The interaction of chloroquine with transport ATPase and acetylcholine esterase in microsomal membranes of rat in vitro and in vivo

Chloroquine, an antimalarial drug has been found to inhibit Na+, K+-ATPase activity in vitro in the microsomal membranes of rat brain on time, temperature and concentration dependent manner. There have been stimulation of Na+,K+-ATPase, Ca+2-ATPase and acetylcholine esterase activities in vivo studies at lower concentration of drug or shorter period of treatment with the drug, whereas higher concentrations or longer periods of treatment lead to inhibition in the microsomal membranes of different organs.     

Activities of enzymes from canine kidney plasma membranes under effects of polyene antibiotics in vivo and in vitro]

The effects of amphotericin B drug containing sodium deoxycholate (DOC) and those of DOC and nistatin on the activities of Na+, K+-ATPase and 5'-nucleotidase of canine kidney plasma membranes were studied. It was found that the activities of Na+, K+-ATPase and 5'-nucleotidase were markedly inhibited only after intravenous injection of amphotericin B, whereas the other agents tested caused no changes in the enzyme activities. Similar results were obtained in vitro. In the presence of amphotericin B the activity of Na+, K+-ATPase was noticeably inhibited already at the antibiotic concentration of 0,1 mkg per mg of membrane protein. It was found that the injection of amphotericin B, DOC and nistatin did not qualitatively or quantitatively affect the phospholipid composition of the plasma membranes. This is indicative of the lack of correlation between the enzyme activities and changes in the phospholipid composition of the plasma membranes under effects of amphotericin B. The pyrimidine derivative--amygluracyl--markedly removes the inhibiting effect of amphotericin B on the enzyme activity of plasma membranes.     

The in vivo inhibition of transport enzyme activities by chloroquine in different organs of rat is reversible

The antimalarial drug chloroquine is found to inhibit Na⁺, K⁺-ATPase, Ca²⁺, Mg²⁺-ATPase, Ca²⁺-ATPase, pNPPase and acetylcholinesterase activities in different organs of rat in vivo when injected for a certain periods of time. The inhibition seems to be due to the changes in the level of phospholipid, cholesterol and the fatty acid of the lipid and the alteration of the fluidity of the microsomal membranes. However, the enzyme activities return to the normal level in about 2–3 weeks after the discontinuation of the drug suggesting that the drug effect is reversible.     

The chlorpromazine inhibition of transport ATPase and acetylcholinesterase activities in the microsomal membranes of rat in vitro and in vivo

Chlorpromazine, an antipsychotic drug, is found to inhibit Na⁺,K⁺-ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na⁺,K⁺-ATPase, Ca^–2-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off.     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+ -ATPase and calmodulin insensitive (Na+ +K+)- and Mg2+ -ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca2+ -ATPase and calmodulin-insensitive (Na+ +K+)- and Mg2+ -ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca2+ -ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca2+ -ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na+ +K+)-ATPase and Mg2+ -ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+-ATPase and calmodulin insensitive (Na+ + K+)- and Mg2+-ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca²⁺-ATPase and calmodulin-insensitive (Na⁺ + K⁺)- and Mg²⁺-ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca²⁺-ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca²⁺-ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Changes in lipid composition and some biologically important enzyme activities in the microsomal membranes of developing toad ovary in different seasons

The microsomal membranes isolated by sucrose density gradient centrifugation from developing toad ovary have been found to differ significantly in lipid composition and various enzyme activities in different seasons. All the enzymes studied, viz. Na+, K(+)-ATPase, delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta HSD) and prostaglandin synthetase, exhibited maximum activity during the breeding season (July-September) at all stages of development (a,b,c & d). The activities of Na+, K(+)-ATPase and delta 5-3 beta HSD increased with development while that of prostaglandin synthetase followed the reverse order. The total phospholipid, cholesterol and fatty acid contents also varied with season and development. The increase in Na+, K(+)-ATPase and delta 5-3 beta HSD activities in the microsomal membranes of toad ovary at breeding season is accompanied with concomitant increase in phospholipid and unsaturated fatty acid contents at different stages in this season, thereby suggesting some correlation between them.     

Modulation of the Ca2+,Mg2+-ATPase Activity of Synaptosomal Plasma Membrane by the Local Anesthetics Dibucaine and Lidocaine

It has been previously shown that local anesthetics inhibit the total Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2(+)-dependent and Mg2(+)-dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2(+)-dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2(+)-dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP-Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+, Mg2(+)-ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2(+)-ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+, Mg2(+)-ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.     

Na+-stimulated ATPase activities in basolateral plasma membranes from guinea-pig small intestinal epithelial cells

Two ATPase activities, a Na+-ATPase and a (Na+ + K+)-ATPase, have been found associated with sheets of basolateral plasma membranes from guinea-pig small intestinal epithelial cells. The specific activity of the former is 10-15% of the latter. The two ATPase activities differ in their affinity for Na+, their optimal pH, their K+ requirement and particularly in their behaviour in the presence of some inhibitors and of Ca2+. Thus the Na+-ATPase is refractory to ouabain but it is strongly inhibited by ethacrynic acid and furosemide, whilst the (Na+ + K+)-ATPase is totally suppressed by ouabain, partially by ethacrynic acid and refractory to furosemide. In addition, the Na+-ATPase is activated by micromolar concentrations of calcium and by resuspension of the membrane preparation at pH 7.8. The Na+-ATPase is only stimulated by sodium and to a lesser extent by lithium; however, this stimulation is independent of the anion accompanying Na+. The latter rules out the participation of an anionic ATPase. The relation between the characteristics of the sodium transport mechanism in basolateral membrane vesicles (Del Castillo, J.R. and Robinson, J.W.L. (1983) Experientia 39,631) and those of the two ATPase activities present in the same membranes, allow us to postulate the existence of two separate sodium pumps in this membranes. Each pump would derive the necessary energy for active ion transport from the hydrolysis of ATP, catalyzed by different ATPase systems.     

Na+-Ca2+ exchange in plasma membranes of crayfish striated muscle

Na+-Ca2+ exchange rates and some physico-chemical properties of the exchanger were studied in crayfish striated muscle membranes enriched in plasma membranes prepared by differential centrifugation of muscle microsomal fraction on discontinuous sucrose density gradient. The lightest subfraction with the highest Na+, K+-ATPase and Mg2+-ATPase activities also showed the highest Na+-Ca2+ exchange rates. A number of physico-chemical characteristics of the Na+-Ca2+ exchanger found in the present experiments were similar to those reported for excitable membranes of mammals, except for the temperature optimum (20 degrees C for the crayfish).     

Erythrocyte-ghost Ca2+-stimulated Mg2+-dependent adenosine triphosphatase in Duchenne muscular dystrophy.

The Ca2+-stimulated Mg2-dependent ATPase activities (Ca2+-ATPase) of erythrocyte-ghost membranes from patients with Duchenne muscular dystrophy (DMD) and carriers of DMD were compared with activities of normal controls. The Ca2+-ATPase activity of DMD-patient ghost preparations was found to follow the same pattern of activation by Ca2+ as the control membranes. However, the Ca2+-ATPase activity in DMD and some DMD-carrier preparations was substantially elevated compared with controls. To characterize further the elevated Ca2+-ATPase activity found in DMD-patient ghost membrane preparations, we estimated kinetic parameters using both fine adjustment and weighting methods to analyse our experimental data. It was established that in both DMD and DMD-carrier preparations the increase in Ca2+-ATPase activity was reflected by a significant increase in Vmax. rather than by any change in Km. The response of the membrane Ca2+-ATPase activity to changes in temperature was also investigated. In all preparations a break in the Arrhenius plot occurred at 20 degrees C, and in DMD and DMD-carrier preparations an elevated Ca2+-ATPase activity was detected at all temperatures. Above 20 degrees C the activation energy for all types of preparation was the same, whereas below this temperature there appeared to be an elevated activation in DMD and DMD-carrier preparations compared with normal controls. The concept that a generalized alteration in the physicochemical nature of the membrane lipid domain may be responsible for the many abnormal membrane properties reported in DMD is discussed.     

Cytochemical localization of ouabain-sensitive, K+ -dependent p-nitrophenylphosphatase and Ca++-stimulated adenosine triphosphatase activities in human parotid and submandibular glands

K+ -dependent p-nitrophenylphosphatase (pNPPase) and Ca++ -stimulated adenosine triphosphatase (ATPase) activities were studied in human parotid and submandibular glands using cytochemical methods at the ultrastructural level. In both glands, only the striated-duct epithelium showed K+ -pNPPase reaction product, thereby indicating the localization of Na+, K+ -ATPase. The precipitate was concentrated on the deep invaginations of the basolateral plasma membranes, in close association with their cytoplasmic surface. Ca++ -ATPase activity was also found on the basolateral plasma membranes, but two striking differences from the K+ -pNPPase distribution were observed: firstly, Ca++ -ATPase appeared in both acinar and ductal cells, and secondly, it was localized on the outer side of the plasma membranes.     

Effect of indomethacin on Ca2+-stimulated adenosine triphosphatase in the synaptic vesicles of rat brain in vitro

1. Indomethacin inhibits calcium-stimulated adenosine triphosphatase (Ca2+-ATPase), calcium, magnesium-stimulated adenosine triphosphatase (Ca2+,Mg2+-ATPase) and magnesium-stimulated adenosine triphosphatase (Mg2+-ATPase) activities in rat brain synaptic vesicles in vitro. 2. The Ca2+-ATPase activity is most strongly affected by this drug all of the activities of ATPases tested. 3. The decrease of Ca2+-ATPase activity by addition of indomethacin is due to a decrease of Vmax. 4. The Ki values for this drug for ATP and Ca2+ in Ca2+-ATPase were 1.13 mM and 0.68 mM, respectively.     

Characterization of sarcolemma from rabbit skeletal muscle: developmental studies

Sarcolemmal membranes were obtained from skeletal muscle of rabbits at different developmental stages. Lipid and protein composition, as well as enzymatic activities, were compared in sarcolemma prepared in vesicular form. During the developmental period no distinct changes in lipid content and composition were found. Some changes in protein pattern and activities of marker enzymes (5′-nucleotidase and ouabain-sensitive Na⁺,K⁺-ATPase) were detected. Also, changes in Mg²⁺-ATPase and Mg²⁺, Ca²⁺-ATPase activities during development were found.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes.

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of (Mg2+ + Na+ + K+)-ATPase than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for (Mg2+ + Na+ +k+)-ATPase and 5'-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome c reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADPH cytochrome c reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome c reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex.

Continuous sucrose density gradient subfractions from bovine adrenal medullary microsomes were found to accumulate 45-Ca-2+ in the presence of ATP and ammonium oxalate mainly in subfractions of intermediate density. (Na-++K-+)-ATPase (plasma membrane marker) and Ca-2+-ATPase activities were also concentrated in these intermediate subfractions but thiamine pyrophosphatase (Golgi apparatus marker) was not. NADH oxidase (endoplasmic reticulum marker) activity was distributed throughout all subfractions. 45-Ca-2+ accumulation in adrenal cortical microsomes was found to rise and fall in parallel with thiamine pyrophosphatase but not with (Na-++K-+)-ATPase or NADH oxidase activities. Accumulation of 45-Ca-2+ in membrane vesicles in these experiments suggests the existence of a calcium transfer mechanism in plasma membranes of the adrenal medulla but not adrenal cortex.     

Inhibition of platelet aggregation by synthetic phosphatidylcholines: possible involvement of vesiculation of platelet plasma membranes

The purpose of this investigation was to determine which enzyme activities are true canine neutrophil plasma membrane markers. Three enzymes thought to be present on plasma membranes were chosen for study: 5'-nucleotidase, magnesium-dependent adenosine triphosphatase (Mg2+-ATPase), and leucine aminopeptidase. Both 5'-nucleotidase and Mg2+-ATPase were found to be ectoenzymes in the canine neutrophil but additional Mg2+-ATPase activity was located intracellularly. An endogenous inhibitor of 5'-nucleotidase was found in the cytosol of canine neutrophils. The specific 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate also inhibited the canine enzyme in intact cells. Leucine aminopeptidase was located solely in the myeloperoxidase-containing granules of the canine neutrophil. Plasma membrane, as identified by the presence of Mg2+-ATPase and 5'-nucleotidase activities, was separated from other cell organelles by Percoll-density gradient centrifugation of a 10 000 X g supernatant of nitrogen cavitated neutrophils.     

Effect of chlorpromazine and gossypol on Ca(2+)-ATPase activity in the microsomal membranes of rat testes.

The microsomal membranes isolated from rat testes have been found to contain a Mg(2+)-dependent and a Mg(2+)-independent Ca(2+)-ATPase. The enzyme activities were inhibited by two contraceptive drugs--gossypol and chlorpromazine. The inhibition by the former was affected by the presence of ligand(s) and not the substrate in the incubation medium, whereas ligand(s)/substrate did not affect the inhibition by chlorpromazine. This may be explained from the fact that the binding of chlorpromazine and ligand(s)/substrate to the enzyme are independent of each other whereas in case of gossypol the ligand(s) compete with the drug at the binding site of the enzyme.     

Insulin stimulates the translocation of Na+/K(+)-dependent ATPase molecules from intracellular stores to the plasma membrane in frog skeletal muscle.

The mechanism of the stimulation of Na+/K+ transport by insulin in frog skeletal muscle was studied. The ouabain-binding capacity in detergent-treated plasma membranes of insulin-exposed muscles was increased 1.9-fold compared with that of controls. Na+/K(+)-ATPase activity was found in an intracellular 'light fraction' (fraction II) prepared by using anion-exchange chromatography. Marker enzyme activities for plasma and Golgi membranes were not detected in this fraction. The specific activity of Na+/K(+)-ATPase in fraction II from insulin-exposed muscles was 58% of that in an identical fraction from control muscles. No significant difference in the protein yield of the plasma membrane preparation was observed between these two groups. In parallel with the decrease in the Na+/K(+)-ATPase activity in fraction II from insulin-exposed muscles, the ouabain-binding capacity in this fraction was also decreased. The addition of saponin to fraction II increased both Na+/K(+)-ATPase activity and ouabain binding, indicating that some of the Na+/K(+)-ATPase is located in sealed vesicles. These findings support the view that insulin stimulates the translocation of Na+/K(+)-ATPase molecules from fraction II to the plasma membrane.     

A direct colorimetric assay for Ca2+ -stimulated ATPase activity

A simple and rapid colorimetric assay for measuring the high affinity Ca2+-ATPase activity in subcellular fractions is presented. With this method a one-step addition of a malachite green/molybdate/polyvinyl alcohol reagent to the assay mixture at the end of the incubation period is all that is required for the spectrophotometric quantification of the phosphomolybdate-malachite green complex. The presence of polyvinyl alcohol allows the quantification of released phosphate without having to separate it from protein. We have validated this assay by characterizing the high affinity Ca2+-ATPase activity in isolated rat liver microsomes. Comparable Ca2+-ATPase activities in rat liver microsomes and adipocyte plasma membranes were found when measured with this colorimetric assay and an isotopic assay. This method is applicable to the measurement of other types of ATPase activities.