Inhibition of hamster sperm Na+, K+-ATPase activity by taurine and hypotaurine

Taurine, hypotaurine and the structural analogue, beta-alanine, were tested for their effects on Na+, K+-ATPase activity of crude homogenates prepared from washed cauda epididymal hamster sperm. Preincubation with 0.1-10 mM taurine or hypotaurine inhibited Na+, K+-ATPase in a dose-dependent manner, while beta-alanine had an inhibitory effect only at 10 mM. The results of this study are the first evidence to demonstrate inhibition of Na+, K+-ATPase activity by taurine and hypotaurine and are discussed in relation to the ability of these compounds to sustain hamster sperm motility and fertility.     

Inhibition of rat cardiac and renal Na+,K+-ATPase by high sodium concentrations and vanadate

In the present study, rat renal Na+,K+-ATPase was found to be more sensitive to inhibition by high Na+ concentrations (100-400 mM) than was rat cardiac Na+,K+-ATPase. K+ was more effective in reversing the inhibition by Na+, of cardiac relative to renal Na+,K+-ATPase. Rat renal Na+,K+-ATPase was also more sensitive than cardiac Na+,K+-ATPase to inhibition by vanadate over this range of Na+ concentrations. These results support the hypothesis that vanadate may selectively regulate Na+,K+-ATPase in the kidney, and they may also help explain the natriuretic and diuretic effects of vanadate in rats. Inhibition of renal Na+,K+ATPase by Na+, may also help explain, in part, the natriuretic and diuretic effects of acute saline loading.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Inhibition of Na+,K+-ATPase in Penaeus indicus postlarvae by lead

The plasma membrane/mitochondrial fractions of Penaeus indicus postlarvae contain Mg2+-dependent ATPase, Na+,K+-stimulated ATPase, Na+-stimulated ATPase and K+-stimulated ATPase. The Na+,K+-activated, Mg2+-dependent ATPase was investigated further in relation to different pH and temperature conditions, and at various concentrations of protein, ouabain, ATP and ions in the incubation medium. In vitro and in vivo effects of lead were studied on the enzyme activity. In vitro lead inhibited the enzyme activity in a concentration-dependent manner with an IC50 value of 204.4 microM. In correlation with in vitro studies, in vivo investigations (both concentration and time dependent) of lead also indicated a gradual inhibition in enzyme activity. A maximum decrease of 85.3% was observed at LC50 (7.2 ppm) of lead for concentration-dependent experiments. In time-dependent studies, the decrease was maximal (81.7%) at 30 days of sublethal exposure (1.44 ppm). In addition, the substrate- and ion-dependent kinetics of Na+,K+-ATPase was studied in relation to in vitro exposure of lead; these studies suggest a non-competitive type of inhibition.     

Inhibition of bovine heart Na+, K+-ATPase by palmitylcarnitine and palmityl-CoA.

The activity of a partially purified bovine heart Na⁺,K⁺-ATPase is inhibited by DL- and L- palmitylcarnitine (I₅₀=44–48μM). Palmitylcarnitine with a I₅₀ of 25μM also markedly inhibits K⁺-phosphatase activity. Palmityl-CoA decreases Na⁺,K⁺-ATPase activity, but to a lesser extent (I₅₀=80μM). Both palmitic acid and hexanoic acid produce 10 to 15% inhibition of activity at concentrations of 70μM and 3–5mM, respectively. These free fatty acids protect the enzyme against inhibition by 40μM palmitylcarnitine. However, at 50μM palmitylcarnitine, the protective effect by hexanoic acid is no longer apparent. Addition of 40μM palmitylcarnitine to the Na⁺,K⁺-ATPase in the presence of varying concentrations of palmityl-CoA produces an additive inhibition of enzyme activity, suggesting two different sites on the enzyme susceptible to inhibition by the two ester forms of the fatty acid.     

Electrogenic ion transport by Na+,K+-ATPase

Experimental data on the ion electrogenic transport by Na+,K+-ATPase available in the literature are analyzed. Special attention is paid to the measurements of unsteady-state electric currents initiated by alternating voltage or rapid introduction of the substrate. In the final part, a physical model of the Na+,K+-ATPase functioning is discussed. According to this model, active transport is carried out by opening and closing of the access channels used for the sodium and potassium exchange between solutions on either side of the membrane. The model explains most of the experimental data, although some details (the channel size, rates of individual transport steps) need further refinement.     

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.     

Effect of glycosphingolipids on erythrocyte Na+,K+-ATPase activity

The total fractions of gangliosides and cerebrosides isolated from the tissue of human brain were studied for their effect on the Na+, K+-ATPase activity of native erythrocytes and their membranes. It is shown that gangliosides depending on time of their preincubation with the enzyme preparation and concentration produce both the activating and inhibiting action and cerebrosides--only the inhibiting one. Gangliosides inhibit the transport ATPase activity noncompetitively with respect to ATP and Na+ and competitively--to K+, cerebrosides inhibit it noncompetitively with respect to all ATPase activators.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Na+,K+ -ATPase activity characteristics in human colon adenocarcinoma

To evaluate the enzyme functional changes the Na+,K+-ATPase activity in membrane fraction of human colorectal adenocarcinoma at II and III cancer stages (according to TNM classification) of varying degrees of differentiation has been investigated. The decrease of the Na+,K+-ATPase activity in comparison with conditionally normal tissue of macroscopically unchanged mucosa was revealed in the tumor membrane preparations. Such changes of the Na+,K+-ATPase activity were higher at low differentiation grade and were less pronounced in moderately and highly differentiated adenocarcinomas. At the same time the changes in Na+,K+-ATPase activity have not been revealed between tumor membrane preparations at studied cancer stages when the degree of differentiation was not taken into account. It is supposed that Na+,K+-ATPase functional specificity occurs in colorectal adenocarcinomas and it is associated with tumor differentiation.     

The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues

Since Na+,K+-ATPase (EC 3.6.1.3) of pig kidney modified with a fluorescent sulfhydryl reagent, N-[p-(2-benzimidazolyl) phenyl]maleimide, at Cys-964 of the alpha-chain showed ATP-dependent, reversible, and dynamic fluorescence changes (Nagai, M., Taniguchi, K., Kangawa, K., Matsuo, S., Nakamura, S., and Iida, S. (1986) J. Biol. Chem. 261, 13197-13202), we studied the conformational change during Na+,K+-ATPase reaction using the modified enzyme. The addition of K+ to the enzyme increased the fluorescence intensity to 2% in the presence of 160 mM Na+ and 3 mM Mg2+ (K0.5 = 16.4 mM). Addition of low concentrations of ATP immediately increased the intensity to 3.2% (K0.5 less than 0.1 microM) to accumulate fully K+-bound enzyme in the presence of 43 mM K+ with Na+ and Mg2+, but further addition of higher concentrations of ATP diminished the increase (K0.5 = 120 microM). After exhaustion of ATP, the fluorescence intensity decreased to -0.4% (K0.5 = 0.3 microM) and -2% (K0.5 = 20 microM), respectively, in the presence of low and high concentrations of ADP produced from ATP. High concentrations of ATP accelerated Na+,K+-ATPase activity with a simultaneous increase in the amount of ADP-sensitive phosphoenzyme irrespective of the modification. Adenylyl imidodiphosphate and ADP accelerated Na+,K+-ATPase activity in the presence of 2.7 microM ATP by decreasing the extent of the fluorescence without affecting the amount of phosphoenzyme, irrespective of the modification. These data suggest that Na+,K+-ATPase activity was accelerated due to the acceleration of the breakdown of K+-bound enzyme by high concentrations of ATP and ATP analogues.     

Inhibition of Na+, K+-ATPase of intact mouse soleus muscle by Mg++

The effect of 10 mM MgCl₂ on the inhibition of respiration by ouabain was investigated with intact mouse soleus muscle preparations. Although ouabain caused a 19.7% inhibition of respiration of soleus muscle incubated in 1 mM MgCl₂ buffer, the response of respiration to ouabain was abolished upon incubation in buffer containing 10 mM MgCl₂. Initial respiration rates were significantly decreased in soleus muscle exposed to 10 mM, as contrasted to 1 mM, MgCl₂.     

Time-dependent effect of leptin on renal Na+,K+-ATPase activity

Leptin, secreted by adipose tissue, is involved in the pathogenesis of arterial hypertension, however, the mechanisms through which leptin increases blood pressure are incompletely elucidated. We investigated the effect of leptin, administered for different time periods, on renal Na(+),K(+)-ATPase activity in the rat. Leptin was infused under anesthesia into the abdominal aorta proximally to the renal arteries for 0.5-3 h. Leptin administered at doses of 1 and 10 microg/min per kg for 30 min decreased the Na(+),K(+)-ATPase activity in the renal medulla. This effect disappeared when the hormone was infused for > or =1 h. Leptin infused for 3 h increased the Na(+),K(+)-ATPase activity in the renal cortex and medulla. The stimulatory effect was abolished by a specific inhibitor of Janus kinases (JAKs), tyrphostin AG490, as well as by an NAD(P)H oxidase inhibitor, apocynin. Leptin increased urinary excretion of hydrogen peroxide (H(2)O(2)) between 2 and 3 h of infusion. The effect of leptin on renal Na(+),K(+)-ATPase and urinary H(2)O(2) was augmented by a superoxide dismutase mimetic, tempol, and was abolished by catalase. In addition, infusion of H(2)O(2) for 30 min increased the Na(+),K(+)-ATPase activity. Inhibitors of extracellular signal regulated kinases (ERKs), PD98059 or U0126, prevented Na(+),K(+)-ATPase stimulation by leptin and H(2)O(2). These data indicate that leptin, by acting directly within the kidney, has a delayed stimulatory effect on Na(+),K(+)-ATPase, mediated by JAKs, H(2)O(2) and ERKs. This mechanism may contribute to the abnormal renal Na(+) handling in diseases associated with chronic hyperleptinemia such as diabetes and obesity.     

Salt,Na+,K+-ATPase and hypertension

Chronic hypertension is characterized by a persistent increase in vascular tone. Sodium-rich diets promote hypertension; however, the underlying molecular mechanisms are not fully understood. Variations in the sodium content of the diet, through hormonal mediators such as dopamine and angiotensin II, modulate renal tubule Na⁺,K⁺-ATPase activity. Stimulation of Na⁺,K⁺-ATPase activity increases sodium transport across the renal proximal tubule epithelia, promoting Na⁺ retention, whereas inhibited Na⁺,K⁺-ATPase activity decreases sodium transport, and thereby natriuresis. Diets rich in sodium also enhance the release of adrenal endogenous ouabain-like compounds (OLC), which inhibit Na⁺,K⁺-ATPase activity, resulting in increased intracellular Na⁺ and Ca²⁺ concentrations in vascular smooth muscle cells, thus increasing the vascular tone, with a corresponding increase in blood pressure. The mechanisms by which these homeostatic processes are integrated in response to salt intake are complex and not completely elucidated. However, recent scientific findings provide new insights that may offer additional avenues to further explore molecular mechanisms related to normal physiology and pathophysiology of various forms of hypertension (i.e. salt-induced). Consequently, new strategies for the development of improved therapeutics and medical management of hypertension are anticipated.     

Vanadate sensitivity of Na+, K+-ATPase from Schistosoma mansoni and its modulation by Na+, K+ and Mg2+

Vanadate inhibitory effects on Na+, K+-ATPases from carcass of Schistosoma mansoni and from lamb kidney outer medulla were compared in the presence of various concentrations of Na+, K+ and Mg2+. Depending on the ionic conditions, the schistosomal Na+, K+-ATPase was 2.4- to 175-fold less sensitive to vanadate than the lamb kidney enzyme. In 100 mM Na+, 3 mM K+ and 3 mM Mg2+, schistosomal Na+, K+-ATPase was surprisingly resistant to vanadate (I50 = 944 microM). The difference in vanadate sensitivity between schistosomal and lamb Na+, K+-ATPases may be due to a species difference in the efficacy of Na+, K+ and Mg2+ in promoting conformational changes between E1 and E2 forms of the enzyme.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

Glucagon stimulation of hepatic Na+, K+-ATPase

Summary In the perfused rat liver administration of glucagon was shown to result in a transiently increased uptake of K⁺, indicating the possible involvement of the Na⁺, K⁺-ATPase. Direct measurement of the activity of Na⁺, K⁺-ATPase revealed a two-fold stimulation of the enzyme by glucagon. The effect of glucagon on the activity of the enzyme was immediate. Simultaneously with the increase in the activity of the Na⁺, K⁺-ATPase, the activity of Mg²⁺-ATPase decreased. In order to evaluate whether the activation of the Na⁺, K⁺-ATPase by glucagon is related to the metabolic effects of the hormone, experimental conditions known to interfere with the activity of the enzyme were employed and glucagon stimulation of Ca²⁺-efflux, mitochondrial metabolism and gluconeogenesis were measured. K⁺-free perfusate, high K⁺ perfusate or ouabain interfered to varying degrees with the glucagon stimulation of these responses. The combination of K⁺-free perfusate and ouabain almost completely abolished the glucagon stimulation of all three parameters. These results demonstrate the glucagon stimulation of Na⁺, K⁺-ATPase and raise the possibility that the activation of the enzyme by glucagon might be a necessary link for the manifestation of its metabolic effects.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.