Neurochemical Evidence that Lysine Inhibits Synaptic Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity and Provokes Oxidative Damage in Striatum of Young Rats In vivo

Lysine (Lys) accumulation in tissues and biological fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and other inherited metabolic disorders. In the present study we investigated the effects of acute administration of Lys on relevant parameters of energy metabolism and oxidative stress in striatum of young rats. We verified that Lys in vivo intrastriatal injection did not change the citric acid cycle function and creatine kinase activity, but, in contrast, significantly inhibited synaptic Na⁺,K⁺-ATPase activity in striatum prepared 2 and 12 h after injection. Moreover, Lys induced lipid peroxidation and diminished the concentrations of glutathione 2 h after injection. These effects were prevented by the antioxidant scavengers melatonin and the combination of α-tocopherol and ascorbic acid. Lys also inhibited glutathione peroxidase activity 12 h after injection. Therefore it is assumed that inhibition of synaptic Na⁺,K⁺-ATPase and oxidative damage caused by brain Lys accumulation may possibly contribute to the neurological manifestations of FH and other neurometabolic conditions with high concentrations of this amino acid.     

Isovaleric Acid Reduces Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity in Synaptic Membranes from Cerebral Cortex of Young Rats

1. Patients affected by isovaleric acidemia (IVAcidemia) suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. The objective of the present study was to investigate the in vitro effects of the metabolites that predominantly accumulate in IVAcidemia, namely isovaleric acid (IVA), 3-hydroxyisovaleric acid (3-OHIVA) and isovalerylglycine (IVG), on important parameters of energy metabolism, such as ¹⁴CO₂ production from acetate and the activities of the respiratory chain complexes I–IV, creatine kinase and Na⁺, K⁺-ATPase in synaptic plasma membranes from cerebral cortex homogenates of 30-day-old rats. 2. We observed that 3-OHIVA acid and IVG did not affect all the parameters analyzed. Similarly, ¹⁴CO₂ production from acetate (Krebs cycle activity), the activities of creatine kinase, and of the respiratory chain complexes was not modified by IVA. In contrast, IVA exposition to cortical homogenates provoked a marked inhibition of Na⁺, K⁺-ATPase activity. However, this activity was not changed when IVA was directly exposed to purified synaptic plasma membranes, suggesting an indirect effect of this organic acid on the enzyme. Furthermore, pretreatment of cortical homogenates with α-tocopherol and creatine totally prevented IVA-induced inhibition on Na⁺, K⁺-ATPase activity from synaptic plasma membranes, whereas glutathione (GSH) and the NO synthase inhibitor N^ω-nitro-l-arginine methyl ester (L-NAME) did not alter this inhibition. 3. These data indicate that peroxide radicals were probably involved in this inhibitory effect. Since Na⁺, K⁺-ATPase is a critical enzyme for normal brain development and functioning and necessary to maintain neuronal excitability, it is presumed that the inhibitory effect of IVA on this activity may be involved in the pathophysiology of the neurological dysfunction of isovaleric acidemic patients.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

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.     

The Study of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity of Rat Brain during Crush Syndrome

Crush syndrome (CS) results from severe traumatic damage to the organism that is characterized by stress, acute homeostatic failure of the tissues, and myoglobinuria with severe intoxication. This leads to an acute impairment of kidneys and heart. The peripheral and central nervous systems are also the subject of significant changes in CS. Na⁺, K⁺-ATPase is a critical enzyme in neuron that is essential for the regulation of neuronal membrane potential, cell volume as well as transmembrane fluxes of Ca⁺⁺ and Excitatory Amino Acids. In the present study, Na⁺, K⁺-ATPase activity of rat brain regions [Olfactory lobes (OL), Cerebral cortex (CC), Cerebellum (CL), and Medulla oblongata (MO)] during CS was investigated. Experimental model of CS in albino rats was induced by 2-h of compression followed by 2, 24, and 48-h of decompression of femoral muscle tissue. In this study, we have observed elevation in Na⁺, K⁺-ATPase activity above normal/control levels in all parts of brain (OL: 34.4%; CC: 1.0%; CL: 3.3% and MO: 45%) during 2-h compression in comparison to controls.     

Clozapine Administration Modifies Neurotensin Effect on Synaptosomal Membrane Na<Superscript>+</Superscript>, K<Superscript>+</Superscript> -ATPase Activity

Na⁺, K⁺-ATPase is inhibited by neurotensin, an effect which involves the peptide high affinity receptor (NTS1). Neurotensin effect on cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase activity of rats injected i.p. with antipsychotic clozapine was studied. Whereas 3.5 × 10⁻⁶ M neurotensin decreased 44% Na⁺, K⁺-ATPase activity in the controls, the peptide failed to modify enzyme activity 30 min after a single 3.0, 10.0 and 30.0 mg/kg clozapine dose. Neurotensin decreased Na⁺, K⁺-ATPase activity 40 or 20% 18 h after 3.0 or 5.6 mg/kg clozapine administration, respectively, and lacked inhibitory effect 18 h after 17.8 and 30.0 mg/kg clozapine doses. Results indicated that the clozapine treatment differentially modifies the further effect of neurotensin on synaptosomal membrane Na⁺, K⁺-ATPase activity according to time and dose conditions employed. Taken into account that clozapine blocks the dopaminergic D2 receptor, findings obtained favor the view of an interplay among neurotensinergic receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase at synaptic membranes.     

Involvement of protons in the ion transport cycle of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase

The effect of pH on electrogenic sodium transport by the Na⁺,K⁺-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na⁺,K⁺-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 mM Na⁺, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence of NaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCl. These results may be explained by involvement of protons in the Na⁺,K⁺-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Modulation of Aspartate Release by Ascorbic Acid and Endobain E,an Endogenous Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitor

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na⁺, K⁺-ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM d-[³H]aspartate (15 min at 37°C), centrifuged, washed, incubated in the presence of additions (60 s at 37°C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5–5.0 mM ascorbic acid, d-[³H]aspartate release was roughly 135–215% or 110–150%, with or without 40 mM KCl, respectively. The endogenous Na⁺, K⁺-ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5–10.0 mM commercial ouabain enhanced roughly 100% d-[³H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50–60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na⁺, K⁺-ATPase, may well modulate neurotransmitter release at synapses.     

Identification of proteins whose interaction with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase is triggered by ouabain

Prolonged exposure of different epithelial cells (canine renal epithelial cells (MDCK), vascular endothelial cells from porcine aorta (PAEC), human umbilical vein endothelial cells (HUVEC), cervical adenocarcinoma (HeLa), as well as epithelial cells from colon carcinoma (Caco-2)) with ouabain or with other cardiotonic steroids was shown earlier to result in the death of these cells. Intermediates in the cell death signal cascade remain unknown. In the present study, we used proteomics methods for identification of proteins whose interaction with Na⁺,K⁺-ATPase is triggered by ouabain. After exposure of Caco-2 human colorectal adenocarcinoma cells with 3 μM of ouabain for 3 h, the protein interacting in complex with Na⁺,K⁺-ATPase was coimmunoprecipitated using antibodies against the enzyme α1-subunit. Proteins of coimmunoprecipitates were separated by 2D electrophoresis in polyacrylamide gel. A number of proteins in the coimmunoprecipitates with molecular masses of 71-74, 46, 40-43, 38, and 33-35 kDa was revealed whose binding to Na⁺,K⁺-ATPase was activated by ouabain. Analyses conducted by mass spectroscopy allowed us to identify some of them, including seven signal proteins from superfamilies of glucocorticoid receptors, serine/threonine protein kinases, and protein phosphatases 2C, Src-, and Rho-GTPases. The possible participation of these proteins in activation of cell signaling terminated by cell death is discussed.     

A possible mechanism for low affinity of silkworm Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase for K<Superscript>+</Superscript>

The affinity for K⁺ of silkworm nerve Na⁺/K⁺-ATPase is markedly lower than that of mammalian Na⁺/K⁺-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K⁺ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na⁺/K⁺-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na⁺/K⁺-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase expressed in the cultured cells showed a low affinity for K⁺ and a high affinity for Na⁺, characteristic of the silkworm nerve Na⁺/K⁺-ATPase. These results suggest that the β subunit is responsible for the affinity for K⁺ of Na⁺/K⁺-ATPase.     

DDT inhibits Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>, Mg<Superscript>2+</Superscript>-ATPase in the Intestinal Mucosae and Gills of Marine Teleosts

SODIUM-potassium-activated, magnesium-dependent, adenosine triphosphatase (Na⁺, K⁺, Mg²⁺-ATPase) is widely accepted as an essential factor in sodium transport¹ and observations on fish substantiate this view. There are concurrent increases, for example, of both Na⁺, K⁺, Mg²⁺-ATPase activity and osmoregulatory sodium transport², in the intestinal mucosae^3,4 and the gills^3,5 of euryhaline teleosts during adaptation to seawater. Furthermore, the gills of stenohaline seawater teleosts, which actively secrete sodium, exhibit higher Na⁺, K⁺, Mg²⁺-ATPase activity than the gills of stenohaline freshwater teleosts, which do not actively secrete sodium^3,5. Na⁺, K⁺, Mg²⁺-ATPase therefore seems to be important in maintaining tissue osmolarity well below that of seawater. It is disquieting to report therefore that Na⁺, K⁺, Mg²⁺-ATPase activity in the intestinal mucosae and gills of marine teleosts is inhibited by the organochlorine insecticide DDT. This observation may help to clarify the unexplained sensitivity of teleosts to DDT⁶.     

Effects of palytoxin on cation occlusion and phosphorylation of the (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.     

Ouabain-sensitive and ouabain-resistant isoforms of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in cerebellar granule cells control MAP kinase activity

Three-hour incubation of rat cerebellar granule cells with 0.1 μM ouabain increases intracellular levels of Ca²⁺ ions and reactive oxygen species (ROS) resulting in pronounced activation of Mitogen-Activated Protein Kinase (MAPK). Higher concentrations of ouabain induce further increases in MAPK activity. The activating effect of ouabain is attenuated by the NMDA-receptor antagonists MK-801 and D-AP5. The data obtained suggest that similar to NMDA receptors ouabain-sensitive and ouabain-resistant isoforms of Na⁺,K⁺-ATPase are actively involved in intracellular signaling cascades controlling proliferative activity of neuronal cells.     

Association Between Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity and the Vulnerability/Resilience to Mood Disorders induced by Early Life Experience

There is increasing evidence that early life events can influence neurodevelopment and later susceptibility to disease. Chronic variable stress (CVS) has been used as a model of depression. The objective of this study was to evaluate the interaction between early experience and vulnerability to chronic variable stress in adulthood, analyzing emotional, metabolic and neurochemical aspects related to depression. Pups were (1) handled (10 min/day) or (2) left undisturbed from day 1 to 10 after birth. When the animals reached adulthood, the groups were subdivided and the rats were submitted or not to CVS, which consisted of daily exposure to different stressors for 40 days, followed by a period of behavioral tasks, biochemical (plasma corticosterone and insulin sensitivity) and neurochemical (Na⁺,K⁺-ATPase activity in hippocampus, amygdala and parietal cortex) measurements. Neonatally-handled rats demonstrated shorter immobility times in the forced swimming test, independently of the stress condition. There was no difference concerning basal corticosterone or insulin sensitivity between the groups. Na⁺,K⁺-ATPase activity was decreased in hippocampus and increased in the amygdala of neonatally-handled rats. CVS decreased the enzyme activity in the three structures, mainly in the non-handled group. These findings suggest that early handling increases the ability to cope with chronic variable stress in adulthood, with animals showing less susceptibility to neurochemical features associated with depression, confirming the relevance of the precocious environment to vulnerability to psychiatric conditions in adulthood.     

Maternal Hypermethioninemia Affects Neurons Number,Neurotrophins Levels,Energy Metabolism,and Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Expression/Content in Brain of Rat Offspring

In the current study, we verified the effects of maternal hypermethioninemia on the number of neurons, apoptosis, nerve growth factor, and brain-derived neurotrophic factor levels, energy metabolism parameters (succinate dehydrogenase, complex II, and cytochrome c oxidase), expression and immunocontent of Na⁺,K⁺-ATPase, edema formation, inflammatory markers (tumor necrosis factor-alpha and interleukin-6), and mitochondrial hydrogen peroxide levels in the encephalon from the offspring. Pregnant Wistar rats were divided into two groups: the first one received saline (control) and the second group received 2.68 μmol methionine/g body weight by subcutaneous injections twice a day during gestation (approximately 21 days). After parturition, pups were killed at the 21st day of life for removal of encephalon. Neuronal staining (anti-NeuN) revealed a reduction in number of neurons, which was associated to decreased nerve growth factor and brain-derived neurotrophic factor levels. Maternal hypermethioninemia also reduced succinate dehydrogenase and complex II activities and increased expression and immunocontent of Na⁺,K⁺-ATPase alpha subunits. These results indicate that maternal hypermethioninemia may be a predisposing factor for damage to the brain during the intrauterine life.     

Rottlerin Inhibits (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)-ATPase Activity in Brain Tissue and Alters <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-δ) to obtain evidence that the activity-dependent distribution of glutamate transporter GLAST is regulated by PKC-δ mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that d-aspartate-induced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na⁺, K⁺)-dependent ATPase (Na⁺, K⁺-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (“metabolic poison”). Rottlerin also inhibited Na⁺, K⁺-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na⁺, K⁺-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na⁺, K⁺-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-δ-catalysed phosphorylation in the process.     

The Administration of Levocabastine,a NTS2 Receptor Antagonist,Modifies Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-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.     

Different domain organization of two main conformational states of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase

The Na⁺/K⁺-ATPase generates an electrochemical gradient of Na⁺ and K⁺, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two principal conformational states, E₁ and E₂. To assess the domain organization of the protein in these conformations, thermal denaturation of Na⁺/K⁺-ATPases from duck salt gland and from rabbit kidney has been studied in the absence and in the presence of Na⁺ or K⁺, which induce the transition to E₁ or E₂. The melting curves for the ion-free forms of the two ATPases have different shapes: the rabbit protein shows one transition at 56.1°C, whereas the duck protein shows two transitions, at 49.8 and 56.9°C. Addition of Na⁺ or K⁺ ions abolishes the difference in thermal behavior between these enzymes, but through opposite effects. The melting curves for the E₂ conformation (K⁺ bound) in both cases exhibit a single peak of heat absorption at ∼63°C. For the E₁ conformation (Na⁺ bound), each melting curve has three peaks, indicating denaturation of three domains. The difference in the domain organization of Na⁺/K⁺-ATPase in the E1 and E2 states may account for the different sensitivity to temperature, proteolysis, and oxidative stress observed for the two enzyme conformations.     

Effect of vanadium on renal Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in diabetic rats: a possible role of leptin

Several researches attempt to protect diabetic patients from the development of nephropathy. Involvement of leptin and renal Na⁺,K⁺-ATPase enzyme in diabetic nephropathy (DN) development is a recent field for researches. Vanadium, as a trace element with insulin mimetic effect, may act synergistically with insulin to protect against the development of DN. Sixty male Sprague Dawley rats were divided into six groups: control group (C), vanadium control group (CV), streptozotocin-induced diabetic group (D), insulin-treated diabetic group (DI), vanadium-treated diabetic group (DV), and combined insulin and vanadium-treated diabetic group. Six weeks later, systolic blood pressure (SBP) was measured and retro-orbital blood samples were collected to estimate glycosylated hemoglobin (HbA_1c), serum sodium (Na⁺) and creatinine, blood urea nitrogen (BUN) and plasma leptin levels. Preparation of microsomal fraction of renal tissue homogenate for estimation of Na⁺,K⁺-ATPase activity was done. The D group showed a significant increase in SBP, HbA_1c, serum Na⁺, creatinine, and BUN levels and Na⁺,K⁺-ATPase activity in microsomal fraction of renal tissue homogenate while plasma leptin level decreased significantly compared with C and CV groups. Both DI and DV groups showed a significant improvement in all the above measured parameters compared with D group while there were no significant changes between the DI and DV groups. Concomitant treatment with insulin and vanadium resulted in a significant improvement in all the measured parameters compared to each alone. Vanadium in combination with insulin ameliorates DN markers and reduces renal Na⁺,K⁺-ATPase overactivity in diabetic rats. An effect that may be partially mediated through correction of hypoleptinemia observed in these animals.