Characterization of ouabain high-affinity binding to rat cerebral cortex. Modulation by melatonin.
High-affinity [3H]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 KD = 3.1 +/- 0.36 nM and a binding site concentration of Bmax = 246.4 +/- 18.4 fmol/mg protein. Kinetic analyses of the association and dissociation curves indicated a kinetic KD = 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 [3H]ouabain binding, the following Ki 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 [3H]ouabain binding increased in a dose-related manner to yield the following Ki 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.
Acuña Castroviejo
D
D
Departamento de Fisiología, Facultad de Medicina, Universidad de Granada, Spain.
del Aguila
C M
CM
Fernández
B
B
Gomar
M D
MD
Castillo
J L
JL
D000818:Animals; D001665:Binding Sites; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008550:Melatonin / Q000494:pharmacology*; D010042:Ouabain / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000494:pharmacology; D013696:Temperature
D010042:Ouabain; D012964:Sodium; D008274:Magnesium; D008550:Melatonin