Jean-Michel Maixent

Modulation of mouse cerebral Na+,K(+)-ATPase activity by oxygen free radicals.

There is increasing evidence that oxygen free radicals (OFR) are involved in cerebral ischaemia–reperfusion injury, possibly via a modulation of Na+,K+-ATPase activity, one of the major membrane pumps responsible for ionic homeostasis. We measured OFR-mediated modulation of this enzymatic activity and examined the roles of lipid and/or protein alterations. Using mouse brain microsomes exposed to UV-C irradiation, our results show a good correlation between activity inhibition and lipoperoxidation estimated by PUFA loss as well as malondialdehyde production. The protective effect of thiourea (OH scavenger) and the lack of effect noted with DTT (thiol protector) suggest that the functionality of the Na+,K+-ATPase is altered by perturbation of membrane integrity rather than by a structural alteration of the protein itself.

Ginkgo biloba extract (EGb 761) protects Na, K-ATPase activity during cerebral ischemia in mice

Neuroprotective drugs such as Ginkgo biloba extract (EGb 761) could prevent the ischemia-induced impairment of the Na,K-ATPase activity. In this study, Na,K-ATPase activity and expression, contents in fatty acids and malondialdehyde, an index of lipoperoxidation, were compared in the ipsilateral (ischemic) and the contralateral (unlesioned) cortices after 1 h of unilateral focal cortices cerebral ischemia in the mouse. EGb 761 (110 mg/kg) was administered daily to half of the animals for 10 days before ischemia. Ischemia significantly reduced Na,K-ATPase activity by about 40% and increased malondialdehyde content; EGb 761 pretreatment abolished these effects. The free radical scavenger properties of EGb 761 are a potential mechanism by which Na,K-ATPase injury and lipoperoxidation are prevented.

Heterogeneous Na+ Sensitivity of Na+,K+‐ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na+ sensitivity of whole brain membrane Na+,K+‐ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10−5 and 10−7M), we were able to discriminate Na+ sensitivity of Na+, K+‐ATPase isoenzymes using nonlinear regression. The ouabain low‐affinity isoform, α1, exhibited high Na+ sensitivity [Ka of 3.88 ± 0.25 mMNa+ and a Hill coefficient (n) of 1.98 ± 0.13]; the ouabain high‐affinity isoform, α2, had two Na+ sensitivities, a high (Ka of 4.98 ± 0.2 mM Na+ and n of 1.34 ± 0.10) and a low (Ka of 28 ± 0.5 mM Na+ and an n of 1.92 ± 0.18) Na+ sensitivity activated above a thresh old (22 ± 0.3 mM Na+); and the ouabain very‐high‐affinity isoform, α3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.

Specific Up-Regulation of Mitochondrial F0F1-ATPase Activity after Short Episodes of Atrial Fibrillation in Sheep

ATPase Activity and Atrial Fibrillation. Introduction: Ventricular fibrillation induced by either digitalis intoxication or electrical stimulation is reported to alter myocardial energy by impairing the sarcolemmal Na,K‐ATPase or the receptor for digitalis and the mitochondrial ATPase synthase or F0,F1‐ATPase. However, little is known about these membrane functions during atrial fibrillation (AF).

Evidence that human endothelial cells express different isoforms of Na,K-ATPase.

Background The catalytic α and smaller β subunits of the plasma membrane Na,K-ATPase occur in various molecular forms (α1, α2, α3, β1 and β2). The α isoforms of the enzyme have varying affinities for ouabain and exist in different tissues with particular distribution patterns. Objective To document the existence of isoforms of the Na,K-ATPase in cultured human umbilical vein endothelial cells. Methods Microsomal fractions were prepared by differential ultracentrifugation from primary cultures of human umbilical vein endothelial cells and from such cells obtained after three passages. Na,K-ATPase activity was assayed using the coupled assay method and sensitivity to ouabain was determined in the presence of varying concentrations of ouabain. Specific antibodies for the various Na,K-ATPase isoforms were used to label these different proteins by immunocytochemistry in endothelial cells and by Western blotting in isolated membranes. Results In plotting the dose–response curves for Na,KATPase activity in response to ouabain we assumed the existence of two independent sites exhibiting different affinities for ouabain (in the μmol/l and the nmol/l ranges). The contribution of low-affinity sites was threefold that of high-affinity sites. After three passages in culture, a specific increase in Na,K-ATPase activity of the high-affinity sites was observed compared with that of cells from primary cultures. Confocal microscopy revealed the existence of β1, β2, and α1 subunit proteins in human umbilical endothelial cells. Staining for α3 isoform was less pronounced and no obvious α2 was detected. Conclusion These findings suggest that human umbilical vein endothelial cells contain β1, β2, a large amount of α1 isoform with an apparently low affinity for ouabain, and a lesser amount of high-affinity sites, which may correspond to the α3 protein.

Antioxidant properties of tea blunt ROS-dependent lipogenesis: beneficial effect on hepatic steatosis in a high fat-high sucrose diet NAFLD obese rat model.

Oxidative stress could trigger lipid accumulation in liver and thus hepatic steatosis. Tea is able to prevent liver disorders, but a direct link between antioxidant capacities and prevention of steatosis has not been reported yet. We aimed to investigate such relationship in a rat model of high fat-high sucrose diet (HFS)-induced obesity and to explore more deeply the mechanisms in isolated hepatocytes. Wistar rats were divided into a control group (standard diet), an HFS group (high fat-sucrose diet) and an HFS+tea group (HFS diet with ad-libitum access to tea drink). Body weight, fat mass, glycemic parameters in blood, lipid and oxidative stress parameters in blood and liver were measured in each group after 14 weeks. Isolated hepatocytes were treated with the reactive oxygen species (ROS) inducer t-BHP in the presence or not of antioxidants (tempol or tea), and superoxide anion production and lipid accumulation were measured using specific fluorescent probes. We reported that the HFS diet highly increased hepatic lipids content, while tea consumption attenuated steatosis and improved the oxidative status (decrease in hepatic oxidative stress, increase in plasma total antioxidant capacity). The role of antioxidant properties of tea in such phenomenon was confirmed in primary cultured rat hepatocytes. Indeed, the increase of mitochondrial ROS production with t-BHP resulted in lipid accumulation in hepatocytes (positive linear regression), and antioxidants (tempol or tea) normalized both. We reported that the antioxidant properties of tea protect rats from an obesogenic HFS diet-induced hepatic steatosis by counteracting the ROS-dependent lipogenesis.

A Quantitative Immunocytochemical Study of Na+, K+-ATPase in Rat Hepatocytes After STZ-induced Diabetes and Dietary Fish Oil Supplementation

Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+, K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na+, K+-ATPase catalytic α1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic α1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the α1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+, K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.

Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model.

Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas’ leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5–12 µM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.

Dietary fish oil promotes positive inotropy of ouabain in the rat heart

We tested the hypothesis that a fish oil (FO) diet promotes positive inotropy of ouabain without increased toxicity. For 2 mo, two groups of adult male rats were fed 1) a regular food diet supplemented with dietary long-chain polyunsaturated fatty acid from FO or 2) a regular food diet (control). The responsiveness to ouabain was evaluated for the two groups in Langendorff-perfused hearts, by31P nuclear magnetic resonance spectroscopy, and on purified membrane-bound Na-K-ATPase. The maximum positive inotropy achieved with ouabain was nearly two times higher in the FO than in the control group and was not associated with significant changes in energetics. Alteration of function and energetic metabolism and inhibition of Na-K-ATPase in response to 3 × 10-4 M ouabain were delayed in the FO group. This study demonstrates that dietary FO, by a cardiac membrane incorporation of n-3 polyunsaturated fatty acid, promotes positive inotropy of ouabain without toxicity and changes in cardiac metabolism.

Mechanism underlying the strong positive inotropic effects of LND-623: specific inhibition of Na, K-ATPase isoforms and exclusion of cellular sites of contractile control.

LND-623 is a new aminosteroid analog of ouabain, with a greater separation between efficacy and toxicity than ouabain. To determine its mechanism of action, we studied its biochemical and physiological effects on human red blood cell sodium transports on different cellular structures regarded as sites of contractile control, and we compared its relative efficacy to ouabain in rat heart preparations and membrane-bound Na, K-ATPase isoenzymes. The response to ouabain was evaluated in Langendorff-perfused hearts and on purified membrane-bound Na, K-ATPase. LND-623 is 6.8-fold more efficient than ouabain in inhibiting the human Na+ pump (IC50= 0.098 ± 0.001 μM vs. 0.67 ± 0.02 μM); (P < 0.0001). LND-623 had no effect on the following cellular functions: Na-Ca exchange, Na-K cotransport, Ca-ATPase, slow calcium channels, adenylate cyclase system, phosphodiesterase, and calcium sensitivity of the contractile protein system. The dose–response curve for the positive inotropic and inhibitory effects on rat cardiac isoenzymes produced by LND-623 were clearly biphasic. The amplitude of the maximum inotropic effect, without any toxic effect, was up to three-fold higher with LND-623 than with the same maximum dose of ouabain used. The strong positive inotropic effect of LND-623 in rats could be related to a specific inhibition of the two rat cardiac isoforms of the Na, K-ATPase.