7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells.

As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 µg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

Inhibition of Na,K-ATPase by external electrical cardioversion in a sheep model of atrial fibrillation.

INTRODUCTION: Electrical external cardioversion commonly used to treat atrial fibrillation (AF) is associated with myocardial membrane damage and disturbances in ionic homeostasis (hemodynamically unstable). The present study was designed to investigate whether alterations in ionic homeostasis observed were due in part to changes in the myocardial activity of Na,K-ATPase. METHODS AND RESULTS: AF was induced by pacing in ten anesthetized sheep divided into two groups. Group I (n = 4) received a single external countershock of 360 J after three episodes of AF lasting 10 minutes. Group II (n = 6) served as controls. Activity, responsiveness to ouabain, and membrane expression of catalytic alpha and beta subunits of Na,K-ATPase in sarcolemmal myocardial membrane fractions were investigated. Membrane fluidity and fatty acid composition, and plasma levels of atrial natriuretic factor (ANF) also were measured. One shock after episodes of AF significantly decreased ventricular Na,K-ATPase activity up to 50% (P < 0.001) without modification of atrial activity at the membrane level. Sites with low affinity to ouabain showed a fivefold lower affinity for ouabain in the cardioversion group than in the control group (IC50 = 7.9 micromol/L vs 40 micromol/L ouabain, P < 0.05). Plasma levels of ANF were significantly increased in the cardioversion group compared with the control group. These changes were independent of membrane modulation in terms of expression of Na,K-ATPase, membrane fluidity, and fatty acid composition. CONCLUSION: This study suggests that left ventricular perturbation of ionic homeostasis subsequent to transthoracic cardioversion could result from inactivation of Na,K-ATPase activity.

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.

Role of lysine and tryptophan residues in the biological activity of toxin VII (Ts gamma) from the scorpion Tityus serrulatus.

Toxin VII (TsVII), also known as Ts gamma, is the most potent neurotoxin in the venom of the Brazilian scorpion Tityus serrulatus. It has been purified to homogeneity using a new fast and efficient method. Chemical modification of TsVII with the tryptophan-specific reagent o-nitrophenylsulfenyl chloride yielded three modified derivatives (residues Trp39, Trp50 and Trp54). Acetylation of TsVII mostly generated the monoacetylated Lys12 derivative. No side reactions were detected, as indicated by endoproteinase Lys-C peptide mapping, Edman degradation and electrospray mass spectrometry. Circular dichroism and fluorimetric measurements showed that none of the chemical modifications altered the overall structure of the derivatives. The acetylation of Lys12 or the sulfenylation of Trp39 or Trp54 led to a loss of both toxicity in mice and apparent binding affinity for rat brain and cockroach synaptosomal preparations. Sulfenylation of Trp50, however, moderately affected the toxicity of TsVII in mice and had almost no effect on its binding properties. A 3-dimensional model of TsVII was constructed by homology modeling. It suggests that the most reactive residues (Lys12 and Trp39 and Trp54) are all important in the functional disruption of neuronal sodium channels by TsVII, and are close to each other in the hydrophobic conserved region.

MDR-related properties of K 562 cells grown in two different culture media.

Using a synthetic substitute, Ultroser HY, for fetal bovine serum to supplement a classical RPMI 1640 culture medium produced changes in the properties of sensitive and of multidrug-resistant K 562 cells. Though no morphological changes were found, a statistically significant decrease in doubling-time was noted. Plasma membranes were more rigid, as reflected by an increase in the order parameter values. Adriamycin cytotoxicity was decreased, as shown by an increase in IC 50 values. The THP-adriamycin uptake, monitored by fluorimetry, was diminished even when the revertant agent verapamil was added. Moreover, the apparent number of Pgp 170 molecules per cell was lower for resistant cells grown with Ultroser HY. Thus Pgp 170 was not involved in the MDR increase induced by Ultroser HY. In conclusion, it must be kept in mind that environmental factors such as media chemical composition influence the MDR phenomenon and that environmental factors may also influence the MDR phenomenon in clinical situations.

Quantitative determination of the MDR-related P-glycoprotein, Pgp 170, by a rapid flow cytometric technique.

Pgp 170 is the main integral membrane protein involved in acquired or de novo multidrug resistance (MDR), frequently implicated in chemotherapeutic failure. Because there is at present no method for quantitating Pgp 170 levels, a new and convenient assay, using flow cytometry with a standard fluorescence curve and MRK 16, a mAb recognizing an external epitope of human Pgp 170, was developed. Assuming a 1:1 stoichiometry, we calculated for the first time the apparent number of Pgp 170 molecules per cell. The method was applied successfully to cells in suspension or grown as monolayers and their mixtures. All quality criteria were checked and proved the suitability of the method for quantifying Pgp 170.

Effects of benfluorex metabolites on membrane fluidity and insulin-related processes.

As little work has dealt with the antihyperglycemic property of benfluorex at the hepatocyte level, we studied the effects of its main metabolites, S422 and S1475, on membrane fluidity and on insulin binding, internalization and action in healthy rat hepatocytes. Both metabolites were effective fluidizing agents. Neither one affected insulin binding. Only S422 favored the bound insulin-receptor internalization process. The metabolites produced no change in basal alpha-aminoisobutyric acid uptake. Only S422 promoted the insulin-stimulated alpha-aminoisobutyric acid uptake in a dose-dependent way. Therefore, our study demonstrated that: (i) the effects of S422 on insulin-related processes in isolated hepatocytes were direct, specific and not due to any membrane fluidizing mechanism; (ii) S422 improved hepatocyte response to insulin at a post-binding level. These results in vitro give an additional explanation, at the cellular level, of the benefit of benfluorex treatment for non insulin-dependent diabetes patients.

In vitro influence of benfluorex and its main metabolites on rat liver microsomal membrane properties.

Benfluorex and its three main metabolites at 30 microM have been shown to inhibit Acyl CoA cholesterol acyl transferase activity in rat liver microsome preparations and to fluidize these membranes, as reflected by a decrease in the lipid order parameter. When drug concentrations were higher (60-200 microM), the compounds differed in their enzymatic inhibition properties but retained the same fluidizing effects. Only the parent compound had a dose-dependent inhibiting effect. These results are discussed with regard to the chemical properties of compounds, in particular their electric charges and their lipophilic characters.

Variations in fluorescence and enzymic properties of bovine dihydrofolate reductase.NADPH complex during the slow conformational change induced by coenzyme binding.

When NADPH was added in large excess to bovine dihydrofolate reductase (H2folate reductase), there was a slow isomerization process between two conformers of the binary complex (B1<-->B2), as shown by changes in the fluorescence properties. Thus, we monitored the time dependence of (a) the quenching of protein intrinsic fluorescence intensity, (b) the polarization state of the fluorescence light emitted by NADPH.H2folate reductase complexes and (c), from a more biological point of view, the enzymic activity of binary complex solutions. The kinetics for these three processes were in good agreement using the same temperature conditions. Furthermore, fluorescence studies provided information on the NADPH environment in the binary complex. As soon as NADPH bound to H2folate reductase, light emitted by the invariant Trp24 residue located within the coenzyme-binding site was quenched by an energy-transfer process. Moreover, Trp57 and/or Trp113 emissions were partially quenched. The subsequent NADPH-bound protein conformational change caused an additional quenching, probably of Trp57 and/or Trp113 emissions. Thus, NADPH.H2folate reductase conformation was modified but no change was observed at the coenzyme-binding site, at least in our fluorescence study. These results were confirmed by polarization measurements. The conformational change, as well as the instantaneous NADPH binding, resulted in a more rigid form of the protein, as shown by an increase in steady-state anisotropy values. Finally, the isomerization process led to a more active enzymic form.

Influence of cetiedil on erythrocyte membrane microviscosity and acetylcholinesterase activity.

Since one of the cellular targets of cetiedil, a vaso-erythroactive drug, is likely to be the erythrocyte membrane, we have studied the influence of this drug on erythrocyte membrane microviscosity and acetylcholinesterase activity. No effect was evidenced on microviscosity, as measured by fluorescence polarization of light emitted by DPH or TMA-DPH labelling of the lipid bilayer. Cetiedil, however, did lower acetylcholinesterase activity, but it did not directly inhibit this enzyme activity. It can therefore be considered as an amphiphilic drug that perturbs membrane properties without affecting the physical state of the erythrocyte membrane.

Fluidity of human erythrocyte ghosts.

The fluidity, defined by its two components, the order parameter, S, and the rotation correlation time, tau c, was studied on healthy human erythrocytes ghosts. We also measured ghost protein, cholesterol and phospholipid contents as well as acetylcholinesterase activities. No statistically significant difference was evidenced between erythrocyte ghosts from men and women. Whereas tau c values did not significantly vary among sample elements, variations of ghost order parameters about the mean were explained at 61% by changes in cholesterol contents and, to a lesser extent, in protein contents. No relationship was evidenced between ghost order parameter values and those of corresponding acetylcholinesterase activities. Liposomes prepared from ghost lipid extracts had much lower order parameter values than did corresponding ghosts. A few experiments were performed in the same way on ghosts from sickle blood. This disease appeared to decrease the bilayer lipid motionnal freedom as an increase of the order parameter values was evidenced.

Conformational change induced by coenzyme binding to bovine liver dihydrofolate reductase: a spectrofluorimetric study.

When NADPH was added in excess to a bovine liver DHFR solution, a fluorescence peak due to an energy transfer mechanism was apparent at 450 nm. It did not vary over time. The intrinsic fluorescence peak of DHFR at 320 nm was quenched and this phenomenon increased over the time-course after NADPH addition. This result was ascribed to a slow DHFR conformational change induced by NADPH binding, which has never been previously described in such a long time scale (more than 30 min). A kinetic scheme accounting for this mechanism has been proposed. Furthermore, this interconversion between two protein conformers led to an increase in the initial apparent rate of the enzymatic reaction catalyzed by DHFR.

Activity measurements in human tissues of the methotrexate molecular target: a novel fluorometric assay.

As DHFR is the main molecular target of MTX, a widely used anticancer drug, its level in human tissues is likely to be one of the factors determining tissue sensitivity towards this drug. Forty-one biopsies were analyzed for their DHFR activity by a convenient spectrofluorometric assay developed in our laboratory; this sensitive method proved to be suitable for measurements in very small human samples. Statistical analysis of the results showed that (i) DHFR activity is not an index of tumorogenicity, at least in the cases studied, (ii) tumorous extracts contain modulators of DHFR activity.

Lipid diet and enterocyte microsomal membrane fluidity in rats.

Rats were fed on diets more or less enriched with n-3 and n-6 unsaturated fatty acids, before removal of the small intestine. The global protein, cholesterol and phospholipid contents of enterocyte microsomes were measured. Fatty acids of the total lipid extracts were determined. Acyl coenzyme A: cholesterol acyl transferase (ACAT) was chosen as the enzyme whose activity reflects metabolic changes induced by lipid diets. Fluorescence measurements using diphenylhexatriene as the membrane probe were performed. As dietary fat may change the fatty acid composition of membranes, the order parameter S calculated from fluorescence measurements was studied with regard to dietary fatty acid composition. The S values, distributed over a large range, were not different between rat groups. They were positively correlated with the ratios of cholesterol and proteins to phospholipids and the molar percentage of saturated fatty acids. ACAT activity was negatively correlated with S. Variations in S values among rats, whatever the diet, could in part be attributed to individual factors.

Influence of the guanine nucleotide phosphorylation state and of Mg2+ ions on the interaction of vinzolidine/tubulin 6 S: a fluorescence quenching study.

The binding of the new vincaalkaloid vinzolidine to tubulin 6 S was investigated by using fluorescence quenching methods. The value of the apparent equilibrium binding constant was found to depend on the phosphorylation state of the guanine nucleotide bound to the tubulin exchangeable site (E-site), with Ka values of 4.9 X 10(4) and 8.19 X 10(4) M-1 for GTP- and GDP-tubulin, respectively. The effect of Mg2+ ions on this binding was more important on GTP-tubulin than on GDP-tubulin, and might be related to the existence of Mg2+ site(s) independent of the nucleotide.

A novel fluorometric assay for quantitative analysis of dihydrofolate reductase activity in biological samples.

In an effort to study the level of dihydrofolate reductase (DHFR), the main molecular target of antifolate drugs, in healthy and malignant tissues of human origin, a new and convenient fluorometric enzymatic assay has been developed. The technique measures the overall decrease in fluorescence emission at 454 nm (lambda ex = 342 nm) due to the contributions from coenzyme oxidation and substrate reduction. This technique was developed by using an enzyme purified from beef liver. All criteria of quality were checked: sensitivity, reproducibility and specificity made it suitable for low activity measurements. It was successfully applied to human tissue crude extracts.

Influence of time and chloride ions on the interaction of cisplatin with human albumin in-vitro.

The interaction of cis-dichlorodiammineplatinum (II) (cisplatin) with human serum albumin (HSA), dissolved in phosphate buffer with or without sodium chloride (0.1 M) has been examined at pH 7.4 and mu = 0.154. Equal volumes of cisplatin and HSA solutions were incubated at 37 degrees C for various times and filterable platinum concentrations versus time measured by flameless atomic absorption spectrophotometry. Binding kinetics differed depending on the buffer solutions used and on the time elapsing between cisplatin dissolution and outset of incubation with HSA. Experimental data were fitted to a theoretical equation used to calculate the number of nucleophilic sites per HSA molecule. Titrations of the HSA sulphydryl group content before and after incubation with a cisplatin solution were made, from which it was shown that the lone SH-group of the HSA macromolecule is involved in cisplatin binding. We also studied HSA's sensitivity towards denaturing agents when it was complexed with cisplatin. This sensitivity was decreased upon cisplatin binding. Also, the binding capacities of HSA and the HSA-Pt(II) complex to both tryptophan and warfarin were compared to determine the possible influence of cisplatin upon the binding to HSA of other drugs; this influence was negligible.

pH-Stat titration method for dihydrofolate reductase activity measurements: application to determination of substrate Michaelis constant and antifolate inhibition constant.

A pH-Stat titration method was developed for measuring dihydrofolate reductase (DHFR) activity; this method permits detection of very low DHFR activities corresponding to 100 pmol of substrate reduced per minute. This value is about ten times lower than those observed using the classical spectrophotometric method. This sensitivity makes it possible to measure the DHFR in crude tissue extracts. With beef liver DHFR, Michaelis constants for the cofactor NADPH and the natural substrate determined by this method were 1.9 +/- 0.3 X 10(-5) and 8.5 +/- 0.5 X 10(-7) M, respectively. The inhibition constant of methotrexate, a competitive inhibitor of dihydrofolate, was 3.4 +/- 1.3 X 10(-11) M.

In vitro plasma binding of some second generation antitumor platinum complexes.

The kinetics of five cisplatin analogs binding to human plasma fractions possessing a molecular weight greater than 50,000 daltons were studied. Each drug solution in plasma ultrafiltrate or phosphate buffer (pH = 7.4, mu = 0.154) was mixed with human plasma and filterable platinum concentrations were measured versus time by atomic absorption spectrophotometry. The only Pt IV compound studied did not bind. All the other Pt II complexes bound, but their binding kinetics were quite different. The experimental data were fitted to a theoretical equation based on the hypothesis that plasma nucleophilic agents possessing a molecular weight greater than 50,000 daltons are able to react with Pt compounds with an apparent second order rate constant. The apparent reaction rate constants and initial concentrations of these nucleophilic agents were calculated. The difference between the respective values obtained for each cisplatin analog could be explained by differences in their chemical formulas. Therefore our result should contribute towards a better understanding of the pharmacokinetics of the cisplatin analogs studied.