Brain Research, 542 (1991) 151-154 151 Elsevier BRES 24543 Two binding sites for [3H]glibenclamide in the rat brain Sylvie Zini, Evelyne Tremblay, Marie-Paule Roisin and Yehezkel Ben-Ari Laboratoire de neurobiologie et neurophysiologie du d~veloppement, INSERM U.29, Paris (France) (Accepted 13 November 1990) Key words: ATP-sensitive potassium channel; Glibenclamide; Sulfonylurea; Binding; Rat brain The binding of [3H]glibenclamide, a potent sulfonylurea which blocks ATP-sensitive potassium channels, was studied in the rat brain. A Scatchard plot of saturation isotherms suggests that [3H]glibenclamide binds in various brain regions to a high- and a low-affinity binding site (K d values of 0.21 nM and 111 nM and B~ values of 41 and 1060 fmol/mg protein, respectively). Competitive binding assays with various unlabelled sulfonylureas showed biphasic displacements of [3H]glibenclamide with pseudo-Hill coefficients significantly different from unity. These data indicate the existence of a heterogeneity of binding sites to [3H]glibenclamidein the rat brain; this may correlate with the variability of effects of sulfonylureas observed from physiological experiments. ATP-sensitive potassium channels (KATP) belong to a class of potassium channels characterized by their ability to be blocked by high intracellular concentrations of ATP 3'9'1s. First described in peripheral tissues including the cardiac muscle is, pancreatic fl cellss and the skeletal muscle 21, they have recently been found in the central nervous system 4 where they may link cell excitability to the metabolic state of the neuron, notably during anoxia 6A6. Sulfonylureas, used for a long time as hy- poglycaemic drugs, produce their therapeutic effects by the blockade of KAXp, thereby increasing electrical activity and releasing insulin from pancreatic fl cells 2°. The blockade of these channels seems closely related with the presence of specific binding sites for sulfonyl- ureas in insulin-secreting cells, cardiac cells and brain membranes ~2'17"2°. In these tissues, glibenclamide, the most potent antidiabetic sulfonylurea, binds with a high affinity with a dissociation constant (Kd) in a 0.1-1 nM range. Although these values show a good correlation with the half-maximum inhibition of 86Rb+ efflux from insulin-secreting cells ~°,H,2°, recent reports suggest that [3H]glibenclamide, or its iodinated analog, can bind to high- and low-affinity binding sites, in canine cardiac membranes 13 and hamster fl cells line 1. We now report that specific high and low affinity binding sites for [3H]glibenclamide also exist in the rat central nervous system. Male Sprague-Dawley rats (180-200 g) were killed by decapitation and the brain was rapidly removed. The cerebral cortex, striatum, hippocampus, hypothalamus, thalamus and cerebellum were dissected out on an ice-cold plate and immediately homogenized in 20 vols. of 0.32 M sucrose, 5 mM Tris-HCl buffer, pH 7.4, 4 °C, with a motor-driven glass-teflon Potter homogenizer (Braun-Melsungen). The homogenate was then centri- fuged at 1000 g for 10 min at 4 °C. The pellet was resuspended in 0.32 M sucrose, 5 mM Tris-HCl buffer, pH 7.4 and then centrifuged at 20,000 g for 15 min at 4 °C and washed again in the same conditions. The final pellet was resuspended in 50 mM Tris-HCl buffer, pH 7.4, 4 °C, to give a protein concentration about 5 mg/ml. Protein concentration was determined according to the method of Bradford 7, using bovine gamma globulin as standard. Equilibrium binding studies were carried out by incubat- ing membranes of the cerebral cortex or other discrete brain region (0.4 mg protein/ml) with [3H]glibenclamide (51 Ci/mmol, CEA, France) in 50 mM Tris-HCl buffer, pH 7.4, for 2 h at 4 °C, in a final volume of 2 ml (two replicates for each determination). Equilibrium was terminated by adding 5 ml of ice-cold 5 mM Tris-HCl buffer. Bound and free [3H]glibenclamide were immedi- ately separated by a rapid filtration through Whatman GF/B filters. The filters were then washed 3 times with 5 ml of ice-cold buffer and the radioactivity was measured by liquid scintillation spectrometry. [3H]Glibenclamide specific binding was defined as the difference between the total binding and the non-specific binding determined in the presence of 1 /~M glibenclamide. Kinetics of association and dissociation as well as displacement studies were performed in the presence of 3 nM Correspondence: S. Zini, Laboratoire de neurobiologie et neurophysiologie du dEveloppement, INSERM U.29, 123, Boulevard de Port-Royal, 75014 Paris, France. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)