CONICET | Buscador de Institutos y Recursos Humanos

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 1×10−8 M glutamate plus 1×10−5 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.

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