[H-3]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. Thi s cardiac glycoside possesses neurotoxic properties attributable to Na+, K-ATPase inhibition, which leads to NM DA receptor activation, thus supporti ng the concept that Na+/K+ gradient impairment has a critical impact on suc h receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [H-3]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thorou ghly washed and stored as pellets ('non-resuspended' membranes) or after re suspension in sucrose ('resuspended' membranes) were employed. Membrane sam ples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinatio ns, with or without spermidine. [H-3]Dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 're suspended' membranes. Endobain E decreased [H-3]dizocilpine binding to 'res uspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. A mong tested conditions, only in 'resuspended' membranes, with 4 nM ligand a nd with I x 10(-8) M glutamate plus I x 10(-5) M glycine, was [H-3]dizocilp ine binding enhanced roughly + 24% by ouabain (I mM). After Triton X-100 me mbrane treatment, which drastically reduces Na+, K+-ATPase activity, the ef fect of ouabain on binding was lost whereas that of endobain E remained una ltered. Results indicate that not only membrane preparation but also treatm ent and storage are crucial to observe direct endobain E and ouabain effect s on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration. (C) 2001 Elsevier Science Lt d. All rights reserved.