ABSTRACT
Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 µg and 250 µg/kg SR 48692. It was observed that the 250 µg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.
Subject(s)
Cerebral Cortex/metabolism , Neurotensin/pharmacology , Ouabain/metabolism , Animals , Male , Piperidines/pharmacology , Protein Binding/drug effects , Pyrazoles/pharmacology , Quinolines/pharmacology , Rats , Rats, Wistar , Receptors, Neurotensin/agonists , Receptors, Neurotensin/antagonists & inhibitors , Receptors, Neurotensin/metabolismABSTRACT
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.
Subject(s)
Central Nervous System/drug effects , Central Nervous System/metabolism , Enzyme Inhibitors/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Male , Ouabain/analogs & derivatives , Ouabain/pharmacology , Rats , Rats, Wistar , Synaptosomes/drug effects , Synaptosomes/enzymologyABSTRACT
Normal operation of the Na+ pump (Na+, K(+)-ATPase) is essential for the maintenance of neurotransmission. Filtration through Sephadex G-50 of a brain soluble fraction allowed the separation of peaks I and II fractions, respectively stimulating and inhibiting synaptosomal membrane Na+, K(+)-ATPase activity. Peaks I and II were isolated from rat cerebral cortex and their effect together with serotonin (5-HT) was studied on ATPase activity by estimating K(+)-p-nitrophenylphosphatase activity in brain cortex synaptosomal membranes. It was observed that 10(-5) or 10(-4) M 5-HT failed to modify either control membrane enzyme activity or peak I activatory effect; on the other hand, such 5-HT concentrations significantly suppressed peak II inhibitory effect. This ability of 5-HT to reverse the inhibitory effect of endogenous factors on Na+, K(+)-ATPase activity could well be a new 5-HT modulatory action within the brain.
Subject(s)
Animals , Male , Female , Rats , In Vitro Techniques , Serotonin , Sodium-Potassium-Exchanging ATPase , Synaptosomes , Cell-Free System , Cerebral Cortex/ultrastructureABSTRACT
Normal operation of the Na+ pump (Na+, K(+)-ATPase) is essential for the maintenance of neurotransmission. Filtration through Sephadex G-50 of a brain soluble fraction allowed the separation of peaks I and II fractions, respectively stimulating and inhibiting synaptosomal membrane Na+, K(+)-ATPase activity. Peaks I and II were isolated from rat cerebral cortex and their effect together with serotonin (5-HT) was studied on ATPase activity by estimating K(+)-p-nitrophenylphosphatase activity in brain cortex synaptosomal membranes. It was observed that 10(-5) or 10(-4) M 5-HT failed to modify either control membrane enzyme activity or peak I activatory effect; on the other hand, such 5-HT concentrations significantly suppressed peak II inhibitory effect. This ability of 5-HT to reverse the inhibitory effect of endogenous factors on Na+, K(+)-ATPase activity could well be a new 5-HT modulatory action within the brain.(AU)
