Changes in [3H]glibenclamide binding to mouse forebrain membranes during morphine tolerance

The characteristics of specific binding of the ATP-sensitive K+ (K-ATP) channel blocker [H-3]glibenclamide to forebrain membranes (P-2 fraction, 4 degreesC) obtained from morphine-naive and -tolerant mice were evaluated. Morphine tolerance was induced by osmotic minipumps that released 45 mg/kg/day of morphine subcutaneously for 6 days. This treatment enhanced the antinociceptive ED50 of morphine without changing its E-max. In morphine-naive animals. (1) both the association and the dissociation of [H-3]glibenclamide were biphasic; (2) [H-3]glibenclamide was displaced by other sulfonylureas (order of potency: glibenclamide > glipizide much greater than tolbutamide) with pseudo-Hill coefficients lower than unity and biphasic Hofstee plots; and (3) Scatchard plots of saturation experiments were curvilinear, showed a Hill coefficient of 0.81 +/- 0.04 and suggested the presence of two binding sites with a K-D of 0.13 and 3.17 nM and a B-max of 12.30 and 84.47 fmol/mg protein, respectively. By contrast, in membranes obtained from morphine-tolerant animals, (1) the Scatchard plots showed only one population of binding sites with a K-D of 0.87 nM and a B-max of 77.99 fmol/mg protein, and the Hill coefficient was very close to unity (0.96 +/- 0.1); (2) competition experiments (using glibenclamide as displacer) showed a pseudo-Hill coefficient of 0.99 +/- 0.04; and (3) dissociation experiments showed only one phase of dissociation. These results suggest that [H-3]glibenclamide binds to two different sites in membranes obtained from morphine-naive animals, but to only one site in morphine-tolerant animals. Consequently, it seems that morphine tolerance in mice involves adaptive changes in K-ATP channels. (C) 2001 Published by Elsevier Science B.V.