Ganglioside protection of the erythrocyte membranes in myocardial ischemia]

Myocardial ischemia was shown to lead to modification of structural and functional organization of rat erythrocyte membranes. Thus, it was found that the activity of Na+, K+-ATP-ase markedly decreased, while accumulation of LPO products and of lysophosphatidylcholine (lyso--PC) took place in erythrocyte membranes of rats subjected to myocardial ischemia. Using nonpenetrating modifier trinitrobenzosulfonic acid, an increase in the content of modified phosphatidylethanolamine in erythrocyte membranes of ischemic rats was revealed as compared to the membranes of control animals. The intravenous administration of gangliosides (30 mg/kg) resulted in partial normalization of Na+, K+(-)ATPase activity, of LPO product and lysoPC content and of transbilayer distribution of lipids.     

Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi

The cholesterol content of human erythrocyte membranes has been modified by incubation of intact cells with sonicated egg phosphatidylcholine/cholesterol vesicles and with egg phosphatidylcholine vesicles. (Na+ + K+)-ATPase ATP hydrolyzing activity was measured as a function of membrane cholesterol content. High membrane cholesterol inhibits the ATPase activity of the enzyme and low membrane cholesterol activates that enzyme activity. The most likely mechanism of inhibition is suggested to comprise direct cholesterol-protein interactions which lead to a low activity conformation. Ouabain binding studies show that the inhibition is not due to a loss of enzyme from the membrane.     

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.     

Thallium inhibition of ouabain-sensitive sodium transport and of the (Na+ plus K+)-ATPase in human erythrocytes.

The influence of Tl+ on Na+ transport and on the ATPase activity in human erythrocytes was studied. 0.1-1.0 mM Tl+ added to a K+-free medium inhibited the ouabain-sensitive self-exchange of Na+ and activated both the ouabain-sensitive 22Na outward transport and the transport related ATPase. 5-10mM external Tl+ caused inhibition of the ouabain-sensitive 22Na efflux as well as the (Na+ plus Tl+)-ATPase. Competition between the internal Na+ and rapidly penetrating thallous ions at the inner Na+-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl+. An increase of the internal Na+ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl+. A protective effect of Na+ was also demonstrated on the (Na+ plus Tl+)-ATPase of fragmented erythrocyte membranes studied at various Na+ and Tl+ concentrations.     

Exaprolol as a modulator of heart sarcolemmal (Na+ + K+)-ATPase. Evidence for interaction with an essential sulfhydryl group in the catalytic centre of the enzyme

Beta-adrenoceptor blocking agents may have, in addition to their primary action, also ancillary effects on the cell membrane. In the present paper the non-specific interaction of exaprolol with the ATPase systems in isolated rat heart sarcolemmal membranes was investigated. When preincubated with sarcolemmal membranes in vitro, exaprolol in concentrations below 10(-4) mol.l-1 had no significant effect on sarcolemmal Mg2+-, Ca2+- and (Na+ + K+)-ATPase activities. At exaprolol concentration of 10(-4) mol.l-1 the Mg2+- and Ca2+-ATPase activities became inhibited whereas the (Na+ + K+)-ATPase activity was markedly stimulated. A kinetic analysis of these interactions revealed a non-competitive inhibition of Mg2+- and Ca2+-ATPase. In the case of (Na+ + K+)-ATPase a synergistic type of stimulation characterized by an exaprolol-induced conversion of an essential sulfhydryl group in the active site of the enzyme to the more reactive [S-] form has been observed thus increasing the affinity of the enzyme to ATP. Exaprolol concentrations exceeding 5 X 10(-4) mol.l-1 induced an overall depression of the investigated enzyme activities.     

Ca2+-Mg2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca2+ +Mg2+ dependent ATPase activities (about 9 mumol/h per mg protein). The Na+ +K+ +Mg2+ dependent ATPase activity was 3.2 mumol/h per mg (+/- 1.0, S.D., n = 3) when microvesicles were prepared according to (1) and 1.5 mumol/h per mg (+/- 1.0, S.D., n = 3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca2+ transport in mitochondria and erythrocyte membranes had no effect on Ca2+ +Mg2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca2+ +Mg2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na+ +K+ +Mg2+ ATPase was decreased. Na+ +K+ +Mg2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na+ +K+ +Mg2+ dependent ATPase activity in the eluted coated microvesicle fraction. It was concluded that Ca2+ +Mg2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.     

Effect of erythropoietin on the different ATPases and acetylcholinesterase of rat RBC membrane

The effect of Ep on different ATPases and acetylcholinesterase of rat RBC membrane was studied. Starvation caused a slight decrease in Mg2+-, Ca2+-, and Na+ + K+-ATPases. However, these enzyme activities were markedly increased on Ep treatment of starved rats. Specific activities of all three ATPases increased linearly with increasing concentration of Ep. Under identical conditions the hormone failed to stimulate the ATPase activity of liver plasma membrane. Desensitization by fluoride of allosteric inhibition of erythrocyte membrane-bound Na+ + K+-ATPase was observed under starvation which showed a return to normal n values on Ep administration. The enzyme from normal animals was inhibited almost completely at 0.1 mM fluoride whereas enzyme from starved and Ep-treated animals showed only about 50% inhibition at that fluoride concentration. Ep increased the acetylcholinesterase activity of normal RBC membrane to a small extent whereas the stimulation was much higher under starvation. The fluoride inhibition curve of this enzyme changed from sigmoidal to hyperbolic under starvation which again changed to allosteric on administration of Ep. These changes were closely correlated to n values. Red blood cells of Ep-treated animals became more susceptible to osmotic shock under the experimental conditions.     

Hypothalamic factor inhibits the (Na,K)ATPase from the extracellular surface. Mechanism of inhibition

We have characterized the effect of a stable small molecule isolated from bovine hypothalamus (Haupert, G. T., and Sancho, J. M. (1979) Proc. Natl. Acad. Sci. 76, 4658-4660) on mammalian (Na,K)ATPase. This hypothalamus-derived inhibitory factor, HIF, has been shown to inhibit ATPase activity of purified dog kidney enzyme reversibly with high affinity (Haupert, G. T., Carilli, C. T., and Cantley, L. C. (1984) Am. J. Physiol. 247, F919-F924). In this report it is shown that HIF inhibits the ouabain sensitive component of 86Rb+ uptake into human red blood cells. HIF also inhibited (Na,K)ATPase activity of unsealed red cell membranes but not that of sealed inside-out vesicles, indicating that HIF is impermeant to red cell membranes and inhibits the (Na,K)ATPase from the extracellular side. In unsealed human red cell membranes, concentrations of HIF which caused 70% inhibition of the (Na,K)ATPase did not inhibit ATP hydrolysis by plasma membrane (Ca2+)ATPase or (Mg2+)ATPase. However, at a similar concentration, HIF was shown to inhibit rabbit muscle sarcoplasmic reticulum (Ca2+)ATPase. HIF also inhibited p-nitrophenylphosphatase activity of unmodified or fluorescein-5'-iso-thiocyanate labeled dog kidney (Na,K)ATPase. As judged by fluorescein fluorescence of the modified enzyme, HIF stabilized the low fluorescent "E2" conformation of the enzyme similar to that stabilized by ouabain. However, unlike ouabain, HIF blocked covalent phosphorylation of dog kidney (Na,K)ATPase by inorganic phosphate. These studies show that HIF is an inhibitor of (Na,K)ATPase which acts from the extracellular side of the membrane by a mechanism similar to but not identical to that of cardiac glycosides.     

Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes

Erythrocyte plasma membranes were isolated from a homogeneous population of human or rabbit erythrocytes fractionated into classes representing young, middle-age and old age in vivo. Lipid analyses of human erythrocyte plasma membranes reveal a decrease of the cholesterol to phospholipid molar ratio, followed by a marked decrease in the activities of the membrane-bound enzymes (Na+,K+)-stimulated ATPase, acetylcholinesterase and NAD+ase from young to old age. Such changes were not observed between young and middle-age rabbit erythrocytes. Incubation of rabbit young erythrocytes with phosphatidylcholine vesicles (liposomes) to obtain partial depletion of their membrane cholesterol, indicated that cholesterol depletion causes a statistically significant decrease of the (Na+,K+)-stimulated ATPase and acetylcholinesterase activities, but the NAD+ase activity remained almost unchanged. The biological significance of these data are discussed in terms of the differences and modifications in the interaction of membrane-bound enzymes with membrane lipids during in vivo ageing of erythrocytes.     

Proteolytic digestion of Cl(-)-ATPase in rat brain synaptosomes

Upon tryptic digestion of synaptosomes, ATPase activities decreased in the order of Cl(-)-ATPase greater than or equal to Na+,K+-ATPase greater than anion-insensitive Mg2+-ATPase. Upon synaptosome treatment with hypotonic solution, Cl(-)-ATPase or anion-insensitive Mg2+-ATPase was slightly inactivated, while Na+,K+-ATPase underwent a much larger degree of inactivation. ATP-Mg inhibited the ATPase digestion in the hypotonic-solution-treated synaptosomes in a concentration-dependent manner, but not in the untreated synaptosomes. These results suggest that trypsin-digestible site of Cl(-)-ATPase are present on both sides of the synaptosomal plasma membrane, and the ATP-Mg binding site of the enzyme is located on the inner surface of the membrane.     

A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo.

The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques. In the closed-loops, 5 mM deoxycholate selectively inactivated (Na+ + K+)-ATPase, and net secretion of Na+ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-0-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5 plus or minus 2.9% per g wet st of gut) greatly exceeded those of taurocholate (4.3 plus or minus 1.0). In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na+ and C1-, and inhibited active glucose transport; concomitantly "total" ATPase, (Na+ + K+)-ATPase, and Mg-2+-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg-2+-ATPase activity. Taurocholate and cholate at 1mM had no effect on transport of (Na+ + K+)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate. These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na+ + K+)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Effect of anthelmintics and phenothiazines on adenosine 5'-triphosphatases of filarial parasite Setaria cervi

S. cervi showed particulate bound Ca2+ ATPase and Na+,K(+)-ATPase activities while Mg2+ ATPase was detected in traces. ATPase of S. cervi was also differentiated from the nonspecific p-nitrophenyl phosphatase activity. Female parasite and microfilariae exhibited higher Ca2+ ATPase and Na+,K(+)-ATPase activities than the male adults and the enzyme Na+,K(+)-ATPase was mainly concentrated in the gastrointestinal tract of the filarial parasite. Na+,K(+)-ATPase of the filariid was ouabain-sensitive while Ca2(+)-ATPase activity was regulated by concentration of Ca2+ ions and inhibited by EGTA. Phenothiazines, viz. trifluoperazine, promethazine and chlorpromazine caused significant inhibition of Ca2+ ATPase and Na+,K(+)-ATPase. Diethylcarbamazine was a potent inhibitor of these ATPases. Mebendazole, levamisole and centperazine also caused significant inhibition of the ATPases indicating this enzyme system as a common target for the action of anthelmintic drugs.     

严重烧伤早期红细胞膜胆固醇含量及Na+-K+-ATP酶活性的变化

目的 :研究严重烧伤患者早期红细胞滤过指数 (EFI)与红细胞膜胆固醇含量、Na K ATPase活性的变化。探讨其在严重烧伤早期中的相互关系及意义。方法 :采用核孔滤膜法测定红细胞滤过指数 (EFI) ,用化学修饰电极法测定红细胞膜胆固醇含量 ,应用定磷法测定红细胞膜Na K ATPase活性。结果 :4 7例严重烧伤早期患者EFI较 6 0例正常对照组下降 (P <0 .0 1) ,红细胞膜胆固醇含量、Na K ATPase活性均高于正常对照组 (P <0 .0 1) ,且红细胞膜胆固醇含量、Na K ATPase活性与EFI呈密切负相关 (rcho =- 0 .871,rATPase =- 0 .80 1,P <0 .0 1)。结论 :严重烧伤早期EFI下降 ,变形性明显减低是导致血液粘度和微循环改变的原因之一 ,红细胞膜胆固醇含量和Na K ATPase活性的变化则是引起EFI下降、变形性减低的重要因素     

Mechanisms of inhibition of active transport ATPases by mercurials.

Inhibition by methylmercury and mercuric chloride of Mg,Ca ATPase and Na,K ATPase activities in human erythrocyte ghosts was correlated with the binding capacity of ghosts for the mercurial. Full inhibition was always reached below saturation of binding capacity, and half-inhibition at levels as low as 10% saturation. Under such conditions, concentrations of free inhibitor were negligibly low, and existing mathematical models of inhibition were not applicable. New inhibitor partition equations were introduced to model the mechanisms of action of mercurials. Up to 7 methylmercury groups were calculated to bind to one Na,K ATPase molecule at non-inhibitory sites, while only one reacted with the inhibitory site. Mg,Ca ATPase showed simple one-hit inhibition (one mercurial per enzyme); further washing of ghosts, however, unmasked a second binding site (cooperative two-hit inhibition). Affinities of mercurials to sites of inhibition were calculated relative to other ligands in erythrocyte membranes: the ratios ranged from 3 : 1 to 50 : 1. The results demonstrated the use of binding capacity assays and inhibitor partition equations to measure and compare the susceptibilities of membrane-bound enzymes to poisoning by mercurials.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy

The protective effects of glutathione monoester (GME) on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion and its sequel were evaluated in rat erythrocyte/erythrocyte membrane. Animals were divided into three groups (n=6 in each): control, BSO and BSO+GME group. Administration of BSO, at a concentration of 4 mmol/kg bw, to the albino rats resulted in depletion of blood GSH level to about 59%. GSH was elevated several folds in the GME group as compared to the control (P<0.05) and BSO (P<0.001) groups. Decreased concentration of vitamin E was found in the erythrocyte membrane isolated from BSO-administered animals. Antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were also found to be altered due to BSO-induced GSH depletion in blood erythrocytes. The SOD and CAT activities in BSO group were significantly lower (P<0.001) than the other groups. Lipid peroxidation index and malondialdehyde (MDA) levels in erythrocytes and their membranes were increased to about 45% and 40%, respectively. The activities of Ca2+ ATPase, Mg2+ ATPase and Na+K+ ATPase were lower than those of control group (P<0.05), whereas the activities of these enzymes were found to be restored to normal followed by GME therapy (P<0.05). Cholesterol, phospholipid and C/P ratio and some of the phospholipid classes like phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin were significantly (P<0.05) altered in the erythrocyte membranes of BSO-administered rats compared with those of control group. These parameters were restored to control group levels in GME-treated group. Oxidative stress may play a major role in the BSO-mediated gamma glutamyl cysteine synthetase (gamma-GCS) inhibition and hence the depletion of GSH. In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in BSO-treated rats. GME potentiates the RBC and blood antioxidant defense mechanisms and decreases lipid peroxidation.     

Properties of (Na+ plus K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi.

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124-132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na+ plus K+)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na+ plus K+)-activated ATPase activity was documented by demonstrating specific cation requirements for Na+ and K+, while the divalent cation, Ca(2+), and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na+ plus K+)-activated ATPase activity averaged 10.07 plus or minus 2.80 mumol Pi/mg protein per h compared to 50.03 plus or minus 11.41 for Mg(2+)-activated ATPase and 58.66 plus or minus 10.07 for 5'-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na+ plus K+)-activated ATPase without any effect on Mg(2+)-activated ATPase. Both (Na+ plus K+)-activated ATPase and Mg(2+)-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na+ plus K+)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na+ plus K+)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.