CHARACTERIZATION OF OUABAIN HIGH-AFFINITY BINDING TO RAT CEREBRAL-CORTEX - MODULATION BY MELATONIN

High-affinity [H-3]ouabain binding to membrane preparations of rat cerebral cortex was examined using a rapid filtration procedure. At 37-degrees-C, binding reached equilibrium in about 60 min. Scatchard analyses of the data at equilibrium revealed a single population of binding sites with a dissociation constant of K(D) = 3.1 +/- 0.36 nM and a binding site concentration of B(max) = 246.4 +/- 18.4 fmol/mg protein. Kinetic analyses of the association and dissociation curves indicated a kinetic K(D) = 4.6 nM, which is in good agreement with the value obtained at equilibrium. When various digitalis compounds were tested for their ability to inhibit [H-3]ouabain bidding, the following K(i) values (nM) were obtained: ouabain (3.9); digoxin (18); acetyl-digitoxin (66). k-strophanthin (95); digitoxin (236). When melatonin was added to the incubation medium, the ability of ouabain to inhibit [H-3]ouabain binding increased in a dose-related manner to yield the following K(i) values (nM): melatonin 10 nM (2); melatonin 20 nM (1.2); melatonin 40 nM (0.8). These data suggest the existence in the rat cerebral cortex of high-affinity ouabain binding sites which may be a locus for the molecular action of melatonin.