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

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 M 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 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. [ 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. © 2010 Elsevier B.V.Fil: Rosin, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Farmacología; ArgentinaFil: López Ordieres, María Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Farmacología; ArgentinaFil: Rodriguez, Georgina Emma. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Farmacología; Argentin

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na+, K+-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [3H]-ouabain binding (to analyze K+ site of Na+, K+-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na+, K+-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [3H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2 mg/kg) and clozapine (3, 10 and 30 mg/kg). Animals were sacrificed 18 h later, cerebral cortices harvested, membrane fractions prepared and high affinity [3H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10 mg/kg clozapine. Rats were administered with neurotensin (3, 10 y 30 μg, i.c.v.) and 60 min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [3H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30 μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [3H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2 mg/kg, i.p.) or clozapine (10 mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na+, K+-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na+, K+-ATPase at central synapses.Fil: Rosin, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: López Ordieres, María Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Rodriguez, Georgina Emma. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Na+, K+-ATPase response to neurotensin is altered by streptozotocin administration

Neurotensin is a basic tridecapeptide which can act as a neuromodulator or a neurotransmitter and binds to a group of receptors. Neurotensin is able to inhibit Na+, K+-ATPase activity, an effect blocked by the presence of antagonist SR48692, suggesting the involvement of high affinity neurotensin (NTS1) receptor. Diverse evidences suggest a relationship between neurotensinergic system and glycemia levels. For this reason, potential Na+, K+-ATPase regulation by neurotensin in brain membranes obtained from rats turned hyperglycaemic was explored. As a model to produce diabetes mellitus, rats were administered with Streptozotocin (STZ), a specific toxic agent to the pancreatic beta cells. Our findings indicated that Na+, K+-ATPase activity in synaptosomal membranes isolated from diabetic rats failed to respond to neurotensin. The treatment decreased the affinity of NTS1 receptor for neurotensin and the expression of Na+, K+-ATPase alpha3 subunit in cerebral cortex. The results led us to conclude that STZ administration alters the response of Na+, K+-ATPase to neurotensin. Such effect seems to involve a decrease in enzyme alpha3 isoform expression and NTS1 receptor affinity for neurotensin.Fil: Ordieres López, María Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias "profesor Eduardo de Robertis"; ArgentinaFil: Rosin, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias "profesor Eduardo de Robertis"; ArgentinaFil: Miño, Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Rodriguez, Georgina Emma. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencias "profesor Eduardo de Robertis"; Argentin

Neurotensin inhibitory effect on [ 3h]-ouabain binding to striatal membranes is inverted by administration of clozapine

Previous work indicates that peptide neurotensin inhibits synaptosomal membrane Na+, K+-ATPase activity. Anatomical and biochemical evidences indicate a relationship between neurotensinergic and dopaminergic systems and that both systems are involved in the action mechanism of antipsychotic drugs. Haloperidol and clozapine are antipsychotic drugs currently employed in therapeutics. They are prototypes for typical and atypical antipsychotic drugs, respectively. The objective of the work was to analyze potential relationships between neuronal Na+, K+-ATPase with neurotensinergic and dopaminergic systems. After the blockade of dopaminergic receptors, an alteration of Na+, K+-ATPase properties and neurotensin binding to rat cerebral cortex membranes was recorded. Herein, the study was extended to rat striatum. Haloperidol (2 mg/kg) and clozapine (10 mg/kg) were administered i.p. to rats and 18 hours later, animals were sacrificed, striatum harvested, membrane fractions prepared and high affinity [3H]-ouabain binding assayed. Basal high affinity [3H]-ouabain binding remained unchanged after haloperidol administration whereas it decreased (- 50%) after clozapine administration. With respect to basal values neurotensin addition (10 micromolar concentration) decreased (- 50%) [3H]-ouabain binding after haloperidol administration whereas it enhanced (+ 100%) binding after clozapine administration. Saturation curves for [3H]-neurotensin binding followed by Scatchard and Hill analysis showed that peptide binding affinity (Bmax value) decreased roughly 70% after clozapine administration but remained unaltered after haloperidol administration. Kd and NH values remained unaltered in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modify Na+, K+-ATPase ouabain site (K+ site) and NTS1 neurotensin receptor at striatum. At the same time, support the notion of an interaction between dopaminergic and neurotensinergic systems and Na+, K+-ATPase at central synapses.Fil: Rosin, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Wies Mancini, Victoria Sofia Berenice. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: López Ordieres, María Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Rodriguez, Georgina Emma. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin