The Effect of Thioridazine and Promazine in Reducing the Potassium Loss from Isolated Perfused Rat Hearts

Electrically stimulated isolated rat hearts were perfused with a Ringer solution containing a low concentration of K⁺. At the end of a control period of 20 min. the cumulative K⁺ loss from the hearts was approximately 30 meq./kg dry weight. This value was used in each experiment as reference (100 per cent) for the total loss found after a subsequent additional test period of 30 min. The addition of promazine and thioridazine to the perfusate at the end of the control period reduced the further K⁺ loss. The values found in the different groups at the end of the test period were: Control group (10 hearts): 154 ± 11.6 per cent; with promazine 10^-5 M added (8 hearts): 103 ± 5.6 per cent; with promazine 2.5 × 10^-5 M added (10 hearts): 74 ± 10.5 per cent and with thioridazine 10^-5 M added (8 hearts): 110 ± 5.8 per cent. In another type of experiments, ouabain 1.5 × 10^-6 M was added at the end of the control period. This drug caused an increased K⁺ loss (181 ± 14.2 per cent) and ventricular fibrillation in 6 out of 8 hearts. Promazine 2.5 × 10^-5 M added before ouabain, prevented the development of fibrillation in another group of 7 hearts, and also markedly reduced the K⁺ loss. It is concluded that phenothiazines reduce the K⁺ permeability of the heart muscle membranes.     

Interactions of surface-active alkyltrimethylammonium salts with the erythrocyte membrane.

The interactions of ahomologue series of surface-active alky Itrimethylammonium salts (C₁₀-C₂₀) with the rat erythrocyte membrane were studied. The surfactants were found to have a biphasic effect on the erythrocyte membrane. At low concentrations they protected or stabilized erythrocytes against hypotonic haemolysis, but at higher concentrations they caused rapid haemolysis. The stabilizing and lytic effect increased with an increase in length of the alkyl chain to maximum activity at about C₁₆. It is suggested that laminar-micellar transitions in the lipid bilayer of the membrane are responsible for the lytic activity of the surfactants. Micellar regions in the lipid bilayer abolish the ability of the membrane to prevent the free exchange of ions, and haemolysis of the cell results from a secondary osmotic effect. The stabilizing effect, on the other hand, is proposed to stem from an expansion of the membrane caused by a fluidizing effect of the surfactants on the lipid bilayer. Binding studies with the C₁₆ homologue revealed that at a concentration causing 50 per cent haemolysis in an isotonic solution there are about 780,000 molecules bound per μm² of the erythrocyte membrane. At a concentration giving 50 per cent protection against hypotonic haemolysis, the number of molecules bound per μm² of the erythrocyte membrane was estimated to be 190,000.     

Propentofylline and postischemic brain edema: Relation to Na+-K+-ATPase activity

Following the bilateral occlusion of common carotid arteries in gerbil, an increase in water content and sodium/potassium ratio as well as the inhibition of Na⁺-K⁺-ATPase was found. The xanthine derivative propentofylline (HWA 285) [3-methyl-1-(5-oxohexyl)-7-propylxanthine] given either before or after cerebral ischemia attenuated the development or postischemic brain swelling and the increase in sodium/potassium ratio and prevented the postischemic reduction of Na⁺-K⁺-ATPase activity. It is concluded that the action of propentofylline on brain edema during ischemia is mediated aside from other possible mechanism(s), by the influence of the drug on Na⁺-K⁺-ATPase activity.     

中药大黄的生化学研究ⅩⅫ蒽醌衍生物对兔肾髓质Na+-K+-ATP酶活性的抑制和利尿作用

家兔实验表明:大黄素、大黄酸以30 mg/kg的剂量灌胃给药,2～4h后尿量、排Na⁺和K⁺量达最高峰,比对照组明显增多。而芦荟大黄素和大黄酚的作用较弱。大黄素、大黄酸和芦荟大黄素对免肾髓质Na⁺-K⁺-ATP酶活性有较强的竞争性抑制作用。     

(Na+-K+)-ATPase inhibitors: Implications for new drug discovery

The (Na⁺-K⁺)-ATPase is a ubiquitous membrane-bound enzyme that actively transports Na⁺ out of the cell in exchange for a smaller ratio of extracellular K⁺. The current report focuses on the role of modifiers of (Na⁺-K⁺)-ATPase activity in the development of new pharmacological agents. More versatile biological test systems are proposed. Possible use of Na⁺-K⁺-ATPase activity modulators in treatment of hypertension and other diseases are discussed. It is concluded that the ubiquitous distribution and crucial role of the enzyme in normal and diseased cell function merits a critical biological and chemical reappraisal of the enzyme beyond the current narrow viewpoint dealing with the application of glycosides in the treatment of congestive heart failure.     

The Comparative Effect of Chlorhexidine and Cetrimonium Bromide on Erythrocyte Membranes

Abstract The effect of chlorhexidine (Chx) and cetrimonium bromide (Ctab) on the haemolysis of erythrocytes has been studied. A concentration dependent release of haemoglobin was observed in isotonic media, with total haemolysis at 80 nmol Chx per 10⁷ cells and 20 nmol Ctab per 10⁷ cells. The rate of haemolysis induced by Chx shows a biphasic pattern in contrast to the uniphasic pattern of Ctab. In concentrations below 10 nmol per 10⁷ cells, Chx produces more haemolysis than Ctab whereas the opposite effect is observed at higher concentrations. Chx and Ctab stabilize the erythrocyte membrane against hypotonic shock. The concentrations of Chx and Ctab giving maximal stabilization are 0.25 nmol per 10⁷ cells and 2 nmol per 10⁷ cells respectively. The normal biconcave disc form of the erythrocytes is converted to cup forms and in-vaginated spheres by Chx and Ctab. The binding of Chx to erythrocytes in isotonic media increases linearly with the total concentration up to about 25 nmol Chx per 10⁷ cells where the curve has a point of inflection. With more than 25 nmol Chx per 10⁷ cells the amount of Chx bound again increases linearly up to 120 nmol per 10⁷ cells. The slope of the curve above the point of inflection is approximately 4 times that of the curve below this point. No level of saturation of the binding is observed at the concentrations of Chx used in this study. The mode of action of Chx on erythrocyte membranes is discussed in the light of the present results.     

DIGITALIS 'RECEPTORS' DURING CHRONIC DIGOXIN TREATMENT

1. The effect of digoxin treatment on Mg-dependent [Na⁺-K⁺]-ATPase (the receptor for cardiac glycosides) was assessed by comparison of intracellular Na⁺-K⁺, ⁸⁶Rb uptake and number of digoxin binding sites in the erythrocytes of 138 patients on long term digoxin and of 133 control subjects. The parameters were also assessed in thirty-two patients followed longitudinally for 1 y. 2. The results indicate that the cells adapt to chronic exposure to ‘therapeutic’ dosage of digoxin with an overcompensatory synthesis of new receptors, a possible mechanism through which the normal intraerythrocytic ionic equilibrium is re-established. 3. The process of synthesis of new receptors appears to be completed in erythrocytes in a period of 4 months after the start of digoxin treatment.     

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.     

The Influence of Potassium Concentration on the Inhibitory Effect of Amiodarone on Guinea-Pig Microsomal Na+-K+-ATPase Activity

Abstract: The effects of varying incubation K⁺ concentration on the inhibitory action of amiodarone on the Mg²⁺-dependent ATP hydrolysis by myocardial Na⁺-K⁺-ATPase (EC 3.6.1.3) were studied in guinea pig heart preparations. In the first part of the study, it was established that the activity of the enzyme increased with growing concentrations up to approximately 20 mM K⁺. The concentration-response relationships for amiodarone were investigated in incubation media containing 2.5, 5.0 and 10 mM K⁺ respectively. Amiodarone exhibited similar concentration-dependent inhibitory effects in the range of 0.01 nM ?80 μM at 2.5 mM, 0.13–150 μM at 5.0 mM and 0.3–700 μM at 10.0 mM K⁺. The corresponding IC50 values were 10.4 ± 3.2 μM at 2.5 mM, 28.3 ± 7.6 μM at 5.0 mM and 33.3 ± 9.2 μM at 10.0 mM K⁺, respectively. Thus, reduction in the K⁺ concentration from the “standard” 5.0 to 2.5 mM was accompanied by a significant right-to-left shift in the inhibitory potency of amiodarone, the effective concentrations being shifted from uM into nM ranges. Increasing K⁺ concentration to 10 mM on the other hand attained opposite but less remarkable effects. The results show that the in vitro inhibition of myocardial Na⁺-K⁺-ATPase activity by amiodarone is related to the K⁺ concentration of the incubation medium. These effects may be pertinent to the mechanism by which this drug interferes with the electrogenic Na⁺/K⁺ pump activity of the enzyme, thereby probably contributing to the mechanism(s) responsible for some of its cardiac actions.     

Comparative effects of substituted phenothiazines and their free radicals on (Na =, K + )-activated adenosine triphosphatase

The inhibitory effects of free radicals of various substituted phenothiazines on (Na⁺,K⁺)-activated ATPase [Mg²⁺-dependent, (Na⁺,K⁺)-activated ATP phosphohydrolase; EC 3.6.1.3] were studied in vitro. Enzyme preparations were obtained from rat brain microsomal fractions after deoxycholic acid and NaI treatments. Free radicals were produced by either ultraviolet (253.7 nm) irradiation or enzymatic oxidation with peroxidase. Without enzymatic or photo-oxidation, phenothiazine derivatives failed to inhibit (Na⁺,K⁺)-activated ATPase activity significantly. Photo-oxidative intermediates of thioridazine, triflupromazine and trifluoperazine were potent inhibitors of (Na⁺,K⁺)-activated ATPase activity. Those from chlorpromazine, perphenazine and promazine were less potent. Peroxidase-hydrogen peroxide treatment of promazine, thioridazine, perphenazine and chlorpromazine produced free radical intermediates which significantly inhibited (Na⁺,K⁺)-activated ATPase activity. The same treatment of triflupromazine and trifluoperazine, however, failed to produce detectable amounts of free radical intermediates. Concomitantly, no inhibition of (Na⁺,K⁺)-activated ATPase activity was observed under these conditions. It was concluded that free radical intermediates of various substituted phenothiazines are differentially potent inhibitors of (Na⁺,K⁺)-activated ATPase, and that the formation of free radicals from substituted phenothiazines is dependent upon the oxidizing conditions and nature of substituent groups.     

Toxic effects of diazinon and its photodegradation products

The toxic effects of diazinon and its irradiated solutions were investigated using cultivated human blood cells (lymphocytes and erythrocytes) and skin fibroblasts. Ultra Performance Liquid Chromatography (UPLC)–UV/VIS system was used to monitor the disappearance of starting diazinon during 115-min photodegradation and formation of its by-products (diazoxon and 2-isopropyl-6-methyl-4-pyrimidinol (IMP)) as a function of time. Dose-dependent AChE and Na⁺/K⁺-ATPase inhibition by diazinon was obtained for all investigated cells. Calculated IC₅₀ (72 h) values, in M, were: 7.5 × 10⁻⁶/3.4 × 10⁻⁵, 8.7 × 10⁻⁵/6.6 × 10⁻⁵, and 3.0 × 10⁻⁵/4.6 × 10⁻⁵ for fibroblast, erythrocyte and lymphocyte AChE/Na⁺/K⁺-ATPase, respectively. Results obtained for reference commercially purified target enzymes indicate similar sensitivity of AChE towards diazinon (IC₅₀ (20 min)-7.8 × 10⁻⁵M), while diazinon concentrations below 10 mM did not noticeably affect Na⁺/K⁺-ATPase activity. Besides, diazinon and IMP induced increasing incidence of micronuclei (via clastogenic mode of action) in a dose-dependent manner up to 2 × 10⁻⁶ M and significant inhibition of cell proliferation and increased level of malondialdehyde at all investigated concentrations. Although after 15-min diazinon irradiation formed products do not affect purified commercial enzymes activities, inhibitory effect of irradiated solutions on cell enzymes increased as a function of time exposure to UV light and resulted in significant reduction of AChE (up to 28–45%) and Na⁺/K⁺-ATPase (up to 35–40%) at the end of irradiation period. Moreover, photodegradation treatment strengthened prooxidative properties of diazinon as well as its potency to induce cytogenetic damage.     

Differentiation of renal Na+-K+-ATPase in control and streptozotocin-induced diabetic mice by G-protein acting toxins and phorbol esters

The specific activity of Na⁺-K⁺-ATPase in the renal medulla and cortex of 50-day-old streptozotocin (STZ)-induced diabetic mice was increased 58% and 50%, respectively, as compared to controls. K_m values of Na⁺ and K⁺ for this enzyme were unaltered, while that of ATP was decreased in diabetic mice. The Na⁺-K⁺-ATPase in control medulla and cortex was activated by both cholera and pertussis toxins, while this effect was abolished in diabetics. Since dibutyryl cAMP stimulates cortical Na⁺-K⁺-ATPase activity in control mice, the activation effect of cholera toxin on this enzyme might be due to its interaction with a G_s-protein and the persistent stimulation of adenylate cyclase activity, while the effect of pertussis toxin might be due to its masking of the inhibitory action of a G_i-protein on adenylate activity. However, the protein kinase C(PKC)-associated Na⁺-K⁺-ATPase might also be quiescent in diabetes, because the stimulating effect of phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA) on this enzyme was abolished in diabetic cortex. In addition, nicardipine and ouabain were found to have differential effects on this enzyme derived from control and diabetic mice.     

Kinetics of catecholamine sensitive Na+-K+ ATPase activity in mouse brain synaptosomes.

The effects of several neurotransmitters on mouse brain synaptosomal ATPase activities were determined in vitro. Both dopamine and norepinephrine activated Na⁺-K⁺ and Mg²⁺ ATPase activities in a dose-dependent manner. Na⁺-K⁺ ATPase was more sensitive to the catecholamines than was Mg²⁺ ATPase activity. Acetylcholine, γ-aminobutyric acid, L-glutamic acid and serotonin were without effect up to 10^?3 M concentration. Chlorpromazine, an antipsychotic agent, which has been shown to block the dopamine-receptor site, totally inhibited the dopamine-stimulated Na⁺-K⁺ and Mg²⁺ ATPase activities in mouse brain synaptosomes. Further, catecholamine-sensitive ATPase activities from mouse brain synaptosomal preparation were determined in relation to the substrate, pH and ionic concentrations in the reaction medium. The results indicate that Na⁺-K⁺ and Mg²⁺ ATPases were activated by dopamine (DA) and norepinephrine (NE) at different concentrations of ATP. Lineweaver-Burk plots reveal that Na⁺-K⁺ ATPase was activated non-competitively to ATP with a K_m value of 5.5 × 10^?4 M, whereas Mg²⁺ ATPase exhibited a mixed type of activation in that K_m was decreased and V_max increased in the presence of DA or NE. A maximum stimulation occurred by catecholamines at the optimum pH of 7.5 for Na⁺-K⁺ and 8.0 for Mg²⁺ ATPase activities. Both catecholamines increased the Na⁺-K⁺ ATPase activity in the presence of Na⁺ and K⁺ in the reaction medium. However, in the absence of Na⁺ ions the K⁺ -ATPase activity was stimulated by DA and NE but in the absence of K⁺ ions the Na⁺ ATPase, was not activated by DA or NE, indicating that the ATPase activity was more sensitive to catecholamines in the presence of K⁺ than Na⁺.     

Inhibition of Human Erythrocyte (Na+-K+)ATPase by Organic Hydroperoxides and Protection by Ascorbic Acid and Butylated Hydroxytoluene

• 1.The in vitro effects of cumene hydroperoxide and t-butyl hydroperoxide on intact human erythrocyte membrane (Na⁺-K⁺)ATPase activities have been studied.
• 2.(Na⁺-K⁺)ATPase activities on erythrocyte membranes decreased in agreement with the results of chemiluminescence experiments.
• 3.Our results demonstrated that the organic hydroperoxides inhibit the activity of (Na⁺-K⁺) ATPase enzyme and that the antioxidants used prevent this inhibition.

     

Effects of T-2 toxin on glucose and tryptophan uptake and intestinal mucosal enzymes

We studied the uptake of d-glucose and l-tryptophan by the small intestine and estimated the activities of the intestinal brush border enzymes (sucrase, lactase, Na⁺-K⁺-ATPase and alkaline phosphatase) and lysosomal enzymes in rats receiving T-2 toxin orally. Considerable decrease occurred in glucose and tryptophan uptake and in brush border sucrase, lactase and (Na⁺ - K⁺)-ATPase. Alkaline phosphatase activity and release of lysosomal enzymes (acid phosphatase and acid ribonuclease) was unchanged.     

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.     

Die Haftung verschiedener Cardenolide am Papillarmuskel und einer mikrosomalen ATPase des Meerschweinchenherzens

Summary In experiments on isolated electrically stimulated guinea pig papillary muscles and on isolated cardiac Na⁺-K⁺-activated ATPase preparations the action and the reversibility of action of 3 different cardenolides-digitoxin, k-strophanthidin and strophanthidin-3-bromoacetate (SBA) (supposed to be an irreversible inhibitor of the transport ATPase)-were studied.The equieffective concentrations for maximum positive inotropic effects (around 90%) were 2×10^–6, 2×10^–5 and 4×10^–5 M, respectively. In washout experiments the positive inotropic action of all these substances was found to be completely reversible: the rates of decline of the positive inotropic effects were about 2.7%/min with digitoxin, 24%/min with strophanthidin and 22%/min respectivety 5.7%/0/min (two components) with SBA.The equieffective concentrations for maximum inhibition (90–95%) of the Na⁺-K⁺-activated ATPase by digitoxin, strophanthidin and SBA were 10^–4, 2×10^–4 and 10^–4 M respectively. In washout experiments (repeated centrifugations) different degrees of reversibility of these inhibitory effects were observed depending upon the experimental conditions. Preincubation of the enzyme with the cardenolides in theabsence of Na⁺, Mg²⁺ and ATP resulted in a persisting inhibition of the Na⁺-K⁺-ATPase of 14% with digitoxin, 10% with k-strophanthidin and- significantly higher (p < 0.05)-33% with SBA. Corresponding experiments with preincubation of the enzyme in thepresence of Na⁺, Mg²⁺ and ATP, however, demonstrated a full reversibility of the inhibitory action of all these substances.These results are in contrast, in certain respects, with those obtained in previous experiments on brain ATPase.It is concluded that SBA is able to inhibit irreversibly only the non-phosphorylated form of the cardiac Na⁺-K⁺-activated ATPase, whereas the phosphorylated intermediate of this enzyme seems to be protected against the irreversible inhibition by this substance. Assuming that the latter state of the enzyme is predominant in the intact heart muscle cell, a complete reversibility of the pharmacological action of SBA would be expected if the inotropic effect is mediated by an inhibition of the enzyme. Our results are compatible with this hypothesis.

Wir danken der Deutschen Forschungsgemeinschaft für die Unterstützung durch Sachbeihilfen und der Volkswagenstiftung für die Geräteausstattung.     

The Effect of Sodium Transport and Calcium Channel Inhibitors on Phorbol Ester-induced Contraction of Bovine Airway Smooth Muscle

Summary: We studied the role of sodium transport and calcium channels in protein kinase C mediated signal-transduction pathways in bovine airway smooth muscle. 4-β phorbol 12,13 dibutyrate (PDB), an activator of protein kinase C, caused dose-related slowly developing contraction in bovine bronchial rings with a peak effect at 60-90 min. 4-α PDB, an inactive analogue, was without effect. Mean peak PDB-induced contraction (measured as a percentage of the maximum response to methacholine) was reduced from 122% to 20% when experiments were carried out in calcium-free fluids + EDTA (10^-3 M). Similar reductions were seen in the presence of nifedipine and verapamil, inhibitors of voltage-dependent calcium channels. Amiloride at high concentrations (10^-3 M) reduced the contractile response to PDB from 87% to 20%, but at a concentration which inhibits the sodium entry channel (10^-6 M), amiloride was without effect. 5-N,N-hexamethylene amiloride (10^-5 M), a specific inhibitor of Na⁺/H⁺ exchange, did not alter the contraction produced by PDB. Frusemide (10^-5 M), an inhibitor of Na⁺-K⁺-Cl^- cotransport, was without effect on PDB contractions. We conclude that phorbol ester-induced contraction of bovine airway smooth muscle is dependent on calcium entry via voltage-dependent calcium channels but is independent of Na⁺ entry, Na⁺/H⁺ exchange or Na⁺-K⁺-Cl^- cotransport.     

Inhibition of human colonic (Na++K+)-ATPase by arachidonic and linoleic acid

Summary The sodium pump, (Na⁺+K⁺)-ATPase, which is involved in the transport of cations and water movement by the colonic mucosa, may be decreased in various diarrhoeal states. In this study, we have measured ³H-ouabain binding and (Na⁺+K⁺)-ATPase activity in human colonic biopsy homogenates and the influence of various inflammatory and antiinflammatory compounds on these parameters. ³H-ouabain binds to one site of high affinity (K _D 1.9±0.2×10^–9 mol/l) with a maximal binding capacity of 7.5±0.8×10¹⁴ binding sites/g protein. Both arachidonic and linoleic acid inhibited (Na⁺+K⁺)-ATPase activity (IC₅₀ arachidonic acid: 7.5×10^–5 mol/l, linoleic acid: 6.5×10^–5 mol/l) and Mg²⁺-ATPase activity (IC₅₀ arachidonic acid: 9×10^–5 mol/l, linoleic acid: 4×10^–5 mol/l). Arachidonic acid inhibited ³H-ouabain binding, (IC₅₀ 3.2×10^–5 mol/l). The following antiinflammatory compounds, at concentrations up to 1×10^–3 mol/l, did not influence ATPase activity directly nor reverse the arachidonic acid-induced inhibition: indomethacin (cyclooxygenase inhibitor), nordihydroguaretic acid (lipoxygenase inhibitor), sulphasalazine and its metabolites: 5-aminosalicylic acid, N-acetylaminosalicylic acid and sulphapyridine.These results indicate that human colonic (Na⁺+K⁺)-ATPase is inhibited by the prostanoid precursors, arachidonic and linoleic acid. From a therapeutic point of view (effect on colonic (Na⁺+K⁺)-ATPase and perhaps diarrhoea), the suppression of the production of these prostanoid precursors by drugs may, therefore, be beneficial in the treatment of inflammatory bowel disease.Supported by DFG (Er65/4-4)     

Effect of vanadate on the renal accumulation of p-aminohippurate in the rabbit kidney tubules in vitro

The possible involvement of Na⁺-K⁺-ATPase on renal organic anion transport systems was investigated by examining the effect of vanadate and ouabain on the uptake of p-aminohippurate (PAH) by rabbit kidney cortical slices. Addition of increasing concentration of vanadate (0.01–0.1 mM) reduced Na⁺-K⁺ transport and renal steady-state aerobic accumulation of PAH to 80–50 per cent of the control value, without any significant decrease in tissue concentration of ATP. The rapid Na⁺-independent component of PAH transport was unaffected by the presence of vanadate (0.1 mM) or ouabain (0.2 mM), whereas the slowly equilibrating component, which makes the largest contribution to the final accumulation of PAH was ca. 50 per cent inhibited, by vanadate and almost abolished by ouabain. The anaerobic uptake of PAH was unaffected by a high concentration of vanadate (1 mM), suggesting that vanadate does not competitively inhibit the transport of organic anions. On the basis of the above-mentioned findings, it is suggested that PAH transport is probably linked in a direct manner to the function of Na⁺-K⁺-ATPase.