MG2-DEPENDENT INHIBITION OF K-ATP BY SULFONYLUREAS IN CRI-G1 INSULIN-SECRETING CELLS()

1 Patch-clamp recording techniques were used to examine the effects of tolbutamide, glibenclamide, meglitinide and thiopentone on K-ATP in C RI-GI insulin-secreting cells in the presence and absence of Mg2+. 2 I n the absence of Mg2+ in the intracellular bathing solution, tolbutami de was significantly less effective when applied either to the intrace llular or to the extracellular surfaces of cell-free patches. Removal of extracellular Mg2+ did not alter the effectiveness of tolbutamide p rovided that Mg2+ was present at the intracellular surface of the patc h. 3 Tolbutamide was also significantly less effective when applied to the intracellular surface of cell-free patches when Mn2+ was used as a replacement for Mg2+. 4 Both the sulphonylurea, glibenclamide and th e non-sulphonylurea derivative, meglitinide also showed Mg2+ dependent inhibitory effects in cell-free patches. In contrast, the barbiturate thiopentone inhibited K-ATP in a Mg2+-independent manner. 5 Whole-cel l I-K(ATP) were used to quantify the effects of tolbutamide and gliben clamide in the presence and absence of intracellular Mg2+. Concentrati on-inhibition curves, in the presence of intracellular Mg2+, resulted in IC50 values of 12.1 mu M and 2.1 nM for tolbutamide and glibenclami de, respectively. In the absence of intracellular Mg2+, the correspond ing IC50 values were 25.3 mM and 3.6 mu M, respectively. The values of IC50 for thiopentone in the presence and absence of intracellular Mg2 + were 69.4 mu M and 69.2 mu M, respectively.6 With respect to the hig h affinity binding sites for [H-3]-glibenclamide in CRI-GI membranes, no significant differences were found between the dissociation constan ts for, or the maximal binding capacities of, [H-3]-glibenclamide in t he presence or absence of Mg2+. 7 In the CRI-G1 insulin-secreting cell line, it is concluded that intracellular Mg2+ does not influence the affinity of the sulphonylureas for the sulphonylurea receptor but that this ion is critically important for the interaction between the sulp honylurea receptor and K-ATP.