Brain Na+, K+-ATPase activity In aging and disease

Na+/K+ pump or sodium- and potassium-activated adenosine 5’-triphosphatase (Na+, K+-ATPase), its enzymatic version, is a crucial protein responsible for the electrochemical gradient across the cell membranes. It is an ion transporter, which in addition to exchange cations, is the ligand for cardenolides. This enzyme regulates the entry of K+ with the exit of Na+ from cells, being the responsible for Na+/K+ equilibrium maintenance through neuronal membranes. This transport system couples the hydrolysis of one molecule of ATP to exchange three sodium ions for two potassium ions, thus maintaining the normal gradient of these cations in animal cells. Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required for the maintenance of the ionic gradients that underlie resting and action potentials which are involved in nerve impulse propagation, neurotransmitter release and cation homeostasis. Protein phosphorylation is a key process in biological regulation. At nervous system level, protein phosphorylation is the major molecular mechanism through which the function of neural proteins is modulted in response to extracellular signals, including the response to neurotransmitter stimuli. It is the major mechanism of neural plasticity, including memory processing. The phosphorylation of Na+, K+-ATPase catalytic subunit inhibits enzyme activity whereas the inhibition of protein kinase C restores the enzyme activity. The dephosphorylation of neuronal Na+, K+-ATPase is mediated by calcineurin, a serine / threonine phosphatase. The latter enzyme is involved in a wide range of cellular responses to Ca2+ mobilizing signals, in the regulation of neuronal excitability by controlling the activity of ion channels, in the release of neurotransmitters and hormones, as well as in synaptic plasticity and gene transcription. In the present article evidence showing Na+, K+-ATPase involvement in signaling pathways, enzyme changes in diverse neurological diseases as well as during aging, have been summarized. Issues refer mainly to Na+, K+-ATPase studies in ischemia, brain injury, depression and mood disorders, mania, stress, Alzheimer´s disease, learning and memory, and neuronal hyperexcitability and epilepsy.Fil: 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; 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; Argentin

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

Neurotensin is a peptide present in mammalian CNS and peripheral tissues, which may play a major role in neurotransmission or neuromodulation, subserving diverse physiological functions. We studied the effect of added neurotensin on ATPase activities in synaptosomal membranes isolated from rat cerebral cortex. Neurotensin at 3 x 10-8-3 x 10-6 M concentration decreased 20-44% Na+, K+-ATPase activity but failed to modify Mg2+-ATPase activity; lower neurotensin concentrations (3 x 10-14-3 x 10-10 M) had no effect on enzyme activities. This inhibitory effect was abolished by neurotensin heating, by enzyme preincubation with neurotensin during periods exceeding 10 min, or by adding 1 x 10-6 M SR 48692, a high affinity neurotensin receptor antagonist. Levocabastine, which blocks low affinity neurotensin receptor, failed to alter enzyme inhibition by the peptide. It is suggested that the sodium pump may be a target for neurotensin effects at neuronal level involving the participation of high affinity neurotensin receptor. © 2000 Elsevier Science Inc.Fil: López Ordieres, María Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. 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 ; Argentin

Kinectics of Na+, K+-ATPase inhibition by calcitonin and neurotensin

The kinetics of interactions between the peptides calcitonin or neurotensin, and synaptosomal membrane Na+, K+-ATPase activity was characterized in this study. Calcitonin is a 32-amino acid peptide produced by the thyroid gland, whose main role is to prevent bone resorption. Calcitonin produced a non-competitive type of inhibition at varied potassium concentrations whereas in the presence of sodium and ATP, calcitonin produced an uncompetitive and competitive type of inhibition, respectively. Neurotensin is a basic tridecapeptide which also inhibits Na+, K+-ATPase activity. Therefore, in the presence of higher sodium concentrations the peptide produced a competitive interaction and it produced a non-competitive type of inhibition at varied potassium and ATP concentrations. In summary, calcitonin and neutotensin behave as enzyme inhibitors but, each one leads to a characteristic type of enzyme inhibition at varied substrate concentrations due to differences in peptide structure and functionality that could be influencing the kinetics of substrate interactions.Fil: 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; Argentin

Neurotensin modulates central muscarinic receptors, an effect which does not involve the high-affinity neurotensin receptor (NTS1)

Neurotensin (NT) is a tridecapeptide distributed in central and peripheral nervous systems, which can behave as a neurotransmitter or neuromodulator at central and peripheral levels. Herein we tested the potential effect of this peptide on quinuclidinyl benzilate ([3H]-QNB) binding to muscarinic receptor in rat CNS membranes. It was observed that NT decreased up to 50-70% ligand binding at 1×10-7M-1×10-5M concentration in cerebral cortex, cerebellum and striatum. In the hippocampus, NT exerted a biphasic effect, behaving as a stimulator in the presence of 1×10-12M-1×10-10M concentration but as an inhibitor at 1×10-8M-1×10-5M concentration. In order to test the involvement of high-affinity NT receptor (NTS1) in NT inhibitory effect, assays were carried out in the presence of 1×10-6M NT and/or SR 48692 (Sanofi-Aventis, U.S., Inc.), a specific antagonist for this receptor, dissolved in dimethylsulfoxide (DMSO) 10% v/v. As controls, membranes incubated with DMSO and/or NT 1×10-6M plus DMSO were processed. It was found that NT+DMSO decreased [3H]-QNB binding to cerebral cortex, cerebellum and hippocampal membranes by 49%, 32% and 53%, respectively. This inhibition was not observed with the DMSO control group. Membrane preincubation with 1×10-6M SR 48692 failed to alter NT effect on binding. SR 48692 at 1×10-6M concentration decreased the binding by 50% only in cerebral cortex membranes, suggesting a possible direct effect of the antagonist on muscarinic receptors in this area. It was therefore concluded that the high-affinity NT receptor may not be involved in ligand binding inhibition to muscarinic receptor by NT.Fil: Schneider, Patricia 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: 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

The administration of levocabastine, a NTS2 receptor antagonist, modifies Na+, K+-ATPase properties

Neurotensin behaves as a neuromodulator or as a neurotransmitter interacting with NTS1 and NTS2 receptors. Neurotensin in vitro inhibits synaptosomal membrane Na+, K+-ATPase activity. This effect is prevented by administration of SR 48692 (antagonist for NTS1 receptor). The administration of levocabastine (antagonist for NTS2 receptor) does not prevent Na+, K+-ATPase inhibition by neurotensin when the enzyme is assayed with ATP as substrate. Herein levocabastine effect on Na+, K+-ATPase K+ site was explored. For this purpose, levocabastine was administered to rats and K+-p-nitrophenylphosphatase (K+-p-NPPase) activity in synaptosomal membranes and [3H]-ouabain binding to cerebral cortex membranes were assayed in the absence (basal) and in the presence of neurotensin. Male Wistar rats were administered with levocabastine (50 μg/kg, i.p., 30 min) or the vehicle (saline solution). Synaptosomal membranes were obtained from cerebral cortex by differential and gradient centrifugation. The activity of K+-p-NPPase was determined in media laking or containing ATP plus NaCl. In such phosphorylating condition enzyme behaviour resembles that observed when ATP hydrolyses is recorded. In the absence of ATP plus NaCl, K+-p-NPPase activity was similar for levocabastine or vehicle injected (roughly 11 μmole hydrolyzed substrate per mg protein per hour). Such value remained unaltered by the presence of 3.5 × 10−6 M neurotensin. In the phosphorylating medium, neurotensin decreased (32 %) the enzyme activity in membranes obtained from rats injected with the vehicle but failed to alter those obtained from rats injected with levocabastine. Levocabastine administration enhanced (50 %) basal [3H]-ouabain binding to cerebral cortex membranes but failed to modify neurotensin inhibitory effect on this ligand binding. It is concluded that NTS2 receptor blockade modifies the properties of neuronal Na+, K+-ATPase and that neurotensin effect on Na+, K+-ATPase involves NTS1 receptor and -at least partially- NTS2 receptor.Fil: Gutnisky, Alicia. 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Farmacología; Argentina. 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

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

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

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

High affinity receptors but not low affinity receptors for neurotensin are involved in neuronal Na/K-ATPase inhibition by the peptide

Neurotensin acts as a neuromodulator or as a neurotransmitter that binds to a group of receptors. Two of the receptors, namely NTS1 and NTS2, bind to neurotensin with high affinity and low affinity, respectively. Neurotensin added in vitro inhibits synaptosomal membrane Na+ , K+ - ATPase activity. This effect seems to be mediated by NTS1 receptor because it is fully blocked by antagonist SR 48692. Herein neurotensin effect was assayed after administration of SR 48692 and levocabastine which are antagonists for NTS1 and NTS2 receptors, respectively. Male Wistar rats were administered by i.p. injection, with 150 µg/kg SR 48692 (Sanofi-Aventis U.S., Inc.) suspended in the vehicle (0.01% Tween 80 in saline solution), 50 μg/kg levocabastine (disolved in saline solution) and the corresponding vehicle solutions. Thirty minutes later, the animals were sacrificed, cerebral cortices removed, separately pooled, and processed to obtain synaptosomal membranes. In membrane samples, Na+ , K+ - and Mg2+-ATPase activities were determined in the absence and presence of 3.5 x 10-6 M neurotensin. Basal Na+ , K+ -ATPase activity in membranes isolated from control rats (vehicle injected) decreased roughly by 60% by the peptide. This effect was entirely prevented by the administration of NTS1 antagonist SR 48692. Administration of levocabastine, which enhanced basal Na+ , K+ - ATPase activity, failed to prevent neurotensin inhibitory effect on this enzyme activity. Mg2+- ATPase activity remained unaltered in all conditions tested. It is concluded that Na+ , K+ - ATPase inhibition by neurotensin seems mediated only by NTS1 receptor because the administration of NTS1 antagonist SR 48692, and not the NTS2 antagonist levocabastine prevented the effect of the peptide.Fil: Alvarez Juliá, Anabel. 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: Gutnisky, Alicia. 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 ; 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 ; Argentin