Influence of lorcainide on microsomal Na+, K(+)-ATPase in guinea-pig isolated heart preparations.

1. The effects of lorcainide on the myocardial Mg2(+)-dependent, Na+ and K(+)-activated adenosine triphosphatase (Na+, K(+)-ATPase) were compared in guinea-pig heart preparations with those of ouabain, a specific inhibitor of the enzyme activity. 2. Both ouabain and lorcainide inhibited the microsomal Na+, K(+)-ATPase activity in a concentration-dependent fashion. Their inhibitory effective ranges were 0.05-100 microM and 0.15-125 microM, respectively, and the concentrations for half maximal inhibition (IC50 values) were 2.1 +/- 0.3 and 33.5 +/- 7.3 microM, respectively. 3. In a second series of experiments, the combined effects of the two drugs on the enzyme activity were studied. In these experiments, lorcainide produced a concentration-dependent potentiation of the inhibitory effects of ouabain on Na+, K(+)-ATPase activity. 4. The present study demonstrates that lorcainide is a potent inhibitor of myocardial Na+, K(+)-ATPase.     

Interaction of beta-eudesmol with Na+,K(+)-ATPase: inhibition of K(+)-pNPPase activity.

The effect of beta-eudesmol, one of the major components in So-jutsu (Atractylodis Lanceae Rhizoma), on K(+)-dependent p-nitrophenyl phosphatase (K(+)-pNPPase) activity was studied. It inhibited K(+)-pNPPase activity with an I50 value of 4.1 x 10(-4) M. The inhibition rate decreased as the K+ concentration was increased, whereas greater inhibition was observed with high concentrations of either Na+ or ATP. The Ki values for Na+ in the presence of 0, 0.1 and 1 mM ATP were 140, 260 and 310 mM, respectively, but with the addition of beta-eudesmol, these values decreased to 90 mM regardless of the ATP concentration. This study on K(+)-pNPPase activity supports the conclusion obtained from the study on Na+,K(+)-ATPase activity (Satoh K et al., Biochem Pharmacol 44: 373-378, 1992) that is, beta-eudesmol interacts with enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P. Furthermore, in the study of the effects of K+ and beta-eudesmol on K(+)-pNPPase activity, it was confirmed that beta-eudesmol prevents the conformational change of Na.E1----K.E2.     

Developmental lead exposure inhibits adult rat retinal, but not kidney, Na+,K(+)-ATPase.

Neonatal lead exposure produces selective rod degeneration and functional deficits in adult hooded rats. Similar alterations occur following retinal exposure to ouabain. This study determined whether there were long-term effects of neonatal lead exposure on rat retinal or renal Na+,K(+)-ATPase (Na,K-ATPase) activity and employed in vitro studies to examine the mechanism of ionic lead (Pb2+)-induced inhibition of retinal Na,K-ATPase. Pups, exposed to lead only via the milk of dams consuming 0, 0.02, or 0.2% lead solutions, had mean blood lead concentrations of 1.2, 18.8, and 59.4 micrograms/dl at weaning, respectively, and 5-7 micrograms/dl as adults. Prior lead exposure produced significant dose-dependent decreases in isolated retinal Na,K-ATPase activity (-11%; -26%) whereas activity in the kidney was unchanged. In contrast, Na,K-ATPase from both isolated control tissues was inhibited by Pb2+. The half-maximal inhibitory dose (I50) of Pb2+ for retinal and renal Na,K-ATPase was 5.21 x 10(-7) and 1.25 x 10(-5) M, respectively. The Hill coefficient of the retina was 0.42 whereas it was 0.88 in the kidney. With MgATP as a substrate, the Pb(2+)-induced inhibition of retinal Na,K-ATPase was competitive and reversible with a Ki of 2.1 x 10(-7) M. Retinal and renal Na,K-ATPase were 20-fold and 1.1-fold more sensitive to inhibition by Pb2+ than by Ca2+, respectively. The Pb(2+)-induced inhibition of retinal Na,K-ATPase was antagonized by Na+, potentiated by Mg2+, not altered by K+ or Ca2+, and prevented by ATP. Kinetic and competition studies with the retinal Na,K-ATPase establish that the Pb(2+)-induced inhibition is complex. The increased sensitivity of retinal, compared to renal, Na,K-ATPase to inhibition following in vivo or in vitro lead exposure may relate to their different alpha subunit composition. This is speculated to play a fundamental role in the target organ toxicity of lead.     

Na+,K(+)-ATPase inhibiting activity of cardiac glycosides from Erysimum cheiranthoides.

We previously reported the isolation of eleven new cardiac glycosides called cheiranthosides I-XI together with two known ones (olitoriside and erysimoside) from the seeds of Erysimum cheiranthoides L. The glycosides were evaluated for their inhibitory activity against Na+,K(+)-ATPase by comparing with typical cardiac glycosides. Two of them, cheiranthoside III and VIII, showed high inhibiting activity which was equivalent to that of digitoxin. Cheiranthoside XI containing a rhamnopyranosyl digitoxopyranosyl moiety and a carboxyl group showed the lowest activity which was similar to that of the inactive aglycone, strophanthidin. Some characteristics in the structure-activity relationship are also discussed.     

Tetrahydroacridine inhibits voltage-dependent Na^＋ current in guinea-pig ventricular myocytes

AIM: To study the effects of tetrahydroacridine (tacrine) on voltage-gated Na^ channels in cardiac tissues. METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na^ current was recorded using whole cell voltage-clamp technique. RESULTS: (1) Tacrine reversibly inhibited Na^ current with an IC50 value of 120μmol/L(95% confidence range: 108-133μmol/L). (2) The inhibitory effects of tacrine on Na^ current exhibited both a tonic nature and use-dependence. (3) Tacrine at 100μmol/L caused a negative shift (about 10mV) in the voltage-dependence of steady-state inactivation of Na^ current, and retarded its recovery from inactivation, but did not affect its activation curve. (4) Intracellular application of tacrine significantly inhibited Na^ current. CONCLUSION: In addition to blocking other voltage-gated ion channels,tacrine blocked Na^ channels in guinea-pig ventricular myocytes. Tacrine acted as inactivation stabilizer of Na^ channels in cardiac tissues.     

Ouabain-like Na+,K(+)-ATPase inhibitory activity of a plasma extract in normal pregnancy and pregnancy induced hypertension.

To estimate the participation of a Na+,K(+)-ATPase-inhibiting plasma factor in pregnancy induced hypertension (PIH), the inhibitory activity and the characteristics of plasma extract eluted with ethanol through a C8 column were examined in normotensive non-pregnant women (N) and women with normal pregnancy (NP) and PIH. There were no differences among the 3 groups in the Na+,K(+)-ATPase activity of erythrocyte ghosts. The heat- and acid-stable plasma extract dose-dependently inhibited Na+,K(+)-ATPase activity with a pattern similar to that of ouabain, but different from that of vanadate. The inhibitory activity of plasma extract was not influenced by polyclonal digoxin antibody which almost completely prevented digoxin-induced inhibition and slightly but significantly reduced the ouabain-induced one. The results indicate that the plasma extract has ouabain-like inhibitory activity on Na+,K(+)-ATPase and that it is not endogenously synthesized digoxin itself, but a substance differing in structure from digoxin. Furthermore, the ouabain-like Na+,K(+)-ATPase inhibitory activity in NP plasma was significantly lower than that in PIH plasma, which was similar to that in N plasma. There were significant relationships between the ouabain-like Na+,K(+)-ATPase inhibitory activities in plasma and the diastolic and systolic blood pressures in NP and PIH groups. The results suggest that the lower ouabain-like Na+,K(+)-ATPase inhibitory activity in plasma probably participates in maintaining the blood pressure within the normal range during pregnancy and its failure may be involved in the genesis of PIH.     

Palytoxin binds to and inhibits kidney and erythrocyte Na+, K+-ATPase

Summary Hog kidney Na⁺, K⁺-ATPase, purified to the microsomal stage and activated with detergent, binds palytoxin, as shown by the nearly complete competition of the toxin with ³H-ouabain. The K _i-values of palytoxin, but not of ouabain, depend on the protein concentration; this indicates additional binding sites for the toxin on kidney membranes. — Palytoxin inhibits the enzymatic activity of the detergent-activated preparation nearly completely (IC₅₀ 8·10^–7 mol/l). Inhibition of ATPase activity and of ouabain binding are promoted by borate, a known activator of palytoxin. — Palytoxin also inhibits the Na⁺, K⁺-ATPase of erythrocyte ghosts in the same dose range.The data are discussed in context with the hypothesis (Chhatwal et al. 1983) that palytoxin raises the cellular permeability by altering the state of Na⁺, K⁺-ATPase or its environment.Part of the thesis (Dr. rer. nat.) of H. Böttinger     

Interaction of bretylium tosylate with guinea-pig myocardial Na(+)-K(+)-ATPase.

1. The effects of bretylium on myocardial Mg(2+)-dependent, Na(+)-K(+)-ATPase (EC 3.6.1.3) activity were compared with those of ouabain in guinea-pig heart preparations. 2. Both ouabain and bretylium inhibited microsomal Na(+)-K(+)-ATPase activity in a concentration-dependent fashion in the range of 0.01-100 and 1.0-4000 microM, respectively. The IC50 values were 1.93 +/- 0.27 microM for ouabain and 2.45 +/- 0.17 mM for bretylium. 3. In another set of experiments, the effects of bretylium on the enzyme activity were tested in combination with 2.5 or 5.0 microM ouabain. 4. The combined effects of the two drugs resulted in a net reduction in the total inhibitory activities of the individual drugs, which became more marked the higher the bretylium concentration. This trend seems to suggest a competitive mode of interaction of the two drugs in their inhibitory actions on Na(+)-K(+)-ATPase activity. 5. The results demonstrate therefore that bretylium is a potent inhibitor of ouabain-inhibited ATP hydrolysis by myocardial Na(+)-K(+)-ATPase. These actions may be pertinent with regard to some of its cardiac actions.     

Gastric H+, K(+)-ATPase inhibition by catechins.

Five catechins, (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate, inhibited gastric H+, K(+)-ATPase activity with IC50 values ranging from 1.7 x 10(-4) to 6.9 x 10(-8) M, with (-)-epigallocatechin gallate as the most potent inhibitor. The intensity of inhibitor activity paralleled the number of phenolic hydroxy groups in the molecule. The inhibition of the enzyme by (-)-epicatechin was competitive with respect to ATP and noncompetitive with respect to K+. These findings suggest that the anti-secretory and anti-ulcerogenic effects of catechins previously reported, are due to their inhibitory activity on gastric H+, K(+)-ATPase.     

Inhibitory effects of ginsenoside Rg1 and Rb1 on rat brain microsomal Na+,K(+)-ATPase activity

Rat brain microsomal Na+, K(+)-ATPase activity was inhibited significantly and rapidly by ginsenoside Rb1. The IC50 of Rb1 for Na+,K(+)-ATPase was 6.3 +/- 1.0 mumol/L. The inhibition was enhanced with increasing the concentration of Rb1 or decreasing that of Na+ and K+. Kinetic analysis revealed that ginsenoside was an uncompetitive inhibitor with respect to ATP. The inhibitory effect of Rg1 on rat brain microsomal Na+,K(+)-ATPase was much weaker than that of Rb1.     

Inotropic effects and Na+,K+-ATPase inhibition of ouabain in isolated guinea-pig atria and diaphragm

Effects of ouabain on force of contraction were compared in electrically driven isolated tissue preparations of guinea-pig left atria and diaphragm. A distinct and steady positive inotropic effect of ouabain was observed in atrial preparations, whereas in diaphragm preparations, ouabain produced only a slight and transient positive inotropic effect, followed by the negative inotropic phase. The transient positive inotropic effect of ouabain was observed even in the absence of extracellular calcium, but was markedly dependent on the extracellular sodium concentration. In vitro [3H]ouabain binding studies revealed that the affinity of Na+,K+-ATPase for ouabain was about eight times higher and tissue concentration of the enzyme was significantly lower in diaphragm than in cardiac tissue. The Ki value for ouabain inhibition of the cardiac Na+,K+-ATPase was also approximately ten times higher than for the diaphragm enzyme. Ouabain-sensitive 86Rb uptake, an estimate of sodium pump activity, was inhibited by ouabain at a time when it produced its transient positive inotropic effect in diaphragm preparations. These results indicate that the lack of a distinct and steady positive inotropic effect of ouabain in diaphragm was due neither to the difference in the ouabain-Na+,K+-ATPase interaction between diaphragm and cardiac tissues nor the failure of sodium pump inhibition by ouabain in diaphragm.     

Antioxidant treatment normalizes renal Na+,K+-ATPase activity in leptin-treated rats

Hyperleptinemia may be involved in the pathogenesis of obesity-associated hypertension, however, the mechanism of hypertensive effect of leptin is incompletely elucidated. Previously, we have demonstrated that chronic hyperleptinemia causes up-regulation of renal Na+,K+-ATPase and decreases urinary Na+ excretion. Herein, we investigated whether antioxidant treatment could correct these abnormalities. The study was performed on male Wistar rats. Leptin administered for 7 days (0.25 mg/kg twice daily sc) increased systolic blood pressure by 20.6%. Leptin had no effect on urine output and creatinine clearance but reduced sodium excretion by 40.1%. Na+,K+-ATPase activity in the renal cortex and medulla was higher in leptin-treated rats by 24.3% and 80.6%, respectively. In addition, hyperleptinemia was associated with an increase in plasma and urinary 8-isoprostanes and reduced urinary excretion of nitric oxide (NO) metabolites and cGMP. Co-treatment with a superoxide dismutase mimetic, tempol, or an NAD(P)H oxidase inhibitor, apocynin (2 mM in the drinking water), prevented leptin-induced blood pressure elevation, normalized plasma and urinary 8-isoprostanes, urinary excretion of sodium, NO metabolites and cGMP, as well as prevented up-regulation of renal Na+,K+-ATPase activity. These data suggest that hyperleptinemia increases renal Na+,K+-ATPase activity and reduces natriuresis by inducing oxidative stress-dependent NO deficiency. Antioxidant treatment is effective in leptin-induced hypertension and should be considered in controlling blood pressure in hyperleptinemic obese individuals.     

Modulatory effect of some steroid hormones, their glucuronides and ouabain-like compounds on Cavia cobaya kidney Na+,K(+)-ATPase activity

1. Ouabain-like compounds (approx. mol. wt 700, 2,000 and 4,000 Da) were purified from plasma of essential hypertensive patients. 2. Dose-response experiments performed with (a) steroid hormones, (b) their glucuronides and (c) ouabain-like compounds, emphasize a modulatory effect [activation of the Na,K-ATPase at very low concentrations of ligand, inhibition at higher levels; apparent Ki: (a) between 1 and 0.5 mM; (b) between 1 and 0.5 microM; and (c) between 10 and 1 nM; maximum enhancement of the enzymatic activity: (a) +20%; (b) +45%; and (c) +100%]. 3. Displacement experiments of [3H]ouabain evidence a high competition of the ligands towards the cardioglycoside. The relative I50s are: (a) between 1 and 0.5 mM; (b) between 10 and 1 microM; and (c) between 10 and 0.01 nM.     

Inhibition of noradrenaline release from cerebrocortical synaptosomes and stimulation of synaptosomal Na+,K(+)-ATPase activity by morphine in rats

The effects of morphine on noradrenaline (NA) release from rat cerebrocortical synaptosomes and on the synaptosomal Na+,K(+)-ATPase activity were determined. Morphine (10(-3)-10(-5) M) caused a dose-related inhibition of enhanced prelabelled [3H]NA release evoked by a high concentration of K+ from synaptosomes and this inhibitory action of morphine was antagonized by the specific antagonist naloxone (10(-4), 10(-5) M). Morphine dose-dependently stimulated the synaptosomal Na+,K(+)-ATPase activity but not Ca2(+)-ATPase activity in the incubation medium containing 2.2 x 10(-6)-4.7 x 10(-7) M free Ca2+, and this stimulatory effect was antagonized by naloxone. These results suggest that morphine may have some role in the suppression of membrane depolarization and/or the release of NA through its stimulatory action on the Na+,K(+)-ATPase activity in rat cerebral cortex.     

Altered cardiac Na+,K+-ATPase activity in prehypertensive spontaneously hypertensive rat

Na⁺,K⁺-ATPase activity, Na⁺-dependent phosphorylation, and [³H]ouabain binding in sarcolemma prepared from 4 week old spontaneously hypertensive rat(SHR) ventricles were compared to the same parameters in sarcolemma from age matched normotensive Wistar-Kyoto (WKY) rat ventricles to examine whether the reduced myocardial Na⁺-pump activity in SHR is an inherited enzymatic defect or a second phenomenon due to sustained hypertension. The total body weights, ventricular weights, and blood pressures were the same for SHR and WKY. No significant differences in sarcolemmal protein content and protein recovery were noted between the two groups. Sarcolemma isolated from SHR ventricles showed significantly less Na⁺, K⁺-ATPase activity and number of phosphorylation sites when, compared to sarcolemma from the WKY ventricles. Equilibrium binding of [³H]ouabain and the tumover number of myocardial Na⁺,K⁺-ATPase, however, were the same for both groups. These results indicate that the low affinity (α, or α₁) isoform for ouabain is reduced in SHR compared to WKY but that the high affinity (α+, or α₂) α isoform is the same in ventricles of SHR and WKY. The reduced amount of isoform of the Na⁺,K⁺-ATPase in, prehypertensive SHR ventricles may play some role in the development of hypertension.