Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

A QSAR study on a series of simplified digitalis-like compounds acting on Na+,K(+)-ATPase

Among the cardiotonics (agents against congestive heart failure), the most important group is of the digitalis cardiac glycosides, but since these compounds suffer from a low therapeutic index, attention has been paid to investigating safer cardiotonic agents through the inhibition of Na+,K(+)-ATPase, the mechanism by which the digitalis cardiac glycosides elicit their action. Recently, a series of perhydroindenes were studied for their Na+,K(+)-ATPase inhibition activity. We report here a QSAR study on them to investigate the physicochemical and structural properties of the molecules that govern their activity in order to rationalize the structural modification to have more potent drugs. A multiple regression analysis reveals a significant correlation between the Na+,K(+)-ATPase inhibition activity of the compounds and Kier's first order valence molecular connectivity index of their R5-substituents and some indicator parameters, suggesting that the R5-substituents of the compounds containing atoms with low valence and high saturation and the R1-substituents having =N-O- moiety will be conducive to the activity.     

Evidence that mammalian lignans show endogenous digitalis-like activities

Enterolactone, a lignan that has been identified in biological samples from man and several mammals, shares with ascorbic acid and cardiac glycosides a gamma-butyrolactone. It displaces 3H-ouabain from its binding sites on cardiac digitalis receptor and inhibits, dose dependently, the Na+, K+-ATPase activity of human and guinea-pig heart. The time dependence of this inhibition resembles that of dihydroouabain, a cardiac glycoside in which the lactone ring does not contain conjugated double bonds. The active concentrations of enterolactone as inhibitor of Na+,K+-ATPase are in the 10(-4) M range and, at those concentrations, the cross-reactivity with antidigoxin antibodies is low. Lignans may contribute to the putative digitalis-like activity found in tissues, blood and urine of several mammals including man.     

The heterogeneity of the Na+, K(+)-ATPase ouabain sensitivity in microsomal membranes of rat colon smooth muscles

The dose dependence of the Na+, K(+)-ATPase ouabain inhibition in the rat colon smooth muscle permeabilized microsomes has been analyzed according to the model of two independent binding sites of inhibitor to determine the activity of separate molecular forms of the enzyme that differ by affinity for cardiac glycosides. The two-phase inhibition curve with moderate content of the high-affinity activity component was revealed. The apparent inhibition constant of the low-affinity component corresponds to the value for the rat kidney microsomal Na+, K(+)-ATPase (alpha1-isoform). The specific role of the alpha2- and alpha1- Na+, K(+)-ATPase catalytic subunit isoforms in colonic smooth muscle electromechanical coupling is considered.     

The calixarenes C-97 and C-107 stimulate influence of ouabain on the Na+,K+-ATPase activity in plasmatic membrane of smooth muscle cells

In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution the authors investigated influence of the calix[4]arenes C-97 and C-107 (codes are shown) on ouabain effect on the Na+,K+-ATPase activity. It was shown that calixarenes in concentration 100 tiM inhibited by 97-98% the enzymatic Na+,K+-ATPase activity, while they did not practically influence on the basal Mg2+-ATPase activity, and suppressed much more effective than ouabain the sodium pump enzymatic activity: in the case of the action of the calixarenes the value of the apparent constant of inhibition I0.5 was < 0.1 microM while for ouabain it was 15-25 microM. The negative cooperative effect was typical of the inhibitory action of calixarenes, as well as ouabain: the value of Hills factor nH = 0.3-0.5 <1. The modelling compound M-3 (0.1 microM 4 microM)--a fragment of the calixarene C-107--did not practically influence the enzymatic activities as Na+,K+-ATPase and basal Mg2+-ATPase. Hence the influence of calixarene C-107 on the Na+, K+-ATPase activity is caused by cooperative action of two fragments M-3 and effect of calixarene bowl, rather than by simple action of the fragment M-3. Calixarenes C-97 and C-107, used in concentration corresponding to values of I0.5 (40 and 60 nM, accordingly), essentially stimulated inhibiting action of ouabain on the specific Na+, K+-ATPase activity in the memrane fraction. Under coaction of ouabain with calixarene C-97 or C-107 there was no additive effect of the action of these inhibitors on the Na+,K+-ATPase activity. Calixarene C-97 brought in the incubation medium in concentration of 10 nM not only led to inhibition of the Na+,K+-ATPase activity relative to control, but also simultaneously increased the affinity of the enzyme for the cardiac glycoside: the magnitudes of the apparent constant of inhibition I0.5 were 21.0 +/- 5.2 microM and 5.3 +/- 0.7 microM. It is concluded, that highly effective inhibitors of the Na+,K+-ATPase activity--calixarenes C-97 and C-107 can enhance the effect of the sodium pump conventional inhibitor--ouabain, increasing the affinity of the enzyme for the cardiac glycoside (on the example of calixarene C-97).     

Isolation and characterization of a cDNA encoding the putative distal colon H+,K(+)-ATPase. Similarity of deduced amino acid sequence to gastric H+,K(+)-ATPase and Na+,K(+)-ATPase and mRNA expression in distal colon, kidney, and uterus.

A series of Northern blot hybridization experiments using probes derived from the rat gastric H+,K(+)-ATPase cDNA and the human ATP1AL1 gene revealed the presence of a 4.3-kilobase mRNA in colon that seemed likely to encode the distal colon H+,K(+)-ATPase, the enzyme responsible for K+ absorption in mammalian colon. A rat colon library was then screened using a probe from the ATP1AL1 gene, and cDNAs containing the entire coding sequence of a new P-type ATPase were isolated and characterized. The deduced polypeptide is 1036 amino acids in length and has an Mr of 114,842. The protein exhibits 63% amino acid identity to the gastric H+,K(+)-ATPase alpha-subunit and 63% identity to the three Na+,K(+)-ATPase alpha-subunit isoforms, consistent with the possibility that it is a K(+)-transporting ATPase. Northern blot analyses show that the 4.3-kilobase mRNA is expressed at high levels in distal colon; at much lower levels in proximal colon, kidney, and uterus; and at trace levels in heart and forestomach. The high mRNA levels in distal colon and the similarity of the colon pump to both gastric H+,K(+)- and Na+,K(+)-ATPases suggest that it is the distal colon H+,K(+)-ATPase. Furthermore, expression of its mRNA in kidney raises the possibility that the enzyme also corresponds to the H+,K(+)-ATPase that seems to play a role in K+ absorption and H+ secretion in the distal nephron.     

Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.

The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.     

Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

The A2 isoform of rat Na+,K(+)-adenosine triphosphatase is active and exhibits high ouabain affinity when expressed in transfected fibroblasts.

The alpha isoforms of the Na+,K(+)-ATPase (Na+ pump) are expressed with developmental and tissue heterogeneity in rodents and possess different sensitivity to inhibition by ouabain. We directly characterized the ouabain sensitivity of the rat A2 (alpha 2) isoform by transfecting NIH 3T3 cells with rat A2. The treated cells exhibit high affinity (40 nM) ouabain binding with a density of 2 pmol/mg protein. 86Rb+ flux studies confirm that A2 is functional in this system and that A2 is inhibited by submicromolar concentrations of ouabain. These findings are consistent with measurements of ouabain affinity in tissues which express the A2 isoform.     

Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K(+)-ATPase alpha subunit isoforms in articular chondrocytes

The catalytic alpha isoforms of the Na+, K(+)-ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1 > alpha 3). The Na+, K(+)-ATPase density of isolated chondrocyte preparations was quantified by specific 3H-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p < 0.05) upregulation of Na+, K(+)-ATPase density and treatment with various pharmacological inhibitors (Brefeldin A, monensin and cycloheximide) significantly (p < 0.001) blocked the upregulation. The subcellular distribution of the Na+, K(+)-ATPase alpha isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K(+)-ATPase alpha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K(+)-ATPase density monitored by 3H-ouabain binding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K(+)-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing alpha 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi complex constitutive secretory pathway and insertion into the plasma membrane.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Na+,K+-ATPase: structure, mechanism, and regulation

Structural organization of alpha- and beta-subunits of Na+,K+-ATPase in the membrane, the enzyme oligomeric structure, and mechanisms of ATP hydrolysis and cation transport are considered. The data on the structure of cation-binding sites and ion-conductive pathways of the pump are reviewed. The properties of isoforms of both subunits are described. Special attention was paid to the ATP modifying effect on Na+,K+-ATPase. To explain the rather complex dependence of the Na+,K+-ATPase activity on ATP concentration, a hypothesis is proposed, which is based on the assumption that the membrane contains the enzyme protomer exhibiting high affinity to ATP and an oligomer having low affinity to the nucleotide and characterized by positive cooperative interactions between subunits. Data on the Na+,K+-ATPase phosphorylation by protein kinases A and C are reviewed.     

Inhibition of the Na+,K(+)-ATPase pump during induction of experimental colon cancer.

During the development of large bowel cancer alterations in colonic epithelial ion transport have been observed some of which result in altered intracellular ionic composition. In many tumors intracellular sodium and potassium become elevated and depressed, respectively. This observation suggests that mechanisms governing intracellular homeostasis for sodium and potassium are no longer tightly regulated. Changes in cell membrane permeability, sodium, potassium-ATPase K(+)-ATPase) pump activity, or both may be responsible for these alterations. It is not known when during initiation and development of cancer such changes may occur. To assess whether there are changes in the Na+, K(+)-ATPase pump early during the induction of large bowel cancer and prior to any notable histological changes, we measured the kinetics of the Na+, K(+)-pump in distal colonic mucosa of CF1 mice one week following only four weekly injections of the carcinogen 1,2-dimethyhydrazine (DMH). The kinetics of the pump were found to be best described by a model of highly cooperative binding. The VMAX of the pump in premalignant mucosa was lower for both sodium and potassium substrate activation (55-65% of control) with little change in other kinetic parameters. Depression of VMAX could not be attributed to an increased barium blockable potassium conductance of the basolateral membrane. Na+,K(+)-ATPase activity was also decreased by 50% in the distal colon of DMH treated mice, but was not affected in the less cancer susceptible proximal colon. These data demonstrate that alterations occur in the Na+,K(+)-pump in premalignant mucosa months before gross tumors develop, and these changes may partially explain the altered levels of Na+ and K+ in the cytoplasm of pre-malignant and malignant colonocytes.     

Endogenous cardiac glycosides, a new class of steroid hormones.

The search for endogenous digitalis has led to the isolation of ouabain as well as several additional cardiotonic steroids of the cardenolide and bufadienolide type from blood, adrenals, and hypothalamus. The concentration of endogenous ouabain is elevated in blood upon increased Na(+) uptake, hypoxia, and physical exercise. Changes in blood levels of ouabain upon physical exercise occur rapidly. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that ouabain is released from adrenal cortex in vivo. Ouabain levels in blood are elevated in 50% of Caucasians with low-renin hypertension. Infusion over several weeks of low concentrations of ouabain, but not of digoxin, induces hypertension in rats. A digoxin-like compound, which has been isolated from human urine and adrenals, as well various other endogenous cardiac glycosides may counterbalance their actions within a regulatory framework of water and salt metabolism. Marinobufagenin, for instance, whose concentration is increased after cardiac infarction, may show natriuretic properties because it inhibits the alpha1 isoform of Na(+)/K(+)-ATPase, the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. In analogy to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. The discovery of ouabain as a new adrenal hormone affecting Na(+) metabolism and the development of the new ouabain antagonist PST 2238 allows for new possibilities for the therapy of hypertension and congestive heart failure. This will lead in turn to a better understanding of the disease on a physiological and endocrinological level and of the action of ouabain on the cellular level as a signal that is transduced to the plasma membrane as well as to the cell nucleus.     

The Na+ pump and intracellular signaling mechanisms

The main properties of Na+ /K(+)-ATPase as a natural receptor for cardiotonic steroids have been discusses. Primary attention is focused on structural and functional differences between the alpha-subunit isoforms of Na+/K(+)-ATPase in different tissues. General information on the role of the Na pump in signaling cascades in kidney epithelial cells, cardiomyocytes and neurons is presented. The data obtained indicate that, in neurons, several alpha-isoforms of Na+/K(+)-ATPase possessing different sensitivity to ouabain may have different signaling functions.     

Ethanol sensitivity of rat brain cortex Na(+), K(+)-ATPase in plasma membrane structural damage induced by sodium dodecyl sulfate

To investigate the role of rat brain cortex Na+, K(+)-ATPase plasma membrane microenvironment in ethanol effect in vitro on membrane the sensitivity of enzyme activity to alcohol was studied under membrane perturbation induced by sodium dodecyl sulfate. The increase of enzyme sensitivity to detergent inactivation in the presence of high ethanol concentrations and to alcohol inhibition after modification by Ds-Na was revealed. It is supposed that Na+, K(+)-ATPase sensitivity to ethanol is dependent on structural state of protein microenvironment in accordance with assumed differences in structural organization of the boundary lipids of the neuronal enzyme isoforms.     

Na+,K(+)-ATPase inhibition by an endogenous peptide, SPAI-1, isolated from porcine duodenum.

SPAI-1, a peptide isolated from porcine duodenum, has been shown to inhibit Na+,K(+)-ATPase in vitro (Araki et al. (1989) Biochem. Biophys. Res. Commun. 164, 496-502). The characteristics of ATPase inhibition by this novel peptide were examined. SPAI-1 inhibited Na+,K(+)-ATPase preparations isolated from various organs of dog or rat or from sheep kidney with similar potency. Three isoforms of rat Na+,K(+)-ATPase had similar sensitivity to inhibition by SPAI-1 although these isoforms had remarkable differences in their sensitivity to the inhibitory effect of ouabain. Ca(2+)-ATPase isolated from the sarcoplasmic reticulum of rabbit skeletal muscle was insensitive to inhibition by SPAI-1. Ouabain-insensitive Mg(2+)-ATPase activity was unaffected by low concentrations of SPAI-1, but was stimulated at high concentrations. SPAI-1 inhibited H+,K(+)-ATPase from hog stomach in concentrations similar to that required for Na+,K(+)-ATPase inhibition. These results indicate that SPAI-1 is a specific inhibitor for monovalent cation transporting ATPases.     

Endogenous digitalis: reality or myth?

Endogenous digitalis is defined as a natural ligand for the digitalis-binding site of the Na+, K(+)-ATPase and is a specific, high-affinity reversible inhibitor of the enzyme activity. Such endogenous digitalis is thought to be involved in sodium homeostasis and blood pressure regulation as a vasoactive and natriuretic substance. The search for endogenous digitalis goes back to the early 1960s. Since then large efforts have been exerted by numerous laboratories worldwide, but little advance has been made until recently except for the identification of nonspecific Na+, K(+)-ATPase inhibitors. Some researchers even doubt the existence of endogenous digitalis. The recognition that assay methodology is associated with many pitfalls and problems has accelerated the rate of recent progress. Chemical identification of endogenous digitalis will be forthcoming in the very near future. In this article, important issues surrounding endogenous digitalis are critically reviewed.