The Na,K-ATPase of nervous tissue

The Na,K-ATPase has been only partially purified from nervous tissue, yet it is clear that two forms (and +) of the catalytic subunit are present. is a component subunit of the glial Na,K-ATPase, which has a relatively low affinity for binding cardiac glycosides and + has been identified as a subunit of the Na,K-ATPase which has relatively high affinity for cardiac glycosides. The + form may also be sensitive to indirect modulation by neurotransmitters or hormones. The ratio of + / changes in the nervous system during development, and + appears to be the predominant species in adult neurones. Changes in Na,K-ATPase activity have been associated with several abnormalities in the nervous system, including epilepsy and altered nerve conduction velocity, but a causal relationship has not been definitively established. Although the Na,K-ATPase has a pivotal role in Na⁺ and K⁺ transport in the nervous system, a special role for the glial Na,K-ATPase in clearing extracellular K⁺ remains controversial.     

Phosphorylation and ubiquitination are necessary for Na,K-ATPase endocytosis during hypoxia

As a cellular adaptative response, hypoxia decreases Na,K-ATPase activity by triggering the endocytosis of its alpha(1) subunit in alveolar epithelial cells. Here, we present evidence that the ubiquitin conjugating system is important in the Na,K-ATPase endocytosis during hypoxia and that ubiquitination of Na,K-ATPase alpha(1) subunit occurs at the basolateral membrane. Endocytosis and ubiquitination were prevented when the Ser 18 in the PKC phosphorylation motif of the Na,K-ATPase alpha(1) subunit was mutated to an alanine, suggesting that phosphorylation at Ser-18 is required for ubiquitination. Mutation of the four lysines surrounding Ser 18 to arginine prevented Na,K-ATPase ubiquitination and endocytosis during hypoxia; however, only one of them was sufficient to restore hypoxia-induced endocytosis. We provide evidence that ubiquitination plays an important role in cellular adaptation to hypoxia by regulating Na,K-ATPase alpha(1)-subunit endocytosis.     

The regulation of the Na, K-ATPase system by neurotransmitters

Factors regulating the activity of synaptosomal Na, K-ATPase have been found in the cytosol of nerve endings. The activatory effect of the factor increases in the presence of neurotransmitters regardless of their direct action on Na, K-ATPase. Synaptosomal Na, K-ATPase is not sensitive to the factor obtained from the cytosol of kidney tissue, or the cytosolic fraction obtained after sedimentation of microsomes. The effect of inhibiting low molecular ET(S) fraction on Na, K-ATPase activity is not mediated through noradrenaline, dopamine and serotonin as well by the system of secondary messengers. Factor stimulated by neurotransmitters activates the Na, K-ATPase system affecting the phosphorylating intermediates of the enzyme and putting the Na, K-ATPase system in the mode of simultaneous transport of Na and K ions.     

Increased brain Na, K-ATPase activity of rats under stress

The activities of Na, K- and Mg-dependent ATPases were measured in crude synaptosomal fractions isolated from the rat brain gray matter. Prolonged (6 h) exposure to emotional painful stress stimulated Na, K-ATPase activity by 40% without affecting that of Mg-ATPase. Preliminary injection of the free radical scavenger ionol presented Na, K-ATPase activation, thus suggesting the involvement of lipid peroxidation initiated in brain tissues under stress in acceleration of NA-pump function. However, model studies with lipid peroxidation induced in vitro by an ascorbate-dependent system in a membranous suspension demonstrated an opposite effect, i. e. fast inhibition of Na, K-ATPase. Possible reasons for the different effects of lipid peroxidation in vivo under stress and on Na, K-ATPase activity in vitro are discussed. It is concluded that activation of Na K-ATPase is a mechanism which is responsible for acceleration of reflex conditioning and for the maintenance of the conditioned reflexes in stress-exposed animals.     

Therapeutic action of an antioxidant in chronic emotional-pain stress in the rat

Chronic emotional pain stress in rats causes disturbances of the cardiovascular system function (increase in arterial pressure and in heart rate), typical of neuroses-like state, and changes of the vegetative nervous system reactivity tested with functional load by two-hour hypokinesis. Increase in spleen weight is observed as well as a tendency to adrenals weight increase, a decrease of Na, K-ATPase activity and activation of lipid peroxidation in cortical and hippocampal homogenates. Administration of F-801 antioxidant according to therapeutic scheme after the end of stress action, restores normal function of the cardiovascular system, normal reactivity of the vegetative nervous system, decreases adrenals weight and increases the weight of thymus and also normalizes ATPase activity and the level of lipid peroxidation. A backward correlation dependence of the Na, K-ATPase activity on the level of malondialdehyde in the brain tissue has been established.     

Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases.

The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.     

Regulation of gene expression by corticoid hormones in the brain and spinal cord

Glucocorticoids (GC) and mineralocorticoids (MC) have profound regulatory effects upon the central nervous system (CNS). Hormonal regulation affects several molecules essential to CNS function. First, evidences are presented that mRNA expression of the 3 and β1-subunits of the Na,K-ATPase are increased by GC and physiological doses of MC in a region-dependent manner. Instead, high MC doses reduce the β1 isoform and enzyme activity in amygdaloid and hypothalamic nuclei, an effect which may be related to MC control of salt appetite. The 3-subunit mRNA of the Na,K-ATPase is also stimulated by GC in motoneurons of the injured spinal cord, suggesting a role for the enzyme in GC neuroprotection. Second, we provide evidences for hormonal effects on the expression of mRNA for the neuropeptide arginine vasopressin (AVP). Our data show that GC inhibition of AVP mRNA levels in the paraventricular nucleus is sex-hormone dependent. This sexual dimorphism may explain sex differences in the hypothalamic–pituitary–adrenal axis function between female and male rats. Third, steroid effects on the astrocyte marker glial fibrillary acidic protein (GFAP) points to a complex regulatory mechanism. In an animal model of neurodegeneration (the Wobbler mouse) showing pronounced astrogliosis of the spinal cord, in vivo GC treatment down-regulated GFAP immunoreactivity, whereas the membrane-active steroid antioxidant U-74389F up-regulated this protein. It is likely that variations in GFAP protein expression affect spinal cord neurodegeneration in Wobbler mice. Fourth, an interaction between neurotrophins and GC is shown in the injured rat spinal cord. In this model, intensive GC treatment increases immunoreactive low affinity nerve growth factor (NGF) receptor in motoneuron processes. Because GC also increases immunoreactive NGF, this mechanism would support trophism and regeneration in damaged tissues. In conclusion, evidences show that some molecules regulated by adrenal steroids in neurons and glial cells are not only involved in physiological control, but additionally, may play important roles in neuropathology.     

Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.     

Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na(+) and K(+) gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100 electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.     

The Na/K-ATPase regulation by neurotransmitters in ontogeny

Activity of the Na/K-ATPase from rat brain synaptic membranes is inhibited by NA (noradrenaline). However, during fractionation of cytozole from nerve endings, two non-homogeneous peaks are found (SF(a), 60-100 kD and SF( i ),;10 kD), which influence the Na/K-ATPase activity, both directly and SF(a) NA-dependently. Joint action of NA and synaptic factors (SF(a) and SF(i)) on the Na/K-ATPase, represents a sum of four different processes: 1) NA, without synaptic factors, inhibits the Na/K-ATPase; 2) At low SF(a) concentrations NA-dependent Na/K-ATPase activatory mechanism is evident; 3) At high SF(a) concentrations NA-independent Na/K-ATPase is activated; 4) The low-molecular SF(i) protein inhibits the Na/K-ATPase. Regulation of the Na/K-ATPase activity by NA, SF(a) and SF( i), obtained in similar conditions from two weeks old and one year old rats, is different. In older rats SF(i) is characterized with strong Na/K-ATPase inhibition; in younger rats SF(i) does not change the Na/K-ATPase activity. The NA- and SF(i) -dependent inhibition and activation ratio is different in young and elder rats. In two week olds NA/SF(i) activatory mechanism is stronger, while in one year olds NA-dependent inhibition of the Na/K-ATPase is prevailing. These experimental data indicate that regulation of the Na/K-ATPase activity has an important role in synaptic transmission and that this process has noteworthy, albeit presently unknown, functional importance in integrative activity of the brain.     

Synthesis and assembly of functional mammalian Na,K-ATPase in yeast.

The yeast Saccharomyces cerevisiae was investigated as an in vivo protein expression system for mammalian Na,K-ATPase. Unlike animal cells, yeast cells lack endogenous Na,K-ATPase. Expression of high affinity ouabain binding sites, ouabain-sensitive ATPase activity, or ouabain-sensitive p-nitrophenylphosphatase activity in membrane fractions of yeast cells was observed to require the expression of both alpha subunit and beta subunit polypeptides of Na,K-ATPase in the same cell. High affinity ouabain binding sites are also expressed at the cell surface of intact yeast cells containing both the alpha subunit and the beta subunit of Na,K-ATPase. These observations demonstrate that both the alpha subunit and the beta subunit of Na,K-ATPase are required for the expression of functional Na,K-ATPase activity and that yeast cells can correctly assemble this oligomeric membrane protein and transport it to the cell surface.     

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.     

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

The heterodimeric Na,K-ATPase has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. We previously showed that the Drosophila Na,K-ATPase is required for septate junction (SJ) formation and that of the three beta-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. Here we show that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, the beta-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimeric beta-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different alpha-subunit isoforms, with only some isoforms from the major alpha-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPalpha catalytic function do not compromise junctional activity, demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat alpha1 isoform has full junctional activity and can rescue Atpalpha-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function.     

Na,K-ATPase is a target of cigarette smoke and reduced expression predicts poor patient outcome of smokers with lung cancer

Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.     

Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.     

Inhibition of Na,K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine, and oleic acid

The effects and modes of action of certain lipid second messengers and protein kinase C regulators, such as sphingosine, lysophosphatidylcholine (lyso-PC), and oleic acid, on Na,K-ATPase and sodium pump were examined. Inhibition of purified rat brain synaptosome Na,K-ATPase by these lipid metabolites, unlike that by ouabain, was subject to membrane dilution (i.e. inhibition being counteracted by increasing amounts of membrane lipids). Kinetic analysis, using the purified enzyme, indicated that sphingosine and lyso-PC were likely to interact, directly or indirectly, with Na+-binding sites of Na,K-ATPase located at the intracellular face of plasma membranes, a conclusion also supported by studies on Na,K-ATPase and 22Na uptake using the inside-out vesicles of human erythrocyte membranes. The studies also showed that ouabain (but not sphingosine and lyso-PC) increased the affinity constant (K0.5) for K+, whereas sphingosine and lyso-PC (but not ouabain) increased K0.5 for Na+. Sphingosine and lyso-PC inhibited 86Rb uptake by intact human leukemia HL-60 cells at potencies comparable to those for inhibitions of purified Na,K-ATPase and protein kinase C. It is suggested that Na,K-ATPase (sodium pump) might represent an additional target system, besides protein kinase C, for sphingosine and possibly other lipid second messengers.     

Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.     

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.