Na,K-ATPase activity in the myocardial sarcolemma in ischemia

The total time-controlled ischemia (up to 45 min) was studied for its effect on the Na,K-ATPase activity, content of nonesterified fatty acids (NEFA) and intensity of lipid peroxidation (LP) in sarcolemmal (SL) preparations and soluble fractions (SF) from the rat and guinea-pig left ventricles. A strong correlation between Na, K-ATPase inhibition and NEFA accumulation was revealed in the SF. On the contrary, changes in the NEFA content and LP level both in SL and SF did not correlate with a decrease in the enzymic activity. Pretreatment with albumin (0.5 mg/ml) induced equally small increase both in the control and in the ischemic SL preparations. It is suggested that the Na,K-ATPase activity during a short period of myocardial ischemia (up to 45 min) may be due to the NEFA accumulation in the cytosolic and/or extracellular space, but not in SL.     

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.     

Impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension

We have observed that, in renal proximal tubular cells, cardiotonic steroids such as ouabain in vitro signal through Na/K-ATPase, which results in inhibition of transepithelial (22)Na(+) transport by redistributing Na/K-ATPase and NHE3. In the present study, we investigate the role of Na/K-ATPase signaling in renal sodium excretion and blood pressure regulation in vivo. In Sprague-Dawley rats, high salt diet activated c-Src and induced redistribution of Na/K-ATPase and NHE3 in renal proximal tubules. In Dahl salt sensitive (S) and resistant (R) rats given high dietary salt, we found different effects on blood pressure but, more interestingly, different effects on renal salt handling. These differences could be explained by different signaling through the proximal tubular Na/K-ATPase. Specifically, in Dahl R rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2, reduced Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-ATPase and NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl S rats. We also studied the primary culture of renal proximal tubule isolated from Dahl S and R rats fed a low salt diet. In this system, ouabain induced Na/K-ATPase/c-Src signaling and redistribution of Na/K-ATPase and NHE3 in the Dahl R rats, but not in the Dahl S rats. Our data suggested that impairment of Na/K-ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt-induced hypertension in the Dahl S rat.     

Inhibition of Na,K-ATPase from chick brain by polyamines.

The amounts of the polyamines putrescine, spermine and spermidine as well as the Na,K-ATPase activity have been determined in the developing chick brain. The amounts of spermine and spermidine per gram fresh weight do not change significantly, the amount of putrescine declines until the 17th day of incubation after which an increase takes place. Spermine is able to inhibit the Na,K-ATPase from chick brain competitively. Half maximal inhibition is achieved at 4 X 10(-5) mol/1 spermine. This polyamine functions as an allosteric inhibitor; the Hill coefficient is 2.2 +/- 0.3. A regulatory effect of spermine on the Na,K-ATPase from chick brain is discussed. In contrast to spermine 1 mmol/1 spermidine inhibits the Na,K-ATPase only slightly, while 1 mmol/1 putrescine does not inhibit the Na,K-ATPase at all.     

Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman

Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ～3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (-4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63) and Ser(68). Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.     

Light-induced changes in activity of Na, K-ATPase from the retinal photoreceptors of vertebrates: possible mechanism

The mechanism of light-induced changes in the activity of Na,K-ATPase from plasma membranes (PM) of photoreceptor cells was studied in vitro. Illumination resulted in inhibition of the ATPase activity and an increase of 18O exchange between water and Pi. The maximum light effect was revealed when the PM contained both the inner segments of the rods (RIS) and rod outer segments (ROS) of the photoreceptor cells. Lipid peroxidation stimulated by the FeSO4+ascorbate system induced a decrease of the ATPase activity. Antioxidants (ionol, Na2SeO3, vitamin E) prevented the effect of the lipid peroxidation products on NA,K-ATPase and the photoinduced changes of the enzyme activity. It is supposed that the photoinduced changes of the Na,K-ATPase activity in vitro are due to lipid peroxidation of photoreceptor PM.     

Effects of anticonvulsive substances on Na,K-ATPase from the synaptic membranes of animal brain]

The distribution pattern of marker enzymes (Na, K-ATPase, acetylcholinesterase) in three fractions of synaptic membranes (SM) of rat brain were studied. The effects of three anticonvulsive agents on Na, K-ATPase from the total fraction of rat brain SM and purified membrane preparation from ox brain were estimated by different methods. Under optimal conditions (Na/K = 5) diphenylhydantoin (DPH) at a concentration of 0,1 mM activates Na, K-ATPase from the total SM fraction only in the absence of ouabain, whereas carbamazepine and pyrroxane taken at the same concentrations have no effect on Na, K-ATPase, irrespective of the type of the enzyme assay. DPH seems to compete with ouabain. Under non-optimal ionic conditions (Na/K = 250) all the anticonvulsive substances studied inhibit Na, K-ATPase of the total SM fraction. The mixture of hydrophobic agents (propylene glycol and ethanol) used to dissolve carbamazepine inhibits Na, K-ATPase from the total SM fraction only under non-optimal conditions. The inhibiting effect of the anticonvulsive substances under study on Na, K-ATPase from the purified membrane preparations is maximal at the concentration of 10(-6) M; at higher concentrations the effect is less pronounced.     

Gender specific influence of fish oil or atorvastatin on functional properties of renal Na,K-ATPase in healthy Wistar and hypertriglyceridemic rats

For better understanding of pathophysiological processes leading to increased retention of sodium as a consequence of hyperlipidemia, the properties of renal Na,K-ATPase, a key enzyme involved in maintaining sodium homeostasis in the organism, were studied. Enzyme kinetics of renal Na,K-ATPase were used for characterization of ATP- and Na(+)-binding sites after administration of fish oil (FO) (30 mg·day(-1)) or atorvastatin (0.5 mg·100 g(-1)·day(-1)) to healthy Wistar rats and rats with hereditary hypertriglyceridemia of both genders. Untreated healthy Wistar and also hypertriglyceridemic female rats revealed higher Na,K-ATPase activity as compared to respective untreated male groups. Hypertriglyceridemia itself was accompanied with higher Na,K-ATPase activity in both genders. Fish oil improved the enzyme affinity to ATP and Na(+), as indicated by lowered values of K(m) and K(Na) in Wistar female rats. In Wistar male rats FO deteriorated the enzyme in the vicinity of the Na(+)-binding site as revealed from the increased K(Na) value. In hypertriglyceridemic rats FO induced a significant effect only in females in the vicinity of the sodium binding sites resulting in improved affinity as documented by the lower value of K(Na). Atorvastatin aggravated the properties of Na,K-ATPase in both genders of Wistar rats. In hypertriglyceridemic rats protection of Na,K-ATPase was observed, but this effect was bound to females only. Both treatments protected renal Na,K-ATPase in a gender specific mode, resulting probably in improved extrusion of excessive intracellular sodium out of the cell affecting thus the retention of sodium in hHTG females only.     

Wortmannin inhibition of forskolin-stimulated chloride secretion by T84 cells

The time- and dose-dependent effects of wortmannin on transepithelial electrical resistance (Rte) and forskolin-stimulated chloride secretion in T84 monolayer cultures were studied. In both instances, maximal effects developed over 2 h and were stable thereafter. Inhibition of forskolin-stimulated chloride secretion, as measured by the short-circuit current (Isc) technique, had an IC50 of 200-500 nM, which is 100-fold higher than for inhibition of phosphatidylinositol 3-kinase (PI3K), but similar to the IC50 for inhibition of myosin light chain kinase (MLCK) and mitogen-activated protein kinases (MAPK). Previous work demonstrated that 500 nM wortmannin did not inhibit the cAMP activation of apical membrane chloride channels. We show here that 500 nM wortmannin has no affect on basolateral Na/K/2Cl-cotransporter activity, but inhibits basolateral membrane Na/K-ATPase activity significantly. The MLCK inhibitors ML-7 and KT5926 were without affect on forskolin-stimulated Isc. Similarly, the p38- and MEK-specific MAPK inhibitors SB203580 and PD98059 did not reduce forskolin-stimulated Isc. In contrast, the non-specific MAPK inhibitor apigenin reduced forskolin-stimulated Isc and basolateral membrane Na/K-ATPase activity similar to wortmannin. In isolated membranes from T84 cells, wortmannin did not inhibit Na/K-ATPase enzymatic activity directly. We conclude that one or more MAPK may regulate the functional expression of basolateral membrane Na/K-ATPase by controlling the abundance of enzyme molecules in the plasma membrane.     

Possibilities of pharmacologic correction of Na, K-ATPase activity in the spinal cord in botulism

The experiments on white rats have shown that gutimin is capable of reactivating Na, K-ATPase of the synaptosomes of the jugular spinal cord in type C botulinic intoxication. Serotonin prevented Na, K-ATPase activity inhibition only in preclinical period of intoxication. Parmidin injection did not prevent suppression of Na, K-ATPase activity either in preclinical period or in skeletal muscle paresis.     

Sodium, and potassium dependent adenosinetriphosphatase from Cavia cobaya kidney: interaction with very low cardiotonic steroid concentrations

We noticed that very low cardiotonic steroid concentrations activate the Na, K-ATPase in a variety of different preparations. In the present research the effect of three cardioactive steroids on the enzymatic activity was tested. The glycosides activated the Na,K-ATPase, while the aglycone strophantidine does not. Ouabain binding studies on various preparations showed the presence of two binding site classes with different affinities. Purification procedures shift the apparent Kd values, while K+ increase them. Accordingly, the activatory and inhibitory effects may be explained by the cardiotonic steroid binding on different sites of the Na,K-ATPase molecule.     

Sulfonylurea binding to a low-affinity site inhibits the Na/K-ATPase and the KATP channel in insulin-secreting cells

We have used hamster insulinoma tumor (HIT) cells, an insulin-secreting tumor cell line, to investigate modulation of the Na/K-ATPase and of the ATP-sensitive K channel (K(ATP)) by the sulfonylurea glyburide. Membrane proteins from cells cultured in RPMI with 11 mM glucose have at least two glyburide receptor populations, as evidenced by high and low binding affinity constants, (K(d) = 0.96 and 91 nM, respectively). In these cells K(ATP) channel activity was blocked by low glyburide concentrations, IC(50) = 5.4 nM. At 12.5 nM glyburide the inhibition developed slowly, tau = 380 s, and caused reduction of channel activity by 75 percent. At higher concentrations, however, inhibition occurred at a fast rate, tau = 42 s at 100 nM, and was almost complete. Na/K- ATPase activity measured enzymatically and electrophysiologically was also suppressed by glyburide, but higher concentrations were needed, IC(50) = 20-40 nM. Inhibition occurred rapidly, tau = 30 s at 50 nM, when maximum, activity was reduced by 40 percent. By contrast, cells cultured in RPMI supplemented with 25 mM glucose exhibit a single receptor population binding glyburide with low affinity, K(d)= 68 nM. In these cells inhibition of the Na/K-ATPase by the sulfonylurea was similar to that observed in cells cultured in 11 mM glucose, but K(ATP) channel inhibition was markedly altered. Inhibition occurred only at high concentrations of glyburide and at a fast rate; maximum inhibition was observed at 100 nM. Based on these data, we propose that glyburide binding to the high affinity site affects primarily K(ATP) channel activity, while interaction with the low affinity site inhibits both Na/K-ATPase and K(ATP) channel activities. The latter observation suggests possible functional interactions between the Na/K-ATPase and the K(ATP) channel.     

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (11-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol P_i/mol of -subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P_i into the -subunit and the loss of activity of the Na,K-ATPase.     

The beta-adrenergic regulation of the Na, K-ATPase activity in the sarcolemma of the heart muscle

Using a sensitive potentiometric method the effect of isoproterenol upon the activity of Na, K-ATPase in cardiomyocytes has been studied. The activity of the enzyme in rat sarcolemma at isoproterenol-induced myocarditis decreases by 42%. A direct action of isoproterenol on the Na, K-ATPase activity in sarcolemma in vitro has been investigated. In the concentration range 10(-9)-10(-3) M (from receptor-binding up to cardiotoxic) a gradual decrease of the activity reaching the complete inhibition at 10(-3) M is revealed. Antagonist of beta-adrenoreceptors propranolol in concentrations required for displacing the agonist (10(-9) M) provides for the recovery of the Na, K-ATPase activity up to 76% of its normal activity. This action transforms into nonspecific inhibition at rising concentration of the antagonist. Possible mechanisms of the beta-adrenergic regulation effect in cardiomyocytes on Na, K-ATPase of the sarcolemma are discussed.     

Some properties of glial membrane Na, K-ATPase]

Na,K-ATPase activity in glial membranes is rather low that in the nerve ending membranes, but is characterized by the same kind of Na+/K+-dependence. Glial Na,K-ATPase is insensitive to acetylcholine (ACh), 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) while norepinephrine activates Na,K-ATPase at low concentrations and inhibits it at high concentrations. Participation of Na,K-ATPase in the regulatory mechanisms of the neuron-neuroglia relations is discussed.     

Regulation of Na,K-ATPase function in the lens

The two cell types in the lens, epithelium and fiber, have a very different specific activity of Na,K-ATPase; activity is much higher in the epithelium. However, judged by Western blot, fibers and epithelium express a similar amount of both Na,K-ATPase alpha and beta subunit proteins. Na,K-ATPase protein abundance does not tally with Na,K-ATPase activity. Studies were conducted to examine whether protein synthesis plays a role in maintenance of the high Na,K-ATPase activity in lens epithelium. An increase of cytoplasmic sodium was found to increase Na,K-ATPase protein expression in the epithelium, but not in the fibers. The findings illustrate the ability of lens epithelium to synthesize new Na,K-ATPase protein as a way to boost Na,K-ATPase in response to cell damage or pathological events. Methionine incorporation studies suggested Na,K-ATPase synthesis may also play a role in day to day preservation of high Na,K-ATPase activity. Na,K-ATPase protein in lens epithelial cells appeared to be continually synthesized and degraded. Experiments with cycloheximide suggest that specific activity of Na,K-ATPase in the lens epithelium may depend on the ability of the cells to continuously synthesize fresh Na,K-ATPase proteins. However, other factors such as phosphorylation of Na,K-ATPase alpha subunit may also influence Na,K-ATPase activity. When intact lenses were exposed to the agonist thrombin, Na,K-ATPase activity was diminished, but the response was suppressed by inhibitors of the Src family of non-receptor tyrosine kinases. Thrombin elicited tyrosine phosphorylation of lens epithelium membrane proteins, including a 100 kDa protein band thought to be the Na,K-ATPase alpha 1 subunit. It remains to be determined whether a tyrosine phosphorylation mechanism contributes to the low activity of Na,K-ATPase in lens fibers.     

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.     

A blood plasma inhibitor is responsible for circadian changes in rat renal Na,K-ATPase activity

Rhythmic changes in activity following a circadian schedule have been described for several enzymes. The possibility of circadian changes in Na,K-ATPase activity was studied in homogenates of rat kidney cortex cells. Male Sprague-Dawley rats were kept on a schedule of 12h light (06:00-18:00 h) and 12 h darkness (18:00-06:00 h) for 2 weeks. At the end of the conditioning period, one rat was killed every 2 h, until completion of a 24 h cycle. Outermost kidney cortex slices were prepared, homogenized and assayed for Na,K-ATPase activity. The whole procedure was repeated six times. Na,K-ATPase activity shows an important oscillation (2 cycles/24 h). Peak activities were detected at 09:00 and 21:00 h, whereas the lowest activities were detected at 15:00 and 01:00-03:00 h. The highest activity was 40+/-3 nmoles Pi mg protein(-1)min(-1) (09:00 h), and the lowest was 79+/-3 nmoles Pi mg protein(-1)min(-1) (15:00 h). The amount of the Na+-stimulated phosphorylated intermediate is the same for the 09:00 h and 15:00 h homogenates. Preincubation of 09:00 h kidney cortex homogenates with blood plasma drawn from rats at either 03:00 h or 15:00 h, significantly inhibited their Na,K-ATPase activity. This inhibition was not seen when the preincubation was carried out with either 09:00 h or 21:00 h blood plasma. The striking oscillation (2 cycles/24 h) of the Na,K-ATPase activity of rat kidney cortex cells is ascribed to the presence of an endogenous inhibitor in blood plasma.