Abstract
The effects of ADP on [3H]glibenclamide binding to membranes and whole cells, the activity of the ATP-sensitive K+ channel (K-ATP channel), intracellular Ca2+ concentration and insulin secretion were studied in a hamster pancreatic beta-cell line, HIT T15. ADP dose-dependently inhibited [3H]glibenclamide binding to membranes and to whole cells in a competitive manner. ADP-agarose also inhibited the binding to whole cells. The activity of the K-ATP channel was assayed by measuring 86Rb efflux from whole cells. ADP inhibited the 86Rb efflux elicited either by diazoxide or by ATP depletion. In the presence, but not in the absence, of extracellular Ca2+, ADP evoked a rapid and sustained increase in intracellular Ca2+ concentration as estimated with the fluorescent dye quin 2. Insulin release from HIT cells was also increased by 0.5-2 mM-ADP in the presence of 0.5 mM-glucose. These effects of ADP on glibenclamide binding, K-ATP channel activity and insulin release were specific for ADP, and were not reproduced by any other nucleotide so far tested. The present findings strongly suggest that ADP and sulphonylureas have common binding sites on the extracellular side of beta-cell plasma membranes, where they inhibit the activity of the K-ATP channel, resulting in an increase in intracellular Ca2+ concentration and insulin release.