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

Abstract

The characteristics of specific binding of the ATP-sensitive K + (K ATP) channel blocker [ 3H]glibenclamide to forebrain membranes (P 2 fraction, 4°C) 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 ED 50 of morphine without changing its E max. In morphine-naive animals, (1) both the association and the dissociation of [ 3H]glibenclamide were biphasic; (2) [ 3H]glibenclamide was displaced by other sulfonylureas (order of potency: glibenclamide>glipizide≫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 [ 3H]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.