[3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain.
An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.
Reinés
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Instituto de Biología Celular y Neurociencias Prof. Eduardo De Robertis, PROBICENE-CONICET, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121, Buenos Aires, Argentina.
Peña
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Rodríguez de Lores Arnaiz
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D000818:Animals; D016291:Dizocilpine Maleate / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism*; D066298:In Vitro Techniques; D008024:Ligands; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D010042:Ouabain / Q000031:analogs & derivatives* / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D008024:Ligands; D016194:Receptors, N-Methyl-D-Aspartate; C119205:endobain E; D010042:Ouabain; D016291:Dizocilpine Maleate; D000254:Sodium-Potassium-Exchanging ATPase