Search

Intracerebroventricular (ICV) infusion of NaCl mimics the effects of a high-salt diet in salt-sensitive hypertension, raising the sodium concentration in the cerebrospinal fluid (CSF [Na]) and subsequently increasing the concentration of an endogenous ouabain-like substance (OLS) in the brain. The OLS, in turn, inhibits the brain [Na.sup.+]-[K.sup.+]-ATPase, causing increases in the activity of the brain renin-angiotensin system (RAS) and blood pressure. The [Na.sup.+]-[K.sup.+]-ATPase [alpha] (catalytic)-isoform(s) that mediates the pressor response to increased CSF [Na] is unknown, but it is likely that one or more isoforms that bind ouabain with high affinity are involved (e.g., the [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2]- and/or [[alpha].sub.3]-subunits). We hypothesize that OLS-induced inhibition of the [[alpha].sub.2]-subunit mediates this response. Therefore, a chronic reduction in [[alpha].sub.2] expression via a heterozygous gene knockout ([[alpha].sub.2] +/-) should enhance the pressor response to increased CSF [Na]. Intracerebroventricular (ICV) infusion of artificial CSF containing 0.225 M NaCl increased mean arterial pressure (MAP) in both wild-type (+/+) and [[alpha].sub.2] +/- mice, hut to a greater extent in [[alpha].sub.2] +/-. Likewise, the pressor response to ICV ouabain was enhanced in [[alpha].sub.2] +/- mice, demonstrating enhanced sensitivity to brain [Na.sup.+]-[K.sup.+]-ATPase inhibition per se. The pressor response to ICV ANG I but not ANG II was also enhanced in [[alpha].sub.2] +/- vs. [[alpha].sub.2]+/+ mice, suggesting an enhanced brain RAS activity that may be mediated by increased brain angiotensin converting enzyme (ACE). The latter hypothesis is supported by enhanced ACE ligand binding in the organum vasculosum laminae terminalis. These studies demonstrate that chronic downregulation of [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2]-isoform expression by heterozygous knockout increases the pressor response to increased CSF [Na] and activates the brain RAS. Since these changes mimic those produced by the endogenous brain OLS, the brain [[alpha].sub.2]-isoform may be a target for the brain OLS during increases in CSF [Na], such as in salt-dependent hypertension. [[alpha].sub.2]-isoform; gene knockout; sodium chloride; brain ouabain-like substance; brain renin-angiotensin system; intracerebroventricular infusion

An interesting feature of [Na.sup.+]-[K.sup.+]-ATPase is that it contains four isoforms of the catalytic [alpha]-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the [[alpha].sub.1]- and [[alpha].sub.2]-isoforms. While knockout mice demonstrated the importance of the [[alpha].sub.1]- and [alpha].sub.2]-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the [Na.sup.+]-[K.sup.+]-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the [Na.sup.+]-[K.sup.+]-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant [[alpha].sub.2]-isoform of [Na.sup.+]-[K.sup.+]-ATPase. Interestingly, animals with the ouabain-sensitive [[alpha].sub.1]-isoform and a ouabain-resistant [[alpha].sub.2]-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the [Na.sup.+]-[K.sup.+]-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH. ouabain; adrenocorticotropic hormone; blood pressure regulation; cardiotonic steroids

The [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2]-subunit isoform modulates contractility in the perinatal mouse diaphragm. Am J Physiol Cell Physiol 287: C1300-C1310, 2004. First published July 14, 2004: doi:10.1152/ajpcell.00231.2004.--This study uses genetically altered mice to examine the contribution of the [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2] catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The [[alpha].sub.2] protein is reduced by 38% in [[alpha].sub.2]-heterozygous and absent in [[alpha].sub.2]-knockout mice, and [[alpha].sub.1]-isoform is upregulated 1.9-fold in [[alpha].sub.2]-knockout. Resting potentials are depolarized by 0.8-4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced [[alpha].sub.2] content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the [[alpha].sub.2]-isoform at a step after membrane excitation, which cannot be restored simply by increasing [[alpha].sub.1] content. [Na.sup.+]/[Ca.sup.2+] exchanger expression decreases in parallel with [[alpha].sub.2]-isoform, suggesting that [Ca.sup.2+] extrusion is affected by the altered [[alpha].sub.2] genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum [Ca.sup.2+]-ATPase, phospholamban, or plasma membrane [Ca.sup.2+]-ATPase. These results demonstrate that the [Na.sup.+]- [K.sup.+]-ATPase [[alpha].sub.1]-isoform alone is able to maintain equilibrium [K.sup.+] and [Na.sup.+] gradients and to substitute for [[alpha].sub.2]-isoform in most cellular functions related to excitability and force. They further indicate that the [[alpha].sub.2]-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2] catalytic subunit; heterozygous mice; knockout mice; resting potential

The effect of the ionophore amphotericin B on sodium transport in the fiber cells of porcine lens epithelium was investigated by measuring the Na+-K+-adenosinetriphosphatase (ATPase) activity of the lens cell plasma membranes. The membranes showed higher Na+ concentration and a two-fold increase in Na+-K+-ATPase activity after 24 hours following exposure to 5 or 10 mumolar of amphotericin B. The results suggest that the ionophore amphotericin B increases Na+-K+-ATPase activity in porcine lens epithelium.

The Na-K-ATPase, which maintains the [Na.sup.+] and [K.sup.+] gradients across the plasma membrane, can play a major role in modulation of skeletal muscle contractility. Although both [[Alpha].sub.1]- and [[Alpha].sub.2]-isoforms of the Na-K-ATPase are expressed in skeletal muscle, the physiological significance of these isoforms in contractility is not known. Evaluation of the contractile parameters of mouse extensor digitorum longus (EDL) was carried out using gene-targeted mice lacking one copy of either the [[Alpha].sub.1]- or [[Alpha].sub.2]-isoform gene of the Na-K-ATPase. The EDL muscles from heterozygous mice contain approximately one-half of the [[Alpha].sub.1]- or [[Alpha].sub.2]-isoform, respectively, which permits differentiation of the functional roles of these isoforms. EDL from the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] mouse shows lower force compared with wild type, whereas that from the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] mouse shows greater force. The different functional roles of these two isoforms are further demonstrated because inhibition of the [[Alpha].sub.2]-isoform with ouabain increases contractility of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] EDL. These results demonstrate that the Na-K-ATPase [[Alpha].sub.1]- and [[Alpha].sub.2]-isoforms may play different roles in skeletal muscle contraction. sodium-potassium-adenosinetriphosphatase; extensor digitorum longus muscle; ouabain; muscle fatigue

Sodium and potassium-activated adenosine triphosphatases (Na,KATPase) are ubiquitous, participate in osmotic balance and membrane potential, and are composed of α, β, and γ subunits. The α subunit is required for the catalytic and transport properties of the enzyme and contains binding sites for cations, ATP, and digitalis-like compounds including ouabain. There are four known a isoforms; three that are expressed in the CNS in a regional and cell-specific manner. The α2 isoform is most commonly found in astrocytes, pyramidal cells of the hippocampus in adults, and developmentally in several other neuronal types. Ouabain-like compounds are thought to be produced endogenously in mammals, bind the Na,K-ATPase, and function as a stress-related hormone, however, the impact of the Na,K-ATPase ouabain binding site on neurobehavioral function is largely unknown. To determine if the ouabain binding site of the α2 isoform plays a physiological role in CNS function, we examined knock-in mice in which the normally ouabain-sensitive α2 isoform was made resistant (a2R/R) while still retaining basal Na,KATPase enzymatic function. Egocentric learning (Cincinnati water maze) was impaired in adult α2R/R mice compared to wild type (WT) mice. They also exhibited decreased locomotor activity in a novel environment and increased responsiveness to a challenge with an indirect sympathomimetic agonist (methamphetamine) relative to WT mice. The α2R/R mice also demonstrated a blunted acoustic startle reflex and a failure to habituate to repeated acoustic stimuli. The α2R/R mice showed no evidence of altered anxiety (elevated zero maze) nor were they impaired in spatial learning or memory in the Morris water maze and neither group could learn in a large Morris maze. These results suggest that the ouabain binding site is involved in specific types of learning and the modulation of dopamine-mediated locomotor behavior. [ABSTRACT FROM AUTHOR]

Intracerebroven- tricular (ICV) infusion of NaCl mimics the effects of a high-salt diet in salt-sensitive hypertension, raising the sodium concentration in the cerebrospinal fluid (CSF [Na]) and subsequently increasing the concentration of an endogenous ouabain-like substance (OLS) in the brain. The OLS, in turn, inhibits the brain Na+-K+-ATPase, causing increases in the activity of the brain renin-angiotensin system (RAS) and blood pressure. The Na+-K+-ATPase α (catalytic)-isoform(s) that mediates the pressor response to increased CSF [Na] is unknown, but it is likely that one or more isoforms that bind ouabain with high affinity are involved (e.g., the Na+-K+-ATPase α2- and/or α3-sub- units). We hypothesize that OLS-induced inhibition of the α2-subunit mediates this response. Therefore, α chronic reduction in α2 expression via a heterozygous gene knockout (α2 +/-) should enhance the pressor response to increased CSF [Na]. Intracerebroventricular (ICV) infusion of artificial CSF containing 0.225 M NaCl increased mean arteriai pressure (MAP) in both wild-type (+/+) and α2 +/- mice, but to a greater extent in α2 +/-. Likewise, the pressor response to ICV ouabain was enhanced in α2 +/- mice, demonstrating enhanced sensitivity to brain Na-K-ATPase inhibition per se. The pressor response to ICV ANG I but not ANG II was also enhanced in α2 +/- vs. α2 +/+ mice, suggesting an enhanced brain RAS activity that may be mediated by increased brain angiotensin converting enzyme (ACE). The latter hypothesis is supported by enhanced ACE ligand binding in the organum vasculosum laminae terminalis. These studies demonstrate that chronic downregulation of Na+-K+-ATPase α2-isoform expression by heterozygous knockout increases the pressor response to increased CSF [Na] and activates the brain RAS. Since these changes mimic those produced by the endogenous brain OLS, the brain α2-isoform may be a target for the brain OLS during increases in CSF [Na], such as in salt-dependent hypertension. [ABSTRACT FROM AUTHOR]

Several disorders have been associated with mutations in Na,K-ATPase α isoforms (rapid-onset dystonia parkinsonism, familial hemiplegic migraine type-2), as well as reduction in Na,K-ATPase content (depression and Alzheimer's disease), thereby raising the issue of whether haploinsufficiency or altered enzymatic function contribute to disease etiology. Three isoforms are expressed in the brain: the α1 isoform is found in many cell types, the α2 isoform is predominantly expressed in astrocytes, and the α3 isoform is exclusively expressed in neurons. Here we show that mice heterozygous for the α2 isoform display increased anxiety-related behavior, reduced locomotor activity, and impaired spatial learning in the Morris water maze. Mice heterozygous for the α3 isoform displayed spatial learning and memory deficits unrelated to differences in cued learning in the Morris maze, increased locomotor activity, an increased locomotor response to methamphetamine, and a 40% reduction in hippocampal NMDA receptor expression. In contrast, heterozygous α1 isoform mice showed increased locomotor response to methamphetamine and increased basal and stimulated corticosterone in plasma. The learning and memory deficits observed in the α2 and α3 heterozygous mice reveal the Na,K-ATPase to be an important factor in the functioning of pathways associated with spatial learning. The neurobehavioral changes seen in heterozygous mice suggest that these mouse models may be useful in future investigations of the associated human CNS disorders. [ABSTRACT FROM AUTHOR]

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic α-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the α1- and α2-isoforms. While knockout mice demonstrated the importance of the α1- and α2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant α2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive α1-isoform and a ouabain-resistant α2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH. [ABSTRACT FROM AUTHOR]

The Na,K-ATPase transports three sodium ions out of the cell and two potassium ions into the cell using ATP hydrolysis for energy. The ion gradient formed by the Na,K-ATPase contributes to the resting membrane potential, maintains cellular excitability and is important for glucose and amino acid uptake in the cell. The α1 catalytic isoform is expressed in virtually all cell types. We have previously examined cardiac physiology of mice lacking one copy of the α1 isoform gene of the Na,K-ATPase. The observation of reduced cardiac contractility in the α1 heterozygous mice was unexpected since mice heterozygous for the α2 isoform displayed enhanced cardiac contractility similar to what would be observed with cardiac glycoside treatment. We further examined hearts from α1 heterozygous mice to identify genomic responses to reduced Na,K-ATPase capacity. Using microarray analyses, we identified groups of genes whose expressions were perturbed in the α1 heterozygous hearts compared to wild-type. Known functional relationships of these genes suggest that multiple biological pathways are altered by α1 hemizygosity including activation of the renin-angiotensin system, changes in genes of energy metabolism and transport and elevated brain natriuretic peptide. This suggests that Na,K-ATPase α1 isoform activity may be required in numerous cellular processes. [ABSTRACT FROM AUTHOR]

This study uses genetically altered mice to examine the contribution of the Na+-K+-ATPase α2 catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The α2 protein is reduced by 38% in α2-heterozygous and absent in α2-knockout mice, and α1-isoform is upregulated 1.9-fold in α2-knockout. Resting potentials are depolarized by 0.8-4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced α2 content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the α2-isoform at a step after membrane excitation, which cannot be restored simply by increasing α1 content. Na+/Ca2+ exchanger expression decreases in parallel with α2-isoform, suggesting that Ca2+ extrusion is affected by the altered α2 genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, or plasma membrane Ca2+-ATPase. These results demonstrate that the Na+- K+-ATPase α1-isoform alone is able to maintain equilibrium K+ and Na+ gradients and to substitute for α2-isoform in most cellular functions related to excitability and force. They further indicate that the α2-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. [ABSTRACT FROM AUTHOR]

This study examined the developmental expression of Na,K-ATPase α- and β-subunit isoforms in different skeletal muscles of the mouse, and the relationship of Na,K-ATPase α2 isoform expression to the developing transverse tubules (t-tubules). We measured Na,K-ATPase and dihydropyridine receptor (DHPR) mRNA and protein in the diaphragm and hindlimb muscles from embryonic day 18.5 (E18.5) to 6 weeks postnatal, using DHPR expression to mark the timing of t-tubule formation. The Na,K-ATPase subunits showed developmental age-dependent and muscle-specific expression that was controlled by both transcriptional and post-transcriptional mechanisms. The α1 isoform is expressed at more constant levels in both diaphragm and hindlimb muscles, while the α2 and β2 isoforms increase postnatally and show greater muscle variation. β1 is the sole expressed β-subunit in the diaphragm throughout development, and in the hindlimb muscles at birth. The Na,K-ATPase α2 subunit is expressed during development when the t-tubules form. These results suggest that the α2 isoform may serve, in part, a physiological role in the muscle t-tubules. [ABSTRACT FROM AUTHOR]

Presents a study which examined the role of sodium-potassium adenosine triphosphatase in skeletal muscle contraction. Materials and methods; Results; Discussion.

Investigates the roles of the alpha-1 (A1) and alpha-2 (A1) isoforms of sodium-potassium-adenosine triphosphatase (Na-K-ATPase) in skeletal muscle contractility. Quantitative analysis of A1 and A2-protein levels in mouse extensor digitorum longus; Physiological functions of the two isoforms in heart.

The alpha(3) isoform of Na(+),K(+)-ATPase is uniquely expressed in afferent and efferent neurons innervating muscle spindles in the peripheral nervous system (PNS) of adult rats, but the distribution pattern of this isoform in other species has not been investigated. We compared expression of alpha(3) Na(+),K(+)-ATPase in lumbar dorsal root ganglia (DRG), spinal roots, and skeletal muscle samples of amphibian (frog), reptilian (turtle), avian (pigeon and chicken), and mammalian (mouse and human) species. In all species studied, the alpha(3) Na(+),K(+)-ATPase isoform was nonuniformly expressed in peripheral ganglia and nerves. In spinal ganglia, only 5-20% of neurons expressed this isoform, and, in avian and mammalian species, these alpha(3) Na(+),K(+)-ATPase-expressing neurons belonged to a subpopulation of large DRG neurons. In ventral root fibers of pigeons, mice, and humans, the alpha(3) Na(+),K(+)-ATPase was abundantly expressed predominantly in small myelinated axons. In skeletal muscle samples from turtles, pigeons, mice, and humans, alpha(3) Na(+),K(+)-ATPase was detected in intramuscular myelinated axons and in profiles of nerve terminals associated with the equatorial and polar regions of muscle spindle intrafusal fibers. These results show that the expression profiles for alpha(3) Na(+),K(+)-ATPase in the peripheral nervous system of a wide variety of vertebrate species are similar to the profile of rats and suggest that stretch receptor-associated expression of alpha(3) Na(+),K(+)-ATPase is preserved through vertebrate evolution.

Experiments were conducted to test the effect of 1 microM ouabain, an Na,K-ATPase inhibitor, on capacitative calcium entry (CCE) and calcium responses elicited by ATP in rat optic nerve astrocytes. In the rat, 1 microM ouabain is sufficient to inhibit the alpha2 Na,K-ATPase, but not the alpha1. Immortalized astrocytes derived from Na,K-ATPase alpha2 homozygous knockout (KO) mice and wild-type (WT) littermates were also used. Cytosolic calcium and sodium concentrations were measured using Fura-2 and SBFI, respectively. The magnitude of the increase in cytosolic calcium concentration during CCE was significantly greater in rat astrocytes exposed to 1 microM ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 microM ouabain, a significantly greater calcium response to ATP was observed. 1 microM ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase alpha2 and alpha1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase alpha1 (rat kidney). In intact astrocytes, 1 microM ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na,K-ATPase alpha2 KO astrocytes, the calcium increase during CCE was significantly higher than in WT cells, as was the magnitude of the calcium response to ATP. In KO astrocytes, but not WT, the cytosolic calcium increase during CCE was insensitive to 1 microM ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase alpha2 isoform has the potential to change calcium signaling and CCE., (Copyright 2003 Wiley-Liss, Inc.)