Modulation by nucleotides of binding sites for [H-3]glibenclamide in rat aorta and cardiac ventricular membranes

Radioligand binding techniques were employed to determine the modulation by nucleotides of the specific [H-3]glibenclamide (Gli) binding to rat aortic and cardiac ventricular preparations. Saturation analysis revealed a singl e binding site with K-D value of 31.3 nM and B-max of 180 fmol/mg wet weigh t in aortic preparations. We also observed that [H-3]Gli bound reversibly a nd specifically to cardiac membranes. Unlabeled glibenclamide displaced [H- 3]Gli-specific binding of cardiac membranes completely with K-I of 54.4 nM. In cardiac membranes, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) (from 0.01-5 mM) concentration depende ntly inhibited [H-3]Gli binding independent of Mg2+. The values of K-I were 0.47, 0.22, and 0.58 mM, respectively. However, in aortic preparations, [H -3]Gli-specific binding was increased by ATP of 5 and 10 null and showed a biphasic response to ADP. At concentrations to 1 mM, ADP inhibited binding; above 5 mM, the specific [H-3]Gli binding was increased. UDP did not alter the binding up to 5 mM. In the presence of Mg2+ (20 mM), the inhibitory ef fects of ATP (0.01-1 mM) or ADP (0.01-5 mM) on the binding in cardiac membr anes were abolished, whereas the facilitatory effects of ATP or ADP in aort ic preparations were strengthened. Analysis of kinetics showed that the tim e of [H-3]Gli association and dissociation in cardiac and aortic preparatio ns was monophasic, The association was delayed with dissociation unchanged by ATP, ADP, and UDP of 1 mM, respectively, in cardiac membranes. In aorta, however, at the same concentration ATP accelerated association and retarde d dissociation and vice versa for ADP. Association and dissociation were no t changed by UDP of 5 mM. We conclude that ATP, ADP, and UDP are all major allosteric modulators of K-ATP channels and they affect the antagonist bind ing to heart (sulfonylurea receptor 2A) and aorta (sulfonylurea receptor 2B ) differently.