mitochondrial function, we have investigated the direct effect of sulphonylureas on exocytosis in pancreatic beta-cells. Tolbutamide (100 mu M) and 100 nM 4-beta-12-phorbolmyristate-13-acetate (PMA), which activates the protein kinase C (PKC) isoforms found in beta-cells, potentiated exocytosis in a non-additive manner. These effects were blocked by down-regulation of PKC. Our data support the idea that tolbutamide can potentiate secretion from beta-cells via a PKC-dependent pathway. Because PKC and sulphonyl ureas can modulate the activity of K-ATP channels, we explored whether the above effects are caused by inhibition of this channel. PMA increased whole-cell K-ATP currents but did not affect their sensitivity to tolbutamide. Downregulation of PKC affected neither the magnitude nor the tolbutamide sensitivity of the K-ATP current. Both tolbutamide and the mitochondrial uncoupler FCCP (1 mu M) mobilized intracellular Ca2+ and prolonged Ca2+ transients elicited by cholinergic mobilization of intracellular Ca2+ stores. Tolbutamide (0.1-0.5 mM), like FCCP, depolarized the mitochondrial membrane potential and activated K-ATP currents. We suggest that sulphonylureas can directly potentiate exocytosis by impairing mitochondrial function and Ca2+ handling, which ultimately leads to activation of Ca2+-dependent enzymes such as PKC.