Non-uniform expression of α subunit isoforms of the Na/K pump in rat dorsal root ganglia neurons

Tissue sections and antibodies selectively recognizing isoforms of the α subunit of the Na⁺/K⁺ pump were used to determine the expression of α₁, α₂ and α₃ pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the α₂ isoform of the Na⁺/K⁺ pump. The α₁ isoform of the Na⁺/K⁺ pump was found in most (77±4%) studied DRG neurons, regardless of cell size. Only 16±7% of the neurons expressed a detectable level of the α₃ Na⁺/K⁺ pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the α₃ positive DRG neurons about 75% coexpressed the α₁ isoform of the Na⁺/K⁺ pump. These data show that the expression of the protein of the α subunit isoforms of the Na⁺/K⁺ pump is not uniform throughout the population of DRG neurons and that α₁ is the predominant isoform in the plasma membrane of these neurons.     

Inhibition of alpha 2/alpha 3 sodium pump isoforms potentiates glutamate neurotoxicity.

Excessive stimulation of neurons by glutamic acid initiates a destructive cascade of ion fluxes, cellular swelling, and death. Homeostatic mechanisms which rectify these disturbances depend largely upon transmembrane ion gradients maintained by Na+,K(+)-ATPase (NaP). We proposed that the neurotoxicity of glutamate is enhanced when the NaP capacity is exceeded, and therefore, that the degree of neuronal death varies inversely with endogenous NaP activity. To test this concept, we directly reduced NaP activity in cultured rat telencephalic cells using either the specific inhibitor ouabain, or dcAMP, and assessed whether these treatments increased glutamate-induced neuronal death. Since rodent NaP catalytic subunits possess both low (alpha 1) and high (alpha 2/alpha 3) affinity for ouabain, we were able to inhibit selectively the alpha 2 (principally glial) and alpha 3 (neuronal) catalytic subunits without affecting the alpha 1 isoform. Brief exposures (5-60 min) to high ouabain concentrations (1-10 mM), which blocks the activity of all three catalytic subunits, killed differentiated neurons but spared glia. In contrast, differential inhibition of the alpha 2/alpha 3 isoforms (by 1 microM ouabain) was not of itself toxic, but produced a supersensitivity to glutamate. [3H]Ouabain binding studies confirmed that the glutamate neurotoxicity observed varied inversely with the degree of NaP inhibition. Further, this relationship was not absolutely dependent upon ouabain, since reductions in alpha 2/alpha 3 pump activity induced by dcAMP also amplified glutamate toxicity. We conclude that inhibition of neuronal NaP with high affinity for ouabain is not lethal to unstimulated cells, but markedly increases susceptibility to glutamate excitotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between excitotoxins and the Na/K-ATPase inhibitor ouabain in causing neuronal lesions in the rat hippocampus

A possible indirect role of glutamate in causing the neuronal death found after intracerebral administration of a low dose of ouabain (0.1 nmol) has been evaluated. This dose of ouabain produces a more extensive neuronal lesion than those caused by glutamate receptor agonists (kainate at an equimolar dose, or NMDA (N-methyl-d-aspartate) at a 50-fold higher dose). The selective glutamate receptor antagonists, dizocilpine (MK-801) and NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), in doses which blocked the direct toxicity of glutamate receptor agonists acting on either the NMDA and non-NMDA classes of glutamate receptor, failed to provide more than a minor protection against ouabain-induced peuronal death in the rat dorsal hippocampus. In contrast, the non-selective glutamate receptor antagonist, kynurenate (100 nmol) reduced the damage by around 70%. The difference in neuroprotection found between the glutamate receptor antagonists suggests that kynurenate may protect by a non-glutamatergic mechanism. Co-administration of ouabain and glutamate receptor agonists (kainate, NMDA or glutamate) resulted in additive rather than synergistic damage to hippocampal neurons. The results suggest that in vivo, ouabain and excitotoxins probably cause neuronal death by independent mechanisms.     

Dependence of Na,K-ATPase and electrogenic component of Em in cultured myotubes on cell fusion

This study was undertaken in order to determine the relation among cell fusion, [3H]ouabain binding and the membrane potential (Em) of cultured rat skeletal muscle. The amount of ouabain bound and the Em both increased with age, the increases being most dramatic following fusion. Inhibition of fusion prevented the developmental increases in both properties of cultured muscle. After fusion, the size of the electrogenic component of Em, determined by the decrease in Em produced by ouabain within 5-10 min, increased independent of the age at which fusion occurred. It is concluded that the increase in Em with age depends on postfusion appearance and activity of Na,K-ATPase.     

The new targets of ouabain in retinal interneurons of Sprague-Dawley rats

Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na⁺, K⁺-ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in an animal model of mania

We evaluated Na⁺,K⁺-ATPase activity in hippocampus of rats submitted to an animal model of mania which included the use of lithium and valproate. In the acute treatment, amphetamine or saline was administered to rats for 14 days, between day 8 and 14, rats were treated with lithium, valproate or saline. In the maintenance treatment, rats were treated with lithium, valproate or saline, between day 8 and 14, amphetamine or saline were administered. Locomotor activity was assessed by open field test and Na⁺,K⁺-ATPase activity was measured. Our results showed that mood stabilizers reversed and prevented amphetamine-induced behavioral effects. Moreover, amphetamine (acute treatment) increased Na⁺,K⁺-ATPase activity, and administration of lithium or valproate reversed this effect. In the maintenance treatment, amphetamine increased Na⁺,K⁺-ATPase activity in saline-pretreated rats. Amphetamine administration in lithium- or valproate-pretreated animals did not alter Na⁺,K⁺-ATPase activity. The findings suggest that amphetamine-induced hyperactivity may be associated with an increase in Na⁺,K⁺-ATPase.     

Inhibition of Na, K-ATPase activity in rat striatum by guanidinoacetate

The aim of this work was to investigate the effect of guanidinoacetate (GAA), the principal metabolite accumulating in guanidinoacetate methyltransferase (GAMT)-deficiency, on Na(+), K(+)-ATPase, Mg(2+)-ATPase and acetylcholinesterase (AChE) activities in striatum of young rats. We also studied the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by guanidinoacetate. Guanidinoacetate did not alter acetylcholinesterase and Mg(2+)-ATPase activities, but significantly inhibited Na(+), K(+)-ATPase activity. The apparent K(m) and V(max) of Na(+), K(+)-ATPase for ATP as substrate were 0.20mM and 0.82nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. K(i) value was 7.18mM, and the inhibition was of the uncompetitive type. The results also showed a competition between guanidinoacetate and argininic acid (AA), suggesting a common binding site for the guanidino compounds (GC) on the enzyme. It is proposed that Na(+), K(+)-ATPase inhibition by guanidinoacetate may be one of the mechanisms involved in the neuronal dysfunction observed in GAMT-deficiency and in other diseases which accumulate guanidinoacetate.     

Rapid decrease of high affinity ouabain binding sites in hippocampal CA1 region following short-term global cerebral ischemia in rat

High affinity [3H]ouabain binding was examined in the hippocampal CA1 region and frontal cortex of rats subjected to 5 min complete cerebral ischemia in a clinical death model, and to subsequent resuscitation. A decrease of Bmax directly after ischemia and its further gradual decrease during 120 min of reperfusion were noted in the ischemia-vulnerable CA1 region, whereas no change of Bmax was observed in frontal cortex. The apparent Kd constant showed insignificant fluctuations in either of the two brain regions. Since ouabain binds with high affinity to the neuronal (alpha +)-form of Na+/K+-ATPase, the results indicate a rapid enzyme loss in CA1 neurons. The high affinity ouabain binding test proved to be a sensitive detector of premorphological changes in nerve cell membranes in ischemia.     

Erythrocyte membrane sodium — potassium adenosine triphosphatase activity in affective disorders

Summary Erythrocyte membrane Na⁺,K⁺-ATPase activity was studied in drug naive patients with bipolar (BP) mania (n=62) and unipolar (UP) depression (n=60) and normal controls (n=66). Compared to controls there was a significantly decreased Na⁺,K⁺-ATPase activity in UP depressives but no change in BP manics. However, lithium treatment caused a significant increase in Na⁺,K⁺-ATPase activity although there was no correlation between plasma lithium levels and enzyme activity. Plasma cortisol correlated inversely with Na⁺,K⁺-ATPase in UP depressives. Interestingly, the lithium responders [<50% Beck Rafaelson's Mania Rating Scale (BRMS) score] showed a significant increase in Na⁺,K⁺-ATPase activity compared to lithium nonresponders (>50% BRMS score). These observations indicate that monitoring of Na⁺,K⁺-ATPase activity during lithium therapy is useful to predict a therapeutic response.     

Evidence for the presence of ‘Ouabain like’ compound in human cerebrospinal fluid

Material extracted and partially purified from human cerebrospinal fluid (CSF) is capable of: a, inhibiting [3H]ouabain binding to rat brain synaptosomes; b, inhibiting the activity of purified pig kidney Na+,K+-ATPase; and c, inhibiting ouabain sensitive induced 86Rb influx to tissue cultured fibroblasts. These results demonstrate the existence of an 'ouabain like' compound (OLC) in human CSF, and are consistent with the hypothesis of the function of this compound as a neuromodulator.     

Effects of inhibitors of Na,K-ATPase on the membrane potentials and neurotransmitter efflux in rat brain slices

The potassium potential EK, of rat brain slices was estimated by determining the uptake of 86Rb+. The ERb was the same for slices prepared from five rostral brain regions, the average value being 66.4 mV. The ERb values in the presence of 20 microM ouabain were only slightly lower than the resting values; increasing concentrations of ouabain above 20 microM resulted in a graded depolarization in all five brain regions. High concentrations (1 mM) of two other inhibitors of Na+,K+-ATPase, dihydro-ouabain and strophanthidin, produced no more depolarization than did 20 microM ouabain. Competitive binding studies indicated that the differential effects were due to the relative binding to brain slices. Erythrosin B, an inhibitor of Na+,K+-ATPase, had no measurable effect on ERb. Intermediate concentrations of the Na+/H+ ionophore monensin slightly hyperpolarized striatal slices, whereas the same monensin concentrations plus 20 microM ouabain, 1 mM strophanthidin or 70 microM erythrosin B resulted in marked depolarization. Measurement of the membrane potential via uptake of methyltriphenylphosphonium cation indicated that ERb was indeed a valid estimation of the membrane potential. EK was measured directly by monitoring 42K+ uptake in striatal slices and was found to be essentially identical to ERb. Uptake of 22Na+ was consistent with the values for ERb or EK. Several conditions that resulted in little or no measurable depolarization of striatal slices did induce efflux of exogenously loaded GABA and dopamine; these conditions included 20 microM ouabain, 1 mM dihydro-ouabain or strophanthidin, and 70 microM erythrosin B. Neurotransmitter efflux in the absence of general cell depolarization was not accompanied by altered rates of respiration or decreased ATP levels.     

Depression of cortical Na, K-ATPase acticvity in rats exposed to hyperbaric oxygen

Extracellular single unit recordings were used to study inhibitory synaptic responses evoked from preoptic-anterior hypothalamic neurones following arcuate-ventromedial stimulation. Intravenously administered methohexitone, pentobarbitone and thiopentone increased the duration of inhibitory synaptic responses by up to 400%. Submaximal responses to iontophoretically applied GABA but not glycine were also potentiated. Recovery from the actions of the short acting barbiturates was observed.     

Inhibition of Na, K-ATPase activity in rat striatum by the metabolites accumulated in Lesch–Nyhan disease

In the present study, we investigated the in vitro effect of hypoxanthine, xanthine and uric acid, metabolites accumulating in tissue of patients with Lesch-Nyhan disease, on Na(+), K(+)-ATPase activity in striatum of neonate rats. Results showed that all compounds significantly inhibited Na(+), K(+)-ATPase activity. We also studied the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by hypoxanthine. The apparent K(m) and V(max) of Na(+), K(+)-ATPase activity for ATP as the substrate and hypoxanthine as the inhibitor were 0.97 mM and 0.69 nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. K(i)-value was 1.9 microM, and the inhibition was of the non-competitive type. We also observed that the inhibitory effects of hypoxanthine, xanthine and uric acid probably occur through the same mechanism, suggesting a common binding site for these oxypurines on Na(+), K(+)-ATPase. Therefore, it is conceivable that inhibition of brain Na(+), K(+)-ATPase activity may be involved at least in part in the neuronal dysfunction characteristic of patients with Lesch-Nyhan disease.     

Presynaptic glutamate receptor — possible involvement of a K channel

Intra-axonal recording was made from the excitatory axon of the lobster walking leg near the nerve terminal and the effects of L-glutamate were studied. Topical application of glutamate to the synapse produced hyperpolarization in the presynaptic membrane with increase in conductance. The glutamate-induced hyperpolarization was reversed to a depolarization at about -100 mV. The reversal potential was not significantly changed by altering the external Cl- but was shifted to more positive values by increasing the external K+. A spider toxin (JSTX), which specifically blocks the postsynaptic glutamate receptor, failed to block the presynaptic glutamate potential. The results suggest that the presynaptic membrane of the lobster neuromuscular synapse has a different type of glutamate receptor from those in the postsynaptic membrane.     

Transport pathways for lithium ions in neuroblastoma × glioma hybrid cells at ‘therapeutic’ concentrations of Li

The pathways of Li⁺ transport in neuroblastoma × glioma hybrid cells were studied at 2 mM external Li⁺. Five components of Li⁺ transport were identified. (1) A Na⁺-dependent Li⁺ countertransport system mediating Li⁺ transport in both directions across the plasma membrane. This transport pathway is insensitive to ouabain or external K⁺. It shows trans-stimulation (i.e. acceleration of Li⁺ extrusion by external Na⁺ and stimulation of Li⁺ uptake by internal Na⁺) and cis-inhibition (i.e. reduction of Li⁺ uptake by external Na⁺). (2) The Na⁺K⁺ pump mediates Li⁺ uptake but not Li⁺ release in cells with physiological Na⁺ and K⁺ content. Li⁺ uptake by the pump in choline media is inhibited by both external Na⁺ and K⁺. In Na⁺ media, external K⁺ exhibits a biphasic effect: in concentrations up to about 1 mM, K⁺ accelerates, and at higher concentrations, K⁺ inhibits, Li⁺ uptake by the pump. (3) Li⁺ can enter the voltage-dependent Na⁺ channel. Li⁺ uptake through this pathway is stimulated by veratridine and scorpion toxin, the stimulation being blocked by tetrodotoxin. Residual pathways comprise (4) a saturable component, which is comparable to basal Na⁺ uptake, and (5) a ouabain-resistant component promoting Li⁺ extrusion against an electrochemical gradient in choline media. The mechanisms for Li⁺ extrusion described here possibly explain how neuronal cells maintain the steady-state ratio of internal to external Li⁺ below 1 during chronic exposure to 1–2 mM external Li⁺.     

Brain Na, K-ATPase activity possibly regulated by a specific serotonin receptor

Na⁺, K⁺-ATPase activity in 6 regions of adult brain was measured after incubation with varying concentrations of serotonin. A concentration-dependent increase in enzyme activity was observed in 4 regions, with cerebral cortex and cerebellum showing the largest response. These results together with previous ones suggest that serotoninmmodulates brain Na⁺, K⁺-ATPase activity through a specific receptor located in target neurons or glial cells.     

Influence of lactate accumulation on Na,K-ATPase activity of ischemic and post-ischemic brain

In this study the cerebral Na+, K+-ATPase activity as well as selected parameters of oxidative metabolism and electrophysiological function were assessed in normoglycemic and hyperglycemic rats which were exposed to ischemia produced by electrocautery of the vertebral arteries and reversible occlusion of the carotid arteries. In hyperglycemic animals 0.5 h of ischemia was associated with massive accumulation of lactate (34 mumol X g-1) and enhanced Na+, K+-ATPase activity (116% control), whereas normoglycemic animals showed more moderate lactate accumulation (17 mumol X g-1) and normal Na+, K+-ATPase activity (102% control). In normoglycemic animals release of the carotid clamps and recirculation for 0.5-1.5 h was associated with a normalization of the lactate levels and a decrease in Na+, K+-ATPase activity (68-72% control). Restituted hyperglycemic animals showed metabolic changes which seemed related to the blood pressure, with hypotensive hyperglycemic animals showing continuing massive lactacidosis (30-35 mumol X g-1) and enhanced Na+, K+-ATPase activity (108-110% control), whereas normotensive hyperglycemic animals showed progressive decreases in lactate level (14-20 mumol X g-1) and normal or mildly suppressed Na+, K+-ATPase activity (88-97% control). These patterns of change suggest that the reperfusion of the post-ischemic hyperglycemic-hyperlactacidotic brain was inadequate or non-homogeneous.     

Inhibition of purified (Na,K)-ATPase activity from porcine cerebral cortex by NO generating drugs

We tested the effects of several nitric oxide (NO) generating compounds on the activity of sodium-potassium adenosine 5′-triphosphatase [(Na⁺,K⁺)-ATPase] purified from porcine cerebral cortex. Sodium nitroprusside (SNP),S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1) and (dl)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) inhibited the (Na⁺,K⁺)-ATPase activity dose dependently. Superoxide dismutase, a NO scavenger, and sulfhydryl (SH) compounds, reduced-form glutathione (rGSH) and dithiothreitol (DTT), prevented the inhibitory action of SNAP, SIN-1 and NOR 3 but not of SNP, when applied simultaneously with NO generating compounds, and this enzyme inhibition could be reactivated by the incubation with these SH compounds but not with SOD. The inhibitory action by SNP was magnified by simultaneous application of DTT. These results suggest that NO generating compounds, SNAP, SIN-1 and NOR 3 but not SNP, may release NO or NO-derived products and may inhibit (Na⁺,K⁺)-ATPase activity by interacting with a SH group at the active site of the enzyme.     

Modulation of glial glutamate transport through cell interactions with the extracellular matrix

Glial glutamate transport plays a pivotal role in maintaining glutamate homeostasis in the central nervous system. Expression of glutamate transporters is highly regulated during brain development, and a number of pathological conditions are associated with deficits in expression and/or function of glutamate transports. While several soluble factors have been shown to regulate the expression of glutamate transporter, the contribution of cell-cell interaction and cell-environmental interaction in the regulation of glutamate transport is unknown. Extracellular matrix (ECM) molecules are essential components in cell-cell and cell-environmental interactions, and the ECM has been shown to play critical role in normal development and during brain pathogenesis. We, therefore, investigated the possibility that ECM molecules may regulate astrocytic glutamate transport. Therefore, we cultured rat cortical astrocytes with different ECMs and determined expression levels of the two astrocytic glutamate transporters GLT-1 and GLAST by Western Blot and determined transporter activity through measurements of 3H-D-aspartate uptake. Astrocytes grown on poly-ornithine or poly-D/L-lysine showed approximately two-fold higher GLT-1 expression than sister cells grown on plastic dishes without ECM. Naturally occurring ECM's, including laminin and collagen, showed a dose-dependent regulation of GLT-1 protein expression. These effects were specific for GLT-1 as GLAST expression was unaffected by different ECMs. Surprisingly, however, none of the examined ECMs altered the apparent glutamate uptake activity. In probing blots side-by-side for expression of Na(+)/K(+)-ATPase, we found that ECMs affected expression of Na(+)/K(+)-ATPase and GLT-1 in a reciprocal fashion. Poly-ornithine, for example, enhanced GLT-1 expression, but reduced expression of Na(+)/K(+)-ATPase. Na(+) transport may, thus, be a limiting factor for glutamate uptake.     

Effect of repeated restraint stress and clomipramine on Na/K-ATPase activity and behavior in rats

Activation of the limbic-hypothalamic-pituitary-adrenal axis (LHPA) and the release of glucocorticoids are fundamental for the adaptive response and immediate survival of an organism in reaction to acute stimuli. However, high levels of glucocorticoids in the brain may produce neuronal injury and a decrease of Na⁺/K⁺-ATPase activity, with effects on neurotransmitter signaling, neural activity, as well as the whole animal behavior. Clomipramine is a tricyclic antidepressant that inhibits the reuptake of serotonin and norepinephrine by indirect actions on the dopaminergic system and LHPA axis. Its chronic use increases the body's ability to cope with stress; however, high doses can potentiate its side effects on memory, learning, and sensory motor function. The purpose of the present study was to compare the effect of repeated restraint stress and clomipramine treatment on Na⁺/K⁺-ATPase activity and on the behavior of male rats. Changes in the behavioral response were evaluated by measuring the memory, learning, anxiety, and exploratory responses. Our results showed that exposure to repeated restraint stress reduced levels of Na⁺/K⁺-ATPase in brain structures and changed short and long-term memory, learning, and exploratory response when compared to the control group. Exposure to clomipramine treatment increased anxiety levels and reduced Na⁺/K⁺-ATPase activity in the cerebral cortex as well as short term memory, learning, and exploratory response. In conclusion, the present results provide additional evidence concerning how repeated restraint stress and clomipramine chronically administered at higher dose levels affect the neural activity and behavior of male rats.