Chronic digoxin treatment on canine myocardial Na+, K+-ATPase

Summary In order to determine whether a prolonged inhibition of cardiac Na⁺, K⁺-ATPase causes a compensatory or adaptive change in this enzyme, the relationships among serum digoxin concentration, binding of digoxin to the enzyme and cardiac Na⁺, K⁺-ATPase and sodium pump activity were studied in dogs chronically treated with digoxin. Digoxin was injected intravenously twice daily up to 4 weeks. Two hours after the injection of a single non-toxic dose of digoxin, Na⁺, K⁺-ATPase and sodium pump activities were inhibited quantitatively in a manner corresponding to the binding of digoxin to the enzyme. The magnitude of sodium pump inhibition was reduced 12 h after the digoxin injection, with simultaneous decreases in serum digoxin concentration and the binding of digoxin to the enzyme. After 1 or 4 weeks of digoxin treatment with non-toxic doses, the relationships among serum digoxin concentration, binding of digoxin to cardiac Na⁺, K⁺-ATPase and the degree of cardiac Na⁺, K⁺-ATPase or sodium pump inhibition remained unchanged. The magnitude of the inhibition was related to serum digoxin concentrations and digoxin binding to cardiac Na⁺, K⁺-ATPase, in a manner similar to that observed after a single digoxin injection. After 4 weeks of digoxin treatment with toxic doses, these relationships were also unaffected. It was concluded that prolonged digoxin treatment fails to alter the inhibition of myocardial Na⁺, K⁺-ATPase by this agent.This work was supported by U.S. Public Health Service Grant HL-16052.     

Inhibitory characteristics of the mycotoxin penicillic acid on (Na+-K+)-activated adenosine triphosphatase

Penicillic acid, a cardioactive mycotoxin produced by various Penicillium molds, is a potent and selective inhibitor of membrane (Na⁺-K⁺)-adenosine triphosphatase (ATPase). A broad range of inhibition of activity by the toxin was demonstrated with a half-maximal concentration equal to 1.8 × 10^?8M. Inhibition was time and pH dependent and complete after 20–30 min preincubation within a narrow range of physiological pH. Kinetic evaluation of cationic substrate activation of (Na⁺-K⁺⁾-ATPase indicated competitive inhibition with regard to Na⁺ concentration and noncompetitive inhibition with regard to K⁺ concentration. Also K⁺ -dependent p-nitrophenyl phosphatase activity was not significantly altered by penicillic acid, and uncompetitive inhibition with regard to ATP activation of the enzyme was demonstrated. Preliminary binding studies indicated that inhibition of ATPase activity could be partially restored by repeated washing and by incubation with dithiothreitol and cysteine. Penicillic acid (high concentrations) impaired [³H]ouabain binding to membrane preparations but this effect was noncompetitive, indicating different sites of action for the two inhibitors. A significant linear correlation between reactive enzyme sulfhydryl content [SH] and ATPase activity in the presence of varying concentrations of toxin also was noted. It is postulated that penicillic acid inhibition of (Na⁺-K⁺) -ATPase occurs via critically accessible membrane thiol receptors regulating Na⁺-dependent phosphorylation of the transport enzyme.     

Effect of methyl isocyanate on rabbit cardiac Na+, K+-ATPase

 The present study describes the effect of methyl isocyanate (MIC) on rabbit cardiac microsomal Na⁺, K⁺-ATPase. Addition of MIC in vitro resulted in dose-dependent inhibition of Na⁺, K⁺-ATPase, Mg²⁺-ATPase and K⁺-activated p-nitrophenyl phosphatase (K⁺-PNPPase). Activation of Na⁺, K⁺- ATPase by ATP in the presence of MIC showed a decrease in V_max with no change in K_m. Similarly, activation of K⁺ PNPPase by PNPP in the presence of MIC showed a decrease in V_max with no change in K_m. The circular dichroism spectral studies revealed that MIC interaction with Na⁺, K⁺-ATPase led to a conformation of the protein wherein the substrates Na⁺ and K⁺ were no longer able to bind at the Na⁺- and K⁺-activation sites. The data suggest that the inhibition of Na⁺, K⁺-ATPase was non-competitive and occurred by interference with the dephosphorylation of the enzyme-phosphoryl complex. Received: 3 November 1994/Accepted: 23 February 1995     

粉防已碱对大鼠心肌微粒体ATPases的作用

体外在常规反应条件下,粉防已碱(Tet)对大鼠心肌微粒体Na⁺,K⁺-ATPase活性无明显影响,但浓度依赖性抑制Mg²⁺-ATPase(IC₅₀=179μmol/L).Tet 10和100μmol/L使哇巴因(Oua)抑制Na⁺,K⁺-ATPase的量效曲线平行右移.Tet 100μmol/L可显著提高低K⁺或高Ca²⁺浓度时的Na⁺,K⁺-ATPase活性,但未能明显增加低Na⁺浓度时该酶的活性.动力学分析提示Tet 100μmol/L增加Na⁺,K⁺-ATPase对ATP的亲和力,但不改变其最大反应速度。     

Action of methanethiol on membrane (Na+ , K+)-ATPase of rat brain.

The action of methanethiol (CH₃SH) on rat brain (Na⁺, K⁺)-ATPase was examined. The results show that CH₃SH acts at several sites on the enzyme system. The effects are characterized by an inhibition of the ATPase activity, but a concurrent stimulation of the associated K⁺-dependent phosphatase. The inhibitory effect of CH₃SH was of an apparently mixed type with respect to the activation of the ATPase by Na⁺ or by ATP suggesting that CH₃SH may inhibit the formation of phosphoenzyme intermediate in the ATPase reaction, and the inhibition may not be fully reversed by increasing Na⁺. Methanethiol inhibited the activation of the ATPase by K⁺ in an apparently uncompetitive manner, whereas it produced a competitive stimulation of the K⁺ activation of the K⁺-dependent phosphatase activity by increasing the affinity of K⁺ for the enzyme. There was no significant change in the apparent K_m for the substrate p-nitrophenyl phosphate for the phosphatase activity. These effects of CH₃SH may be relevant to its toxicity, and offer a possible molecular site of its action with implications for the encephalopathy of hepatic failure.     

Effects of aldehydes on sodium plus potassium ion-stimulated adenosine triphosphatase of mouse brain.

The biogenic aldehydes, 3,4-dihydroxyphenylglycolaldehyde and 3,4-dihydroxyphenylacetal-dehyde, derived from norepinephrine and dopamine, respectively, as well as acetaldehyde, porpionalde-hyde, benzaldehyde and phenylacetaldehyde, inhibited both Na⁺ + K⁺-activated ATPase and Mg²⁺-ATPase. In addition, K⁺ ion-dependent p-nitrophenylphosphatase activity was inhibited by these compounds. The Na⁺ + K⁺-ATPase was much more sensitive than Mg²⁺-ATPase of K⁺-activated phosphatase to inhibition by various aldehydes. The inhibition of Na⁺ + K⁺-ATPase by aldehydes was reversible and was non-competitive with ATP or K⁺ as the variable substrate or activator respectively. Addition of cysteine or mercaptoethanol protected the enzymes from inhibition by aldehydes. The concentrations of aldehydes which produced marked inhibition of Na⁺ + K⁺-ATPase ranged from 2 × 10^?2 M to 6 × 10^?6 M for acetaldehyde and 3,4-dihydroxyphenylglycoladehyde respectively. All aldehydes, including acetaldehyde, were more potent inhibitors of Na⁺ + K⁺-ATPase activity than was ethanol.     

Effect of ethanol on the kinetics of rat brain (Na+ + K+) ATPase and K+-dependent phosphatase with different alkali ions.

(Na⁺ + K⁺)-dependent ATPase [(Na + K)-ATPase] and K⁺-dependent p-nitrophenyl phosphatase [pNPPase] activities in rat brain heavy microsomal fractions were studied in the presence of 120 mM Na⁺ and varied concentrations of K⁺, Rb⁺, Cs⁺, Li⁺ or NH₄⁺. Scatchard and Hill plots indicated non-hyperbolicity (cooperativity) with all except Li⁺, which supported a considerably lower activity than any of the other ions tested. Addition of 0.22 M ethanol to the incubation mixtures produced a formally competitive inhibition of ATPase activity with K⁺, Rb⁺ and Cs⁺, a non-competitive inhibition with Li⁺, and a mixed inhibition with NH₄⁺. The changes in pNPPase activity generally followed a similar but less clear-cut pattern. The values of the Hill constants were not changed for either enzyme activity. The findings are interpreted as evidence that ethanol inhibits ATPase activity by inducing conformational changes which alter the consequences of ion binding to the various receptor sites.     

Effect of penicillic acid on adenosine triphosphatase activity in the mouse.

The effect of penicillic acid on mouse tissue ATPase was examined both in vitro and in vivo. Penicillic acid inhibited in vitro the (Na⁺, K⁺)-activated ATPase in the 13,000g (B) fraction in a dose-dependent manner with calculated I50 values of 2.5 and 3.3 × 10^?5m for brain and kidney, respectively. Similar inhibition in the microsomal fraction also was observed with I50 values of 6.0 and 1.0 × 10^?5m for brain and kidney tissues, respectively. Brain and kidney (Na⁺, K⁺)-ATPase activity also was inhibited by penicillic acid in vivo. An inhibition time response of (Na⁺, K⁺)-stimulated ATPase in brain and kidney tissue from mice receiving 80 mg/kg penicillic acid in saline was noted while a dose-dependent effect was demonstrated in the brain microsomal fraction and in the kidney B and microsomal fractions. Mg²⁺-Activated ATPase activity was not significantly impaired except for oligomycin-insensitive Mg²⁺-ATPase in the brain B fraction. Thus, in vitro and in vivo results suggested a possible correlation between inhibition of (Na⁺, K⁺)-ATPase activity and penicillic acid-mediated toxicity.     

Epigallocatechin-3-gallate is an inhibitor of Na,K-ATPase by favoring the E1 conformation

Four catechins, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin, inhibited activity of the Na⁺,K⁺-ATPase. The two galloyl-type catechins were more potent inhibitors, with IC₅₀ values of about 1 μM, than were the other two catechins. Inhibition by epigallocatechin-3-gallate was noncompetitive with respect to ATP. Epigallocatechin-3-gallate reduced the affinity of vanadate, shifted the equilibrium of E₁P and E₂P toward E₁P, and reduced the rate of the E₁P to E₂P transition. Epigallocatechin-3-gallate potently inhibited membrane-embedded P-type ATPases (gastric H⁺,K⁺-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase) as well as the Na⁺,K⁺-ATPase, whereas soluble ATPases (bacterial F₁-ATPase and myosin ATPase) were weakly inhibited. Solubilization of the Na⁺,K⁺-ATPase with a nonionic detergent reduced sensitivity to epigallocatechin-3-gallate with an elevation of IC₅₀ to 10 μM. These results suggest that epigallocatechin-3-gallate exerts its inhibitory effect through interaction with plasma membrane phospholipid.     

Biochemical basis for the low sensitivity of the rat heart to digitalis

Summary The concentration of cardiac glycosides to produce positive inotropic effects in the rat heart is markedly higher than that in other species. Such a low digitalis sensitivity of the rat heat is attributed to the low affinity of cardiac Na⁺, K⁺-ATPase for digitalis in this species. In the present study the biochemical cause which is responsible for the formation of the unstable complex between the glycosides and Na⁺, K⁺-ATPase or positive inotropic, receptor in the rat heart was examined using Na⁺, K⁺-ATPase preparations obtained from rat hearts, guinea-pig hearts and rat brains as well as isolated, electrically stimulated atrial preparations obtained from these animals. Monensin, which alters transmembrane Na⁺ movements without interacting with the cardiotonic sites on Na⁺, K⁺-ATPase, had equivalent potencies in guinea-pig and rat hearts. Cassaine, which lacks a lactone ring but interacts with cardiotonic sites on Na⁺, K⁺-ATPase, increased the force of contraction in guinea-pig hearts at low, but in rat hearts only at high, concentrations. AY-22,241 (Actodigin) and prednisolone-3,20-bisguanylhydrazone (PBGH) bind to cardiotonic sites on Na⁺, K⁺-ATPase and had a similar spectrum as cassaine in these two species. Actodigin has an altered lactone ring resulting in a marked reduction of the inotropic potency, and PBGH is devoid of this structure. With the latter agent, the rabbit was as insensitive as the rat, although both rabbit and guinea-pig are equally sensitive to digitalis. K⁺ delayed the development of the positive inotropic action of ouabain with a minimal effect on the plateau response in guinea-pig hearts. In rat hearts, however, K⁺ markedly lowered the plateau response without affecting the time course of the response. These results indicate that the low sensitivity of the rat heart to digitalis is due to a difference in the glycoside binding sites on Na⁺, K⁺-ATPase; but the difference cannot be explained by the lack of a lactone ring complementary binding sites. The difference seems to result from the absence of lipid barrier which regulates the rate of release of cardiac glycosides from their binding sites on Na⁺, K⁺-ATPase.This work was supported by U.S. Public Health Service grant, HL-16052 and by the Michigan Heart Association     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Activation by reduced glutathione of methotrexate transport into isolated rat liver cells

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na⁺-free, Na⁺ and K⁺-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP²⁺) and ATP.It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K⁺. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na⁺/K⁺ ratio, both effects were partially overcome by GSH. After treatment by DIP²⁺, Na⁺/K⁺ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K⁺ and accumulation of Na⁺ Here neither effect could be reversed by GSH; consequently, high cellular amounts of K⁺ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake.It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.     

Mechanism of action of AZD0865, a K+-competitive inhibitor of gastric H+,K+-ATPase

AZD0865 is a member of a drug class that inhibits gastric H⁺,K⁺-ATPase by K⁺-competitive binding. The objective of these experiments was to characterize the mechanism of action, selectivity and inhibitory potency of AZD0865 in vitro. In porcine ion-leaky vesicles at pH 7.4, AZD0865 concentration-dependently inhibited K⁺-stimulated H⁺,K⁺-ATPase activity (IC₅₀ 1.0 ± 0.2 μM) but was more potent at pH 6.4 (IC₅₀ 0.13 ± 0.01 μM). The IC₅₀ values for a permanent cation analogue, AR-H070091, were 11 ± 1.2 μM at pH 7.4 and 16 ± 1.8 μM at pH 6.4. These results suggest that the protonated form of AZD0865 inhibits H⁺,K⁺-ATPase. In ion-tight vesicles, AZD0865 inhibited H⁺,K⁺-ATPase more potently (IC₅₀ 6.9 ± 0.4 nM) than in ion-leaky vesicles, suggesting a luminal site of action. AZD0865 inhibited acid formation in histamine- or dibutyryl-cAMP-stimulated rabbit gastric glands (IC₅₀ 0.28 ± 0.01 and 0.26 ± 0.003 μM, respectively). In ion-leaky vesicles at pH 7.4, AZD0865 (3 μM) immediately inhibited H⁺,K⁺-ATPase activity by 88 ± 1%. Immediately after a 10-fold dilution H⁺,K⁺-ATPase inhibition was 41%, indicating reversible binding of AZD0865 to gastric H⁺,K⁺-ATPase. In contrast to omeprazole, AZD0865 inhibited H⁺,K⁺-ATPase activity in a K⁺-competitive manner (K_i 46 ± 3 nM). AZD0865 inhibited the process of cation occlusion concentration-dependently (IC₅₀ 1.7 ± 0.06 μM). At 100 μM, AZD0865 reduced porcine renal Na⁺,K⁺-ATPase activity by 9 ± 2%, demonstrating a high selectivity for H⁺,K⁺-ATPase. Thus, AZD0865 potently, K⁺-competitively, and selectively inhibits gastric H⁺,K⁺-ATPase activity and acid formation in vitro, with a fast onset of effect.     

The gastric proton pump,a target for neuroleptics and antidepressant drugs?

The antisecretory action of the antidepressant drugs trimipramine, doxepin and nortriptyline was studied in two different in-vitro test systems; the isolated and enriched guinea-pig parietal cell and the purified H⁺/K⁺-ATPase preparation. The effect of the antidepressants was compared with that of the neuroleptic agents chlorpromazine, triflupromazine, trifluoperazine, haloperidol, fluspirilene and with that of the tricyclic anticholinergic agent pirenzepine. All neuroleptics and antidepressants inhibited acid formation in intact parietal cells with IC₅₀ values in the nanomolar range. The inhibitory potency for each compound was identical regardless of whether histamine or db-cAMP was used as stimulant. Isolated H⁺/K⁺-ATPase, measured in the presence of 5 mmol litre^?1 KCl, was inhibited by all psychotropic drugs with IC₅₀ values in the micromolar range. EGTA did not affect the inhibitory potency at the H⁺/K⁺-ATPase, indicating that the action of the drugs does not depend on their calmodulin blocking activity. Pirenzepine was ineffective in both test systems. Kinetic studies done with nortriptyline, chlorpromazine and haloperidol showed a competitive type of inhibition with respect to K⁺ at low inhibitor concentrations. This competitive type was changed to a mixed type of inhibition with increasing inhibitor concentrations, demonstrating cooperative effects between drug binding and K⁺ activation of the enzyme. From these data it is suggested that antidepressants and neuroleptics act by an allosteric mechanism of action, and that the lipid solubility is a significant factor to establish enzyme inhibition.     

Cardiac glycosides: Correlations among Na+, K+-ATPase,sodium pump and contractility in the guinea pig heart

Summary Relationships among positive inotropic response to cardiac glycosides, Na⁺,K⁺-ATPase inhibition and monovalent cation pump activities were studied using paced Langendorff preparations of guinea-pig heart. Na⁺,K⁺-ATPase activity was estimated from the initial velocity of (³H)-ouabain binding in ventricular homogenates, and cation pump activity from ouabain-sensitive ⁸⁶Rb uptake of ventricular slices. These parameters were assayed in control, ouabain- or digitoxintreated hearts either at the time of inotropic response to the cardiac glycosides or during the course of drug washout. Development and loss of the inotropic response during ouabain or digitoxin perfusion and washout was accompanied by reduction and subsequent recovery of the initial ouabain binding velocity, respectively. If homogenates from glycoside-treated hearts were incubated at 37°C for 10 min during ouabain-binding studies, the levels of binding were not different from those of control hearts, indicating a rapid dissociation of the glycosides from cardiac Na⁺,K⁺-ATPase in this species. Despite differences in the time course of the loss of inotropic responses produced by ouabain or digitoxin, the relationship between Na⁺,K⁺-ATPase inhibition and inotropic responses were similar. Inotropic responses to digitoxin during perfusion, and subsequent los during washout, also were accompanied by a reduction and subsequent recovery of ⁸⁶Rb uptake. A correlation between inhibition of cation pump activity and positive inotropy has hitherto not been demonstrated. Thus, it appears that with cardiac glycosides, a relationship exists among contractility, cardiac Na⁺,K⁺-ATPase and monovalent cation pump activities.     

Inhibition of Sarcolemma ATPases by some Membrane-stabilizing Drugs

Abstract: Rat sarcolemma preparations were incubated with some membranes stabilizers to study their effects on the (Na⁺,K⁺)- and Ca²⁺-ATPase activity. The drugs inhibited the enzymes with the same order of potency as in the earlier observed muscle contractures: chlorpromazine> dibucaine> propranolol> tetracaine> procaine (range 0.1–3.6 mM) (Røed & Brodal 1979). The contracture inducing effect of the stabilizers is suggested to be caused by a membrane depolarization due to the (Na⁺,K⁺)-ATPase inhibition. Ouabain inhibited the (Na⁺,K⁺)-ATPase activity in purified plasma membrane, but did not inhibit the sarcolemma located ATPases or induce any contracture.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Effects of quinidine on some reactions and ion translocations catalyzed by the Na+, K+ -ATPase complex

Inhibitory effects of quinidine on the Na⁺,K⁺-ATPase activity have been reported before. Here, the results of more detailed studies of the effects of quinidine on the various reactions and ion translocations catalyzed by the Na⁺,K⁺-ATPase complex are presented. Quinidine, in a dose-related fashion, inhibits the Na⁺,K⁺-ATPase and the K⁺-dependent p-nitrophenylphosphatase activities of enzyme preparations obtained from the beef heart, the human red cell and the rat brain. These effects of the drug are partially antagonized by increasing K⁺ concentrations. The sensitivity of the K⁺-dependent p-nitrophenylphosphatase to quinidine is greater than the sensitivity of the Na⁺,K⁺-ATPase to the drug. Quinidine, unlike cardiac glycosides, does not stimulate the p-nitrophenylphosphatase activity in the absence of K⁺. Inhibitory effects of quinidine on the Na⁺-dependent phosphorylation of the enzyme complex by ATP, and the K⁺-stimulated breakdown of the phosphoenzyme are demonstrated. Experiments on the effects of quinidine on Rb⁺ influx in intact human red cells, and on Na⁺ efflux in ATP-filled ghosts of red cells, show that the drug inhibits the coupled transports of Na⁺ and K⁺ that are catalyzed by the Na⁺,K⁺-ATPase complex. Although these studies demonstrate similar effects of quinidine and ouabain on the Na⁺,K⁺-ATPase complex, they also indicate subtle differences between the mechanism and the nature of the interaction of the two drugs with the enzymic transport system.     

Transport ATPase--the different modes of inhibition of the enzyme by various mercury compounds.

The effects of a number of mercurials on the partial reactions of Na⁺, K⁺-ATPase were studied. Results of these studies allow a classification of the mercurials into two groups: selective modifiers of the enzyme and unspecific inhibitors. Thimerosal, ethylmercury and methylmercury were shown to selectively inhibit Na⁺, K⁺-ATPase while not affecting K⁺-NPPase, Na⁺-ATPase, ADP-ATP exchange reaction and phosphoenzyme formation. In contrast, p-chloromercuribenzoic acid, p-chloromercuriphenylsulfonic acid, mersalyl and mercuric chloride inhibited Na⁺, K⁺-ATPase as well as several partial reactions of the enzyme in a parallel manner. The selective modification of Na⁺, K⁺-ATPase by compounds such as ethylmercury and methylmercury makes these agents useful probes of the reaction mechanism of the enzyme. Whether this mode of inhibition of the enzyme is related to the mechanism of toxicity of short-chain alkylmercury compounds remains to be determined.     

Mechanism of inhibition of rat brain (Na + K)-adenosine triphosphatase by 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT).

Inhibition of an (Na + K)-ATPase preparation from rat brain by DDT was five times as effective when the insecticide was particulate than when solubilized by the surfactant, Corexit 7664. The surfactant reduced binding of DDT to the membranes by about 5 fold. Inhibition correlated with the amount of DDT bound rather than with its concentration or physical state. In the absence of the surfactant, maximal inhibition and binding occurred when the membranes contained about 350 pmoles/μg of protein, an amount equal to 12 per cent of the protein content of the membranes. At low concentrations most of the DDT present is bound by the membranes, hence its effect decreases as membrane concentration increases. The kinetics of inhibition of the (Na + K)-ATPase by DDT, allenthrin and DDE were examined by varying the concentrations of inhibitor, Na⁺, or K⁺, while holding the concentration of the others constant. Inhibition by all of these compounds was potentiated by increased K⁺, and the increment in the inhibition caused by K⁺ was reversible by increased Na⁺ concentrations. Hence the effectiveness of DDT is dependent on the membrane concentration and the relative concentrations of Na⁺ and K⁺. The concentration of DDT required for maximal inhibition suggests that the inhibition is not caused by binding to a specific site on the enzyme, but is the result of indirect alterations of the membrane which interfere with allosteric transitions of the (Na + K)-ATPase mediated by Na⁺ and K⁺.