María Graciela López Ordieres

Effect of angiotensin-(1–7) on ATPase activities in several tissues

The present investigation was undertaken to determine whether Ang-(1-7) is able to modify ATPase activities in membrane fractions prepared from several tissues. In the presence of 10(-6) M Ang-(1-7), total (Na , K+, Mg2+)-ATPase activity decreased 31% in rat atrium and 13% in sheep atrium but was unmodified in sheep liver, rat ventricle or crude brain membranes. In rat brain synaptosomal membranes, Ang-(1-7) at 10(-8) and 10(-7) M concentrations activated Na+, K+-ATPase 20 and 24%, respectively. Rat kidney Na+, K+-ATPase activity decreased roughly 40-70% with 10(-10)-10(-6) M Ang-(1-7)), but increased 22% with 10(-12) M peptide concentration, thus indicating a biphasic effect. Our findings showing that ATPase from several tissues responds differently to Ang-(1-7) are attributable to enzyme tissue specificity.

Neurotensin inhibits neuronal Na+,K+-ATPase activity through high affinity peptide receptor.

Fil: Lopez Ordieres, Maria Graciela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Biologia Celular y Neurociencia ; Argentina

A Study of Calcitonin Effect on Synaptosomal Membrane Enzymes

Abstract RodrIGuez de Lores Arnaiz, G. and M. G. LOPez Ordieres. A study of calcitonin effect on synaptosomal membrane enzymes. 18(4) 613–615, 1997.—Calcitonin is a hormone peptide produced by the thyroid gland, whose best described role is to prevent bone reabsorption. It also participates in other biological functions, even at central nervous system level. We studied the effect of added calcitonin on ATPase and acetylcholinesterase activities in synaptosomal membranes isolated from rat cerebral cortex. Calcitonin at 10 −7 − 10 −5 M concentration decreased 20–40% Na + , K + -ATPase and 15–25% K + - p -nitrophenylphosphatase activities, and at 10 −6 − 10 −5 M reduced 20–30% Mg 2+ - p -nitrophenylphosphatase activity. However, this peptide failed to modify Mg 2+ − and Ca 2+ − ATPase or acetylcholinesterase activities. Results suggest that the sodium pump may be a target for calcitonin effects at neuronal level. Thus, calcitonin inhibition of sodium/potassium transport through synaptic membranes supports a regulatory role of this peptide on neurotransmission.

K+-p-nitrophenylphosphatase inhibition by neurotensin involves high affinity neurotensin receptor: influence of potassium concentration and enzyme phosphorylation

Abstract Neurotensin (NT), a 13-amino acid peptide, is widely distributed in the brain and peripheral tissues of several mammalian species including man. In adult rat brain NT can bind to two distinct sites, one of high and the other of low affinity, corresponding to NT 1 and NT 2 receptor, respectively; structurally unrelated to these two, a third NT receptor (NT 3 ) has been described. We have previously shown that Na + , K + -ATPase is inhibited by NT when using ATP as substrate. In order to determine whether K + -stimulated dephosphorylation of this enzyme is involved, we tested NT effect by using p -nitrophenylphosphate, a non-natural substrate. K + - p -nitrophenylphosphatase activity was inhibited 42% by NT at 8.6×10 −6 M using an incubation medium containing 2 mM KCl but was unaffected in the presence of 5 or 20 mM KCl; however, with such KCl concentrations, NT was enabled to inhibit enzyme activity (≅35%) provided a suitable ATP:NaCl mixture (0.6:45.0 mM) was added. Mg 2+ - p -nitrophenylphosphatase activity remained unaltered at all conditions tested. Since SR 48692, a selective non-peptide NT 1 antagonist, abolished NT effect, involvement of NT 1 receptor in enzyme inhibition is suggested.

The inhibitory effect of neurotensin on synaptosomal membrane Na+, K+-ATPase is altered by antipsychotic administration

Abstract Synaptosomal membrane Na + , K + -ATPase is inhibited by neurotensin, an effect which involves its high affinity receptor (NTS1) [Lopez Ordieres MG, Rodriguez de Lores Arnaiz, G. Peptides 2000; 21:571–576.]. Herein, the effect of neurotensin on synaptosomal membrane Na + , K + -ATPase of rats 18 h after i.p. administration of antipsychotic haloperidol (2 mg/kg) or clozapine (10 mg/kg) was studied. Basal enzyme activity after these treatments did not differ from that in vehicle-treated rats. It was observed that 3.5×10 −6 M neurotensin reduced roughly 40% cerebral cortex Na + , K + -ATPase from vehicle-injected rats, produced no effect on the enzyme from rats injected with haloperidol but enhanced 26% that from rats injected with clozapine. The peptide decreased 40% striatal Na + , K + -ATPase from vehicle-injected rats or from rats injected with clozapine, whereas it failed to alter this enzyme activity from rats injected with haloperidol. Haloperidol and clozapine (1×10 −6 M) added in vitro failed to alter Na + , K + -ATPase activity in cerebral cortex synaptosomal membranes. Results obtained after antipsychotic administration may well offer an alternative explanation for the particular side effects recorded in therapeutics by typical (haloperidol) versus atypical (clozapine) antipsychotic drugs.

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 [(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.

Neurotensin Effect on Na+, K+-ATPase Is CNS Area- and Membrane-Dependent and Involves High Affinity NT1 Receptor

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.

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

HighlightsAntipsychotics modify neurotensin effect on [3H]‐ouabain binding to brain membranes.Antipsychotics modify [3H]‐neurotensin binding to brain membranes.Antipsychotics modify the kinetics for [3H]‐neurotensin binding to brain membranes. Abstract 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 &mgr;g, 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 &mgr;g 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.