We have investigated the effects of cholesterol and ω-3 fatty acids eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA) on Na, K-ATPase activity in human endothelial cells (HUVEC). Cultured HUVEC were incubated for 18 h with pure egg phosphatidylcholine (PC), or cholesterol-enriched liposomes (4 mg PC/ml). EPA and DHA α-tocopherol-acetate were emulsified with PC and incubated with HUVEC (10 mM). Na, K-ATPase and 5′-nucleotidase activities were determined using the coupled assay method on microsomal fractions obtained from cultured cells using non treated cells as control. Cholesterol enrichment significantly reduced both Na, K-ATPase and 5′-nucleotidase activities by a similar level (−40%), whereas pure phospholipid liposomes inhibited this activity only by 22%. The dose–response curves of Na, K-ATPase activity were all biphasic assuming the presence of two independent sites exhibiting different affinities for ouabain of nM and μM respectively. The cholesterol induced inhibitory effect was greater for low affinity sites (−54%) as compared to that of the high affinity sites (−24%) whereas ω-3 fatty acids reduced the activity of both sites by 22%. Short term effects of EPA and DHA on Na, K-ATPase activity were determined by incubating microsomal fractions from untreated cells with various concentrations of free fatty acids (from 1 to 200 μM) for 20 min. Both EPA and DHA significantly reduced Na, K-ATPase activity but inhibition by EPA seems to be more effective than DHA. These results suggest that cholesterol and ω-3 fatty acids reduce Na, K-ATPase activity in HUVEC.

Background The catalytic alpha and smaller beta subunits of the plasma membrane Na,K-ATPase occur in various molecular forms (alpha(1), alpha(2), alpha(3), beta(1), beta(2)). The alpha 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,K-ATPase activity in response to ouabain we assumed the existence of two independent sites exhibiting different affinities for ouabain tin the mu mol/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 beta(1), beta(2), and alpha(1) subunit proteins in human umbilical endothelial cells, Staining for alpha(3) isoform was less pronounced and no obvious alpha(2) was detected.Conclusion These findings suggest that human umbilical vein endothelial cells contain beta(1), beta(2), a large amount of alpha(1) isoform with an apparently low affinity for ouabain, and a lesser amount of high-affinity sites, which may correspond to the alpha(3) protein. (C) 1998 Rapid Science Ltd.

Annals of the New York Academy of SciencesVolume 834, Issue 1 p. 656-657 Immunological Identification of Na,K-ATPase Isoforms in Nonfailing and Failing Myocardiuma ODILE BARBEY, ODILE BARBEY Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorALAIN GERBI, ALAIN GERBI Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorKARINE ROBERT, KARINE ROBERT Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorVINCENT MAYOL, VINCENT MAYOL Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorSANDRINE PIERRE, SANDRINE PIERRE Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorFRANCK PAGANELLI, FRANCK PAGANELLI Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorJEAN-MICHEL MAIXENT, Corresponding Author JEAN-MICHEL MAIXENT Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceTo whom correspondence should be addressed. Tel: (33) 91.69.88.81; fax: (33) 91.09.05.06.Search for more papers by this author ODILE BARBEY, ODILE BARBEY Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorALAIN GERBI, ALAIN GERBI Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorKARINE ROBERT, KARINE ROBERT Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorVINCENT MAYOL, VINCENT MAYOL Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorSANDRINE PIERRE, SANDRINE PIERRE Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorFRANCK PAGANELLI, FRANCK PAGANELLI Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceSearch for more papers by this authorJEAN-MICHEL MAIXENT, Corresponding Author JEAN-MICHEL MAIXENT Cardiac Research Laboratories, School of Medicine, University Aix-Marseille, 15 Bld Dramard, F-13326 Marseille, Cedex 15, FranceTo whom correspondence should be addressed. Tel: (33) 91.69.88.81; fax: (33) 91.09.05.06.Search for more papers by this author First published: 17 December 2006 https://doi.org/10.1111/j.1749-6632.1997.tb52342.xCitations: 4 a This work was supported by grants from Procter & Gamble Pharmaceuticals France F91165 Longjumeau Cedex, France and the Assistance Publique à Marseille, Marseille, France. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume834, Issue1Na/K-ATPase and Related Transport ATPases: Structure, Mechanism, and RegulationNovember 1997Pages 656-657 RelatedInformation