Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

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 [3H]-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 1×10−4M concentration. On the other hand, the single addition of 1×10−6M, 1×10−5M and 1×10−4M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [3H]-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 [3H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased Kd value whereas failed to modify Bmax value, indicating a competitive type interaction of the peptide at Na+, K+-ATPase ouabain site. At variance, SR 48692 decreased Bmax value whereas it did not modify Kd value. [3H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30min earlier with 100μg and 250μg/kg SR 48692. It was observed that the 250μg/kg SR 48692 dose led to 19% decrease in basal [3H]-ouabain binding. After SR 48692 treatments, addition of 1×10−6M led to additive or synergic effect. Results suggested that [3H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.