Calmodulin-stimulated plasma membrane (Ca2+ + Mg2+)-ATPase: inhibition by calcium channel entry blockers

Calcium channel entry blockers representing different structural classes were studied for their effects on human erythrocyte basal and calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. Effects on the activity of (Mg2+)-ATPase and (Na+ + K+)-ATPase were also assessed. Of the four Ca2+ entry blockers tested, only verapamil and diltiazem specifically inhibited the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase activity, the basal enzyme activity being unaltered by these drugs. Other membrane-associated ATPases were not affected. Calmodulin concentration effect curves showed the inhibition by verapamil (10(-3) M) and diltiazem (10(-3) M) to be non-competitive. This concentration inhibited the calmodulin-dependent increment (5.1 nM calmodulin) of the ATPase activity by 35 and 36% respectively. Similarly, both drugs inhibited the Ca2+-activation process of calmodulin-stimulated activity in a non-competitive manner, decreasing Vmax by 23 and 17% respectively. Basal (Ca2+ + Mg2+)-ATPase activity was not affected by verapamil or diltiazem at any calcium concentration. In contrast, cinnarizine non-specifically inhibited all four membrane ATPases including calmodulin-stimulated (Ca2+ + Mg2+)-ATPase activity at concentrations above 3 X 10(-6) M. Nifedipine was without effect on any of the four membrane ATPases. From this we conclude that certain calcium channel entry blockers can inhibit calmodulin-regulated plasma membrane Ca2+-pump ATPase. Therefore, this identifies an additional functional low affinity receptor in the plasma membrane for some of the calcium channel entry blockers.     

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.     

Isoproterenol inhibition of potassium release from rat parotid gland

The mechanism of isoproterenol-induced inhibition of potassium release from rat parotid slices has been determined. Spontaneous potassium release from the slices was significantly inhibited by isoproterenol at concentrations above 10(-6) M. This isoproterenol effect was completely abolished in the presence of propranolol (10(-5) M) and ouabain (10(-3) M) and was abolished during Na+-exclusion from the incubation medium. Isoproterenol caused an enhancement of the microsomal Na+, K+-ATPase activity at concentrations above 10(-5) M, and this activity was inhibited by propranolol (10(-5) M). The stimulatory effect of isoproterenol on the Na+, K+-ATPase exhibited a strong correlation with the inhibition of potassium release on each dose of isoproterenol. Moreover, dibutyryl cyclic AMP at concentrations above 10(-4) M inhibited potassium release in a dose-dependent manner and cyclic AMP caused an enhancement of the microsomal Na+, K+-ATPase activity. These results suggest that the inhibitory effect of isoproterenol on potassium release is clearly derived from the elevated Na+, K+-ATPase activity and that it may in part be mediated by cyclic AMP.     

Inhibition of Na+,K+-ATPase activity by phospholipase A2 and several lysophospholipids: possible role of phospholipase A2 in noradrenaline release from cerebral cortical synaptosomes

p-Bromophenacyl bromide (PBPB), quinacrine and indomethacin, which inhibit phospholipase A2 (PLA2; EC 3.1.1.4) activity in several tissues, caused a dose-dependent inhibition of prelabelled [3H]noradrenaline ([3H]NA) release evoked by high concentrations of K+ from rat cerebral cortical synaptosomes. Release of prelabelled [3H]NA was caused by natural lysophosphatidic acid (LPA; 10(-6)-10(-5) g mL-1) and lysophosphatidylcholine (LPC; 10(-6)-10(-5) g mL-1) and synthetic LPA (6 x 10(-6), 2 x 10(-5) M) and LPC (6 x 10(-6), 2 x 10(-5) M), but not by natural lysophosphatidylserine (LPS; 10(-5) g mL-1), lysophosphatidylethanolamine (LPE; 10(-5) g mL-1) and lysophosphatidylinositol (LPI; 10(-5) g mL-1). The release evoked by natural LPA and LPC could be inhibited only marginally by PBPB and quinacrine. Phosphatidic acid (PA)-specific and phosphatidylcholine (PC)-specific PLA2 activities from rat cerebral cortical synaptosomes were stimulated in incubation medium containing high concentrations of K+ or calcium ionophore A23187. Low concentrations of PLA2 (10(-6)-10(-8) g mL-1, from bee venom) inhibited the synaptic membrane Na+,K+-ATPase activity in incubation media with intracellular levels of free Ca2+. Several lysophospholipids (LPLs), metabolites of the PLA2 type, also inhibited the synaptic membrane Na+,K+-ATPase activity in a dose-dependent manner. The minimum effective concentrations of natural LPA, LPC, LPS, LPI and LPE were 10(-6), 4.7 x 10(-6), 10(-5), 4.7 x 10(-5) and 4.7 x 10(-5) g mL-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo and in vitro effects of aldrin on rat brain synaptosomal Mg2+ and Na+,K(+)-adenosine triphosphatase

Aldrin, a chlorinated hydrocarbon, inhibited rat brain synaptosomal membrane-bound Na+,K(+)-adenosine triphosphatase (ATPase) and Mg2(+)-ATPase activities under in vivo and in vitro conditions. Na+,K(+)-ATPase was non-competitively inhibited whereas Mg2(+)-ATPase was inhibited uncompetitively. Arrhenius plots of both these ATPases without aldrin under in vivo and in vitro conditions were found to be linear. In the presence of aldrin, on the other hand, Arrhenius plots of the same ATPases were nonlinear. Slopes of Arrhenius plots of both ATPases under in vivo and in vitro condition were changed with change in temperature with aldrin. The activation energy (AE) of Na+,K(+)-ATPase and Mg2(+)-ATPase activities were changed over the temperature range 15-40 degrees in the presence of aldrin. These results thus suggest that aldrin increases the lipid fluidity of the synaptosomal membrane which may be a cause of inhibition of neuronal membrane-bound Na+,K+ and Mg2(+)-ATPase activities.     

Effect of hydroxychlorodiphenyl ethers (chlorinated pre-and isopredioxins) on erythrocyte membrane adenosinetriphosphatase activity

The effect of hydroxychlorodiphenyl ethers (HO-ClX-DPEs; chlorinated pre- and isopredibenzodioxins), contaminants of technical chlorophenol preparations, on human erythrocyte membrane-bound adenosinetriphosphatases (ATPases) has been investigated. Both 2- and 3-HO-Cl9-DPE inhibited the Na+ + K+-activated, Mg2+-dependent ATPase (Na+, K+, Mg2+-ATPase). The Mg2+-dependent ATPase (Mg2+-ATPase) was stimulated at lower concentration of these compounds, but at higher concentrations there was a gradual decrease in the extent of stimulation, 2-Hydroxy-21, 41, 41-trichlorodiphenyl ether (2-HO-Cl3-DPE; Irgasan DP-300; Triclosan) was not as effective as the nonachloro compounds at inhibiting Na+, K+, Mg2+-ATPase and was inactive at stimulating Mg2+-ATPase. Pure pentachlorophenol (PCP) caused both inhibition of Na+, K+, Mg2+-ATPase and stimulation of Mg2+-ATPase, although each effect required a higher concentration of PCP than was needed for the HO-CL9-DPEs. The possible relationship of the effects of HO-CL x-DPEs on human erythrocyte membrane ATPase activities to the potent hemolytic activity of these compounds is discussed.     

Effect of metavanadate on the uptake and release of noradrenaline in rat brain cerebral cortex slices

The effect of vanadium (as VO3-) on the uptake and release of tritiated noradrenaline ([3H]NA) was studied in vitro in rat cerebral cortex slices. Vanadate inhibited [3H]NA uptake and the inhibition was dependent upon concentration and on incubation time. The IC50 value (20 min incubation) was 8 X 10(-5) M of vanadate. Inhibition of Na+, K+ -ATPase activity by VO3-, chelation of noradrenaline or autooxidation of catecholamine by this oxyanion might contribute to the decrease of [3H]NA uptake. Vanadate inhibited also the release of [3H]NA in a time- and concentration-dependent fashion.     

Sympathetic mechanisms in diet-induced thermogenesis: modification by ciclazindol and anorectic drugs.

1 The sympathetic noradrenergic activation of brown adipose tissue and the biochemical mechanisms involved in diet-induced thermogenesis were studied in rats. 2 A close correlation was found between brown adipose tissue Na+, K+-adenosinetriphosphatase (Na+, K+-ATPase) activity in vitro and in vivo measurements of resting oxygen consumption (VO2). The effects of noradrenaline on in vitro NA+, K+-ATPase activity in brown adipose tissue and in vivo VO2 could be mimicked by a variety of agents. These included beta-adrenoceptor agonists and agents known to induce the release of noradrenaline or inhibit the noradrenaline uptake process. The pharmacological evidence suggests that dopaminergic mechanisms may also be involved in the control of thermogenesis. 3 Amphetamine did not increase VO2 in rats without causing associated increases in locomotor activity. Ciclazindol at doses of 3-30 mg/kg intraperitoneally increased VO2 but did not appear to increase locomotor activity or evoke any other signs of CNS stimulation including lengthening of time to sleep onset or stereotypy. Separation of metabolic and CNS effects occurred only at the lowest dose of mazindol used (0.3 mg/kg i.p.). These results are probably a reflection of (a) the relative abilities of these drugs to inhibit brain and brown adipose noradrenaline uptake processes and (b) the relatively high accumulation of ciclazindol in brown adipose. 4 Of the drugs tested, only ciclazindol was a more potent inhibitor of the noradrenaline uptake system in brown adipose tissue (BAT) than in brain. Kinetic analysis also revealed that the actions of ciclazindol on the NA uptake system and Na+, K+-ATPase in BAT differed from those of mazindol. 5 These findings suggest that ciclazindol may produce an energy wasting effect in rodents without causing overt CNS stimulation; the implications of these findings in terms of human obesity are discussed.     

Inhibition by p-nitrophenylphosphate of acetylcholine release induced by Na+-deprivation

The effect of p-nitrophenylphosphate (p-NPP) on the release of acetylcholine evoked by drugs and ionic environments known to inhibit Na+, K+-ATPase was studied in isolated cortical slices of rat brain and longitudinal muscle strip of guinea-pig ileum. p-NPP inhibited the release of acetylcholine induced by sodium deprivation provided that the circumstances were in favour of the function of the K+-activated part of ATPase. However, it failed to antagonize the increase in the acetylcholine release elicited by omission of K+ or by administration of ouabain. Therefore it is concluded that the K+-stimulated phosphatase moiety of the Na+, K+-ATPase might be involved in the release of acetylcholine.     

Evidence against an involvement of Na+, K+-ATPase in antiarrhythmic mechanism of phenytoin

Phenytoin (Diphenylhydantoin, DPH) did not activate Na+,K+-ATPase activity prepared from both canine cardiac and renal tissues at any ratio of NA+ to K+ in standard assay medium and this drug failed to relieve the inhibitory effect of ouabain on Na+,K+-ATPase. With the Na+,K+-ATPase partially purified from the cardiac tissue the maximum number of ouabain binding sites was 50 pmol ouabain per mg enzyme and the dissociation constant (Kd) was 4 X 10(-8) mol/l. Scatchard analysis of ouabain binding to Na+,K+-ATPase indicates that DPH did not significantly alter these parameters. The release of ouabain from Na+,K+-ATPase and ouabain complex was also not significantly influenced by DPH which indicates that the antiarrhythmic action of DPH against digitalis-induced arrhythmia is not due to a simple displacement of ouabain from Na+,K+-ATPase molecules.     

Electrogenic Na+/Ca2+-exchange regulation by alpha2-receptor in peripheral sympathetic nerve

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release was measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5)M; corticosterone, 5 x 10(-5)M) and after blocking the MAO-enzyme by pargyline (1,2 x 10(-4)M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the stimulation-induced release of [3H]NA in a tetrodotoxin (TTX, 10(-7)M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5)M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2,5 to 0,25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3)M). Direct correlation was obtained between the extent of VSCC-inhibition and the transmitter release enhancing-effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7)M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e. in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5)M) significantly inhibited the nerve-evoked release of NA in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions, in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7)M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6)M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs, brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be inhibited by presynaptic alpha2-receptor activation.     

Inhibition of Na+,K(+)-ATPase activity by beta-eudesmol, a major component of atractylodis lanceae rhizoma, due to the interaction with enzyme in the Na.E1 state.

beta-Eudesmol, a major component of the crude drug "So-jutsu" (Atractylodis Lanceae Rhizoma), inhibited Na+, K(+)-ATPase activity most strongly among the various kinds of phosphatases examined. It also inhibited Ca(2+)-ATPase and H+, K(+)-ATPase, but to a lesser extent. Its effect on Mg(2+)-ATPase was minute. No effects on H(+)-ATPase or alkaline and acid phosphatase activities were observed. The effects of beta-eudesmol on horse kidney Na+, K(+)-ATPase were studied in detail, and the following results were obtained: (1) beta-eudesmol inhibited the Na+, K(+)-ATPase activity with an I50 value of 1.6 x 10(-4) M. The mode of its inhibition was uncompetitive with respect to ATP; (2) it prevented the stimulation of enzyme activity by Na+. The inhibition gradually increased in accord with the increase of Na+ concentration, and it was constant when Na+ was higher than 6.3 mM; (3) it did not alter the K+ concentration necessary for half-maximal activation (K0.5 for K+); and (4) it inhibited the enzyme activity with a mode of action different from ouabain. Phosphorylation of enzyme with [gamma-32P]ATP was inhibited by beta-eudesmol with an I50 of 1.4 x 10(-4) M. The inhibition was greater in 1 M NaCl than in 0.1 M NaCl. It had no effects on dephosphorylation steps, i.e. none of the non-specific, the ADP-sensitive (Na.E1-P----Na.E1) and the K(+)-dependent (E2-P----K.E2) dephosphorylation processes were affected. These results suggest that beta-eudesmol, a relatively specific inhibitor of Na+, K(+)-ATPase, interacts with the enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P.     

Effect of some Pteridine compounds on the Na+ + K+)-ATPase and on the cardiac glycoside receptor of human heart).

Pteridine compounds are known to block Na+-reabsorption and K+-secretion in epithelial cells (salivary duct of the rat), which actively transport Na+ and K+ against an electrochemical gradient. Furthermore, there have been reports on antagonistic effects of these substances in digitalis induced arrhythmias. Therefore the actions of triamterene (Jatropur, Dyrenium), the sulfuric acid ester and the methylether of p-hydroxytriamterene (OH-triamterene) and OH-triamterene on specific [3H] g-strophanthin (ouabain) binding and Na+ + K+)-ATPase activity of isolated human cardiac cell membranes were investigated. Triamterene, the sulfuric acid ester and the methylether of OH-triamterene inhibit (Na+ + K+)-ATPase activity only at very high concentrations (10(-5)--10(-4) M). OH-Triamterene does not inhibit this enzyme at concentrations lower than 10(-3) M. The specific binding of [3H] g-strophanthin to human cardiac cell membranes is inhibited half maximally at relatively high concentrations, too (10(-5)--10(-4) M). These results are rather indicative of unspecific effects due to membrane sites of action other than the (Na+ + K+)-ATPase or the cardiac glycoside receptor.     

Trimetazidine inhibits Na+,K(+)-ATPase activity, and overdrive hyperpolarization in guinea-pig ventricular muscles.

The effect of trimetazidine on Na+,K(+)-ATPase activity or the Na+,K+ pump was studied in guinea pig ventricular muscles with the use of biochemical and electrophysiological methods. The effect of trimetazidine on enzyme activity was compared with that in the liver, jejunum and kidney obtained from the same species. Na+,K(+)-ATPase activity in the heart and liver was significantly and concentration dependently decreased by trimetazidine (above 1.5 x 10(-5) M). Even the highest concentration (1.5 x 10(-4) M) of trimetazidine failed to decrease the Na+,K(+)-ATPase activity in the jejunum and kidney. The membrane potential was recorded in the ventricular muscle with a microelectrode. The hyperpolarization which followed 1-min overdrive stimulation (3.3 Hz) was decreased by trimetazidine (1.5 x 10(-4) M), but the depolarization during the stimulation was not affected by this drug. Ouabain, a potent Na+,K+ pump inhibitor, markedly decreased the overdrive hyperpolarization and increased the depolarization during the stimulation (10(-7), 5 x 10(-7), 10(-6) M). Therefore, the effect of trimetazidine and ouabain on the Na+,K+ pump-mediated alteration in the resting potential is different, suggesting that trimetazidine has additional direct membrane effects, e.g. a decrease in K+ conductance. In conclusion, trimetazidine inhibits Na+,K(+)-ATPase activity and thus the Na+,K+ pump in the ventricular muscles but with an inhibitory effect about 300 times less than that of ouabain. Trimetazidine inhibited the Na+,K(+)-ATPase in the liver as well, but not that in jejunum and kidney.     

In vitro effects of pyrethroids on rat brain and liver ATPase activities

In vitro sensitivity of rat brain and liver ATPases to pyrethroid insecticides, belonging to three categories based on the structural configuration, was studied. Rat brain and liver P2 fractions were prepared by the conventional centrifugation method, and rat brain synaptosomes were prepared by Ficoll-sucrose gradient centrifugation method. Na+, K+-ATPase and oligomycin-sensitive and -insensitive Mg2+- ATPases were determined in brain P2 fraction, whereas in liver P2 fraction only oligomycin-sensitive and -insensitive Mg2+-ATPase activities were determined. [3H]Ouabain binding studies were carried with rat brain synaptosomes. Most of the pyrethroid compounds tested inhibited brain and liver oligomycin-sensitive Mg2+-ATPases at micromolar concentrations. Type II compounds were more effective as compared to Type I compounds. Oligomycin-insensitive Mg2+-ATPase was not affected by any of the compounds tested except deltamethrin, which showed significant effect on liver enzyme. Na+, K+-ATPase of brain was less sensitive to these pyrethroids as compared to oligomycin-sensitive Mg2+-ATPase. [3H]Ouabain binding to rat brain synaptosomes was not affected significantly by these pyrethroid insecticides. These results suggest that inhibition of oligomycin-sensitive Mg2+-ATPase may be involved in the toxicity of pyrethroid compounds.     

Interaction of catecholamines and ethanol on the kinetics of rat brain (Na+ + K+)-ATPase

The effects of catecholamines (CA) and ethanol (EtOH), singly and in combination, on the kinetics of rat brain (Na+ + K+)-ATPase were studied. Addition of 0.05 M EtOH alone did not change Vmax or Km for K+, Na+, Mg2+ and ATP. Addition of 0.1 mM dopamine (DA) or noradrenaline (NA) alone stimulated the enzyme activity in presence of vanadium-containing ATP as substrate, but not with vanadium-free ATP except in the presence of high Mg2+ : ATP ratios. CA alone decreased the Km slightly for K+ and by about 50% for ATP, increased it for Mg2+ and did not change it for Na+. However, the combination of DA or NA + EtOH produced a marked inhibition which was competitive for K+, and uncompetitive or mixed for Mg2+, Na+ and ATP. The inhibitory effect of NA + EtOH was abolished in 20 mM K+. These findings suggest that NA sensitizes the enzyme to EtOH inhibition at physiological K+ concentrations, by conformational change away from the outwardly facing K+-binding E2P for to the inwardly facing Na+-binding E1P form.     

Effects of mercuric chloride on [3H]dopamine release from rat brain striatal synaptosomes

Electrophysiological studies employing amphibian neuromuscular preparations have shown that mercuric chloride (HgCl2) in vitro increases both spontaneous and evoked neurotransmitter release. The present study examines the effect of HgCl2 on the release of [3H]dopamine from synaptosomes prepared from mammalian brain tissue. Mercuric chloride (3-10 microM) produces a concentration-dependent increase in spontaneous [3H]dopamine release from "purified" rat striatal synaptosomes, in both the presence and absence of extra-synaptosomal calcium. The effects of HgCl2 on transmitter release from amphibian neuromuscular junction preparations resemble those produced by the Na+, K+-ATPase inhibitor ouabain. Experiments were performed to determine whether the HgCl2 effects on mammalian synaptosomal dopamine release are a consequence of Na+, K+-ATPase inhibition. Na+, K+-ATPase activity in lysed synaptosomal membranes is inhibited by HgCl2 (IC50 = 160 nM). However, mercuric chloride in the presence of 1 mM ouabain still increased [3H]dopamine release. The specific inhibitor of Na+-dependent, high-affinity dopamine transport, RMI81,182 inhibited ouabain-induced [3H]dopamine release whereas it had no effect on HgCl2-induced [3H]dopamine release. These data suggest that augmentation of spontaneous [3H]dopamine release by HgCl2 probably is not mediated by an inhibition of Na+, K+-ATPase and HgCl2 does not act directly on the dopamine transporter.     

Stimulation of neuronal Na+, K+-ATPase by calcium

The effect of calcium on ATP-phosphohydrolase activity of rat brain homogenates has been investigated. In both the presence and absence of the chelating agent EDTA, free calcium within the concentration range 1.2 x 10(-7) to 5.0 x 10(-4) moles/l consistently affected only the activity of Na+, K+-ATpase; the activities of Mg2+-ATPase and Na+-ATPase were essentially unchanged by Ca2+; Ca2+-ATPase could not be demonstrated. In either the presence or absence of EDTA, concentrations of free-Ca2+ above 3 x 10(-6) moles/l caused an inhibition of Na+,K+-ATPase activity. In the presence of EDTA, concentrations of free-Ca2+ below 3 x 10(-6) moles/l were ineffective at altering Na+, K+-ATPase activity but, in the absence of EDTA, free-Ca2+ in this concentration range caused a marked stimulation of the enzyme. Evidence is presented to show that the stimulation of Na+, K+-ATPase by calcium is modulated by the regulatory protein calmodulin. Since the stimulation occurs over the range of concentrations at which calcium would be expected to be encountered within the cell, it is suggested that this is the major physiological effect of calcium on Na+, K+-ATPase.     

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.     

Inhibition of H+,K+ -ATPase by hinesol, a major component of So-jutsu, by interaction with enzyme in the E1 state

Hinesol, a major component of the crude drug "So-jutsu" (Atractylodis Lanceae Rhizoma), strongly inhibited H+,K+-ATPase activity with a IC50 value of 5.8x10(-5) M. It also inhibited Na+,K+-ATPase, Mg2+-ATPase, Ca2+-ATPase, and H+-ATPase activities, although the inhibition rate was lower. No effects on alkaline or acid phosphatase activities were observed. The mechanism by which hinesol inhibited H+,K+-ATPase activity was studied in detail. The inhibition was uncompetitive with respect to ATP, and it increased as the Mg2+ concentration was raised, whereas it was not affected by the K+ concentration. The activity of K+-dependent p-nitrophenyl phosphatase (K+-pNPPase), a partial reaction of H+,K+-ATPase, was inhibited by hinesol noncompetitively with respect to pNPP (IC50 value of 1.6x10(-4) M), and competitively with respect to K+, whereas it was not affected by the Mg2+ concentration. These results suggest that hinesol is a relatively specific inhibitor of H+,K+-ATPase. It appears that hinesol reacts with enzyme in the E1 state in the presence of ATP and Mg2+ and forms the complex hinesol-H+ E1-ATP or hinesol x E1-P, blocking the conformational change to the E2 state. Furthermore, hinesol enhanced the inhibitory effect of omeprazole on H+,K+-ATPase, and the inhibitory site of hinesol was different from that of omeprazole. The effect of So-jutsu as an anti-gastric ulcer agent may be ascribed to the inhibitory effect of hinesol on H+,K+-ATPase activity.