Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.     

Nitric oxide synthase inhibition by L-NAME prevents the decrease of Na+,K+-ATPase activity in midbrain of rats subjected to arginine administration

In the present study we investigated the effect of acute administration of L-arginine on Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities and on some parameters of oxidative stress (chemiluminescence and total radical-trapping antioxidant parameter-TRAP) in midbrain of adult rats. We also tested the effect of L-NAME on the effects produced by arginine. Sixty-day-old rats were treated with an acute intraperitoneal injection of saline (group I, control), arginine (0.8 g/kg) (group II), L-NAME (2 mg/kg) (group III) or arginine (0.8 g/kg) plus L-NAME (2 mg/kg) (group IV). Na⁺,K⁺-ATPase activity was significantly reduced in the arginine-treated rats, but was not affected by other treatments. In contrast, Mg²⁺-ATPase activity was not altered by any treatment. Furthermore, chemiluminescence was significantly increased and TRAP was significantly decreased in arginine-treated rats, whereas the simultaneous injection of L-NAME prevented these effects. These results demonstrate that in vivo arginine administration reduces Na⁺,K⁺-ATPase activity possibly through free radical generation induced by NO formation.     

The Anticonvulant Effect of Cooling in Comparison to α-Lipoic Acid: A Neurochemical Study

Brain cooling has pronounced effects on seizures and epileptic activity. The aim of the present study is to evaluate the anticonvulsant effect of brain cooling on the oxidative stress and changes in Na⁺, K⁺-ATPase and acetylcholinesterase (AchE) activities during status epilepticus induced by pilocarpine in the hippocampus of adult male rat in comparison with α-lipoic acid. Rats were divided into four groups: control, rats treated with pilocarpine for induction of status epilepticus, rats treated for 3 consecutive days with α-lipoic acid before pilocarpine and rats subjected to whole body cooling for 30 min before pilocarpine. The present findings indicated that pilocarine-induced status epilepticus was accompanied by a state of oxidative stress as clear from the significant increase in lipid peroxidation (MDA) and superoxide dismutase (SOD) and significant decrease in reduced glutathione and nitric oxide (NO) levels and the activities of catalase, AchE and Na⁺, K⁺-ATPase. Pretreatment with α-lipoic acid ameliorated the state of oxidative stress and restored AchE to nearly control activity. However, Na⁺, K⁺-ATPase activity showed a significant decrease. Rats exposed to cooling for 30 min before the induction of status epilepticus revealed significant increases in MDA and NO levels and SOD activity. AchE returned to control value while the significant decrease in Na⁺, K⁺-ATPase persisted. The present data suggest that cooling may have an anticonvulsant effect which may be mediated by the elevated NO level. However, brain cooling may have drastic unwanted insults such as oxidative stress and the decrease in Na⁺, K⁺-ATPase activity.     

Role of the Na+,K+-ATPase α2 isoform in the positive inotropic effect of ouabain and marinobufagenin in the rat diaphragm

It was found that ouabain and marinobufagenin, specific inhibitors of Na⁺,K⁺-ATPase, increased the contraction of the isolated rat diaphragm by ～15% (positive inotropic effect) at EC₅₀ = 1.2 ± 0.3 nM and 0.3 ± 0.1 nM, respectively, which was indicative of the participation of the ouabain-sensitive Na⁺,K⁺-ATPase α2 isoform. Analysis of the dose-response curves for the effect of ouabain on the resting membrane potential of muscle fibers in the absence and in the presence of 100 nM acetylcholine (hyperpolarizing the membrane) showed the presence of two pools of Na⁺,K⁺-ATPase α2 that differed in affinity for ouabain. Only the high-affinity pool (IC₅₀ ～ 9 nM) mediates the hyperpolarizing effect of nanomolar concentrations of acetylcholine. Most likely, it is this pool of that is involved in the positive inotropic effect of ouabain, which can be a mechanism of regulation of the muscle function by circulating endogenous inhibitors of Na⁺,K⁺-ATPase.     

Changes of sodium and ATP affinities of the cardiac (Na,K)-ATase during and after nitric oxide deficient hypertension

In the cardiovascular system, NO is involved in the regulation of a variety of functions. Inhibition of NO synthesis induces sustained hypertension. In several models of hypertension, elevation of intracellular sodium level was documented in cardiac tissue. To assess the molecular basis of disturbances in transmembraneous transport of Na⁺, we studied the response of cardiac (Na,K)-ATPase to NO-deficient hypertension induced in rats by NO-synthase inhibition with 40 mg/kg/day N^G-nitro-L-arginine methyl ester (L-NAME) for 4 four weeks. After 4-week administration of L-NAME, the systolic blood pressure (SBP) increased by 36%. Two weeks after terminating the treatment, the SBP recovered to control value. When activating the (Na,K)-ATPase with its substrate ATP, no changes in K_m and V_max values were observed in NO-deficient rats. During activation with Na⁺, the V_max remained unchanged, however the K_Na increased by 50%, indicating a profound decrease in the affinity of the Na⁺-binding site in NO-deficient rats. After recovery from hypertension, the activity of (Na,K)-ATPase increased, due to higher affinity of the ATP-binding site, as revealed from the lowered K_m value for ATP. The K_Na value for Na⁺ returned to control value. Inhibition of NO-synthase induced a reversible hypertension accompanied by depressed Na⁺-extrusion from cardiac cells as a consequence of deteriorated Na⁺-binding properties of the (Na,K)-ATPase. After recovery of blood pressure to control values, the extrusion of Na⁺ from cardiac cells was normalized, as revealed by restoration of the (Na,K)-ATPase activity. (Mol Cell Biochem 000: 000-000, 1999)     

Postnatal Nitric Oxide Inhibition Modifies Neurotensin Effect on ATPase Activity

We have previously showed that peptide neurotensin inhibits neuronal Na⁺, K⁺-ATPase activity, an effect which involves high affinity neurotensin receptor. Nitric oxide (NO) acts as a neurotransmitter or as a neuromodulator when it is synthesized by neuronal nitric oxide synthase. Neurotensin effect on Na⁺, K⁺-ATPase activity was evaluated in cortical synaptosomal membranes isolated from rats injected at 3, 4 and 5 postnatal days with saline (control) or N (ω)-nitro-l-arginine methyl esther (L-NAME), a nitric oxide synthase inhibitor. Assays were carried out at two stages: juvenile (35 days) and adult (56 days) ages. In an open field task, results recorded in juvenile rats markedly differed from those obtained in adult rats. The presence of neurotensin at 3.5 × 10⁻⁸–3.5 × 10⁻⁶ M concentration decreased 16–34% Na⁺, K⁺-ATPase activity in membranes purified from control animals. At variance, the peptide failed to alter this enzyme activity in membranes obtained after L-NAME treatment. After administration of L-NAME, [³H]-ouabain binding to membranes isolated from adult male rats decreased 64% in the presence of 1.0 × 10⁻⁶ M neurotensin, a peptide concentration which only slightly decreased binding to membranes isolated from juvenile rats. It is postulated that early postnatal NO dysfunction may exert a permanent change in neurotensin system that influence later Na⁺, K⁺-ATPase response to neurotensin.     

Contribution of intracellular ATP to cisplatin resistance of tumor cells

Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na⁺,K⁺-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na⁺,K⁺-ATPase α₁ subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na⁺,K⁺-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na⁺,K⁺-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na⁺,K⁺-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.     

Functional role of the N-terminus of Na,K-ATPase α-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na⁺,K⁺-ATPase α-subunit shows some homology to that of Shaker-B K⁺ channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na⁺,K⁺-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the α-subunit appears to act like an inactivation gate and performs a critical step in the Na⁺,K⁺-ATPase pumping function.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

Effect of Glutamate and Inhibitors of Various Pathways of Free Radical Production on Na+,K+-ATPase Activity and Accumulation of Lipids Peroxidation Products in Rat Brain Cortex Synaptosomes

It has been shown that treatment of the rat brain cortex synaptosomes with glutamate produced both a significant reduction in Na⁺,K⁺-ATPase activity and accumulation of products of lipid peroxidation (LPO) like malone dialdehyde, dienoic conjugates, and Schiff bases. A suppression of different routes of free radical production in cytosol by quinacrine, indomethacin, and allopurinol (blockers of phospholipase A₂, cyclooxygenases, and xanthine oxidases, respectively) as well as by MK-801 (a antagonist of MDA-receptors) prevented or lowered significantly the effect of glutamate on Na⁺,K⁺-ATPase activity. No significant effect of glutamate on the Na⁺,K⁺-ATPase activity was also observed in the presence of L-NAME (inhibitor of NO-synthase). Inhibitors of the arachidonate and NO-synthase pathway of free radical production also prevented accumulation of LPO end products in the rat brain cortex under the effect of glutamate. In the presence of rotenone and olygomycin (blockers of mitochondrial electron transport and ATP synthase, respectively), glutamate led to even a greater inactivation of Na⁺,K⁺-ATPase and accumulation of malone dialdehyde. The data obtained suggest that at early stages of ischemia the neurotoxic effect of glutamate is due to an inflow of calcium ions through NMDA receptors and activation of different pathways of free radical production in cytosol of nerve cells. At these stages, protective functions of mitochondria appear to predominate due to their ability to accumulate calcium ions and to prevent an excessive increase of the cytosol calcium concentration under the effect of excitatory amino acids.     

Quantitative calculation of the role of the Na+,K+-ATPase in thermogenesis

The Na⁺,K⁺-ATPase is accepted as an important source of heat generation (thermogenesis) in animals. Based on information gained on the kinetics of the enzyme's partial reactions we consider via computer simulation whether modifications to the function of the combined Na⁺,K⁺-ATPase/plasma membrane complex system could lead to an increased body temperature, either through the course of evolution or during an individual's lifespan. The enzyme's kinetics must be considered because it is the rate of heat generation which determines body temperature, not simply the amount of heat per enzymatic cycle. The results obtained indicate that a decrease in thermodynamic efficiency of the Na⁺,K⁺-ATPase, which could come about by Na⁺ substituting for K⁺ on the enzyme's extracellular face, could not account for increased thermogenesis. The only feasible mechanisms are an increase in the enzyme's expression level or an increase in its ion pumping activity. The major source of Na⁺,K⁺-ATPase-related thermogenesis (72% of heat production) is found to derive from passive Na⁺ diffusion into the cell, which counterbalances outward Na⁺ pumping to maintain a constant Na⁺ concentration gradient across the membrane. A simultaneous increase in both Na⁺,K⁺-ATPase activity and the membrane's passive Na⁺ permeability could promote a higher body temperature.     

Regulation of Human and Pig Renal Na+,K+-ATPase Activity by Tyrosine Phosphorylation of Their α1-Subunits

Modulation of the physiologically influential Na⁺,K⁺-ATPase is a complex process involving a wide variety of factors. To determine the possible effects of the protein tyrosine phosphatase (PTP) inhibitors dephostatin and Et-3,4-dephostatin on human and pig, renal cells and enzymatic extracts, we treated our samples (15 min–24 h) with those PTP inhibitors (0–100 μM). PTP inhibitors were found to possess a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in both human and pig samples. The inhibition was similarly demonstrated on all cellular, microsomal fraction and purified Na⁺,K⁺-ATPase levels. Despite rigorous activity recovery attempts, the PTP inhibitors’ effects were sustained on Na⁺,K⁺-ATPase activity. Western blotting experiments revealed the expression of both α₁- and β₁-subunits in both human and pig tissues. α₁-Subunits possessed higher tyrosine phosphorylation levels with higher concentrations of PTP inhibitors. Meanwhile, serine/threonine residues of both α₁- and β₁-subunits demonstrated diminished phosphorylation levels upon dephostatin treatment. Accordingly, we provide evidence that Na⁺,K⁺-ATPase can be regulated through tyrosine phosphorylation of primarily their α₁-subunits, using PTP inhibitors.     

Na+K+-ATPase Activity and K+ Channels Differently Contribute to Vascular Relaxation in Male and Female Rats

Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones’ effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K⁺) channels and Na⁺K⁺-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K⁺ channels and Na⁺K⁺-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 µM) and the following K⁺ channels blockers: tetraethylammonium (TEA, 2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 µM) and charybdotoxin (ChTX, 0.1 µM). The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K⁺ channel (K_v) participates more in males. Inhibition of the three types of Ca²⁺-activated K⁺ channels induced a greater reduction in R_max in females than in males. The functional activity of the Na⁺K⁺-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUAincubation. OUA reduced K⁺-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na⁺K⁺-ATPase functional activity. L-NAME reduced K⁺-induced relaxation only in the female group, suggesting that nitric oxide (NO) participates more in their functional Na⁺K⁺-ATPase activity. These results suggest that the K⁺ channels involved in the gender-based vascular relaxation differences are the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in females and K_v in males and in the K⁺-induced relaxation and the Na⁺K⁺-ATPase vascular functional activity is greater in males.     

Role of Trace Elements,Oxidative Stress and Immune System: a Triad in Premature Ovarian Failure

Cadmium is an environmental pollutant closely linked with cardiovascular diseases that seems to involve endothelium dysfunction and reduced nitric oxide (NO) bioavailability. Knowing that NO causes dilatation through the activation of potassium channels and Na⁺/K⁺-ATPase, we aimed to determine whether acute cadmium administration (10 μM) alters the participation of K⁺ channels, voltage-activated calcium channel, and Na⁺/K⁺-ATPase activity in vascular function of isolated aortic rings of rats. Cadmium did not modify the acetylcholine-induced relaxation. After L-NAME addition, the relaxation induced by acetylcholine was abolished in presence or absence of cadmium, suggesting that acutely, this metal did not change NO release. However, tetraethylammonium (a nonselective K⁺ channels blocker) reduced acetylcholine-induced relaxation but this effect was lower in the preparations with cadmium, suggesting a decrease of K⁺ channels function in acetylcholine response after cadmium incubation. Apamin (a selective blocker of small Ca²⁺-activated K⁺ channels—SK_Ca), iberiotoxin (a selective blocker of large-conductance Ca²⁺-activated K⁺ channels—BK_Ca), and verapamil (a blocker of calcium channel) reduced the endothelium-dependent relaxation only in the absence of cadmium. Finally, cadmium decreases Na⁺/K⁺-ATPase activity. Our results provide evidence that the cadmium acute incubation unaffected the calcium-activated potassium channels (SK_Ca and BK_Ca) and voltage-calcium channels on the acetylcholine vasodilatation. In addition, acute cadmium incubation seems to reduce the Na⁺/K⁺-ATPase activity.     

Salt-inducible kinase 1 is present in lung alveolar epithelial cells and regulates active sodium transport

Salt-inducible kinase 1 (SIK1) in epithelial cells mediates the increases in active sodium transport (Na⁺, K⁺-ATPase-mediated) in response to elevations in the intracellular concentration of sodium. In lung alveolar epithelial cells increases in active sodium transport in response to β-adrenergic stimulation increases pulmonary edema clearance. Therefore, we sought to determine whether SIK1 is present in lung epithelial cells and to examine whether isoproterenol-dependent stimulation of Na⁺, K⁺-ATPase is mediated via SIK1 activity. All three SIK isoforms were present in airway epithelial cells, and in alveolar epithelial cells type 1 and type 2 from rat and mouse lungs, as well as from human and mouse cell lines representative of lung alveolar epithelium. In mouse lung epithelial cells, SIK1 associated with the Na⁺, K⁺-ATPase α-subunit, and isoproterenol increased SIK1 activity. Isoproterenol increased Na⁺, K⁺-ATPase activity and the incorporation of Na⁺, K⁺-ATPase molecules at the plasma membrane. Furthermore, those effects were abolished in cells depleted of SIK1 using shRNA, or in cells overexpressing a SIK1 kinase-deficient mutant. These results provide evidence that SIK1 is present in lung epithelial cells and that its function is relevant for the action of isoproterenol during regulation of active sodium transport. As such, SIK1 may constitute an important target for drug discovery aimed at improving the clearance of pulmonary edema.     

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons

Binding to Na⁺,K⁺-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na⁺,K⁺-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na⁺,K⁺-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na⁺,K⁺-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein–protein interactions between Na⁺,K⁺-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na⁺,K⁺-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na⁺,K⁺-ATPase.     

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.     

Differential expression of Na+, K+-ATPase α-1 isoforms during seawater acclimation in the amphidromous galaxiid fish Galaxias maculatus

Inanga (Galaxias maculatus) is an amphidromous fish with a well-known capacity to withstand a wide range of environmental salinities. To investigate the molecular mechanisms facilitating acclimation of inanga to seawater, several isoforms of the Na⁺, K⁺-ATPase ion transporter were identified. This included three α-1 (a, b and c), an α-2 and two α-3 (a and b) isoforms. Phylogenetic analysis showed that the inanga α-1a and α-1b formed a clade with the α-1a and α-1b isoforms of rainbow trout, while another clade contained the α-1c isoforms of these species. The expression of all the α-1 isoforms was modulated after seawater exposure (28 ‰). In gills, the expression of the α-1a isoform was progressively down-regulated after seawater exposure, while the expression of the α-1b isoform was up-regulated. The α-1c isoform behaved similarly to the α-1a, although changes were less dramatic. Physiological indicators of salinity acclimation matched the time frame of the changes observed at the molecular level. A 24-h osmotic shock period was highlighted by small increases in plasma osmolality, plasma Na⁺ and a decrease in muscle tissue water content. Thereafter, these values returned close to their pre-exposure (freshwater) values. Na⁺, K⁺-ATPase activity showed a decreasing trend over the first 72 h following seawater exposure, but activity increased after 240 h. Our results indicate that inanga is an excellent osmoregulator, an ability that is conferred by the rapid activation of physiological and molecular responses to salinity change.