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The expression of Na(+), K(+)-ATPase α3 subunit and synaptosomal membrane Na(+), K(+)-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na(+), K(+)-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris-HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na(+), K(+)-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na(+), K(+)-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na(+), K(+)-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na(+), K(+)-ATPase inhibitors modify differentially the expression of Na(+), K(+)-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.

Phosphoinositide (PI) metabolism is enhanced in neonatal brain by activation of neurotransmitter receptors and by inhibition of the sodium pump with ouabain or endogenous inhibitor termed endobain E. Peptide neurotensin inhibits synaptosomal membrane Na(+), K(+)-ATPase activity, an effect blocked by SR 48692, a selective antagonist for high-affinity neurotensin receptor (NTS1). The purpose of this study was to evaluate potential participation of NTS1 receptor on PI hydrolysis enhancement by sodium pump inhibition. Cerebral cortex miniprisms from neonatal Wistar rats were preloaded with [(3)H]myoinositol in buffer during 60 min and further preincubated for 0 min or 30 min in the absence or presence of SR 48692. Then, ouabain or endobain E were added and incubation proceeded during 20 or 60 min. Reaction was stopped with chloroform/methanol and [(3)H]inositol-phosphates (IPs) accumulation was quantified in the water phase. After 60-min incubation with ouabain, IPs accumulation values reached roughly 500% or 860% in comparison with basal values (100%), if the preincubation was omitted or lasted 30 min, respectively. Values were reduced 50% in the presence of SR 48692. In 20-min incubation experiments, IPs accumulation by ouabain versus basal was 300% or 410% if preincubation was 0 min or 30 min, respectively, an effect blocked 23% or 32% with SR 48692. PI hydrolysis enhancement by endobain E was similarly blocked by SR 48692, being this effect higher when sample incubation with the endogenous inhibitor lasted 60 min versus 20 min. Present results indicate that PI hydrolysis increase by sodium pump inhibition with ouabain or endobain E is partially diminished by SR 48692. It is therefore suggested that NTS1 receptor may be involved in cell signaling system mediated by PI turnover.

Previous studies showed that endobain E, an endogenous Na+, K+-ATPase inhibitor, decreases dizocilpine binding to NMDA receptor in isolated membranes. The effect of endobain E on expression of NMDA receptor subunits in membranes of rat cerebral cortex and hippocampus was analyzed by Western blot. Two days after administration of 10 mul endobain E (1 microl = 29 mg fresh tissue) NR1 subunit expression enhanced 5-fold and 2.5-fold in cerebral cortex and hippocampus, respectively. NR2A subunit expression increased 2-fold in cerebral cortex and 1.5-fold in hippocampus. The level of NR2B subunit raised 3-fold in cerebral cortex but remained unaltered in hippocampus. NR2C subunit expression was unaffected in either area. NR2D subunit enhanced 1.6 and 2.1-fold for cerebral cortex and hippocampus, respectively. Results indicate that endogenous Na+, K+-ATPase inhibitor endobain E differentially modifies the expression of NMDA receptor subunits.