Cholesterol and omega-3 fatty acids inhibit Na, K-ATPase activity in humanendothelial cells

We have investigated the effects of cholesterol and omega-3 fatty adds eico sapentaenoic acid (EPA) and docosahexenoic acid (DHA) on Na, K-ATPase activ ity in human endothelial cells (HUVEC). Cultured HUVEC were incubated for 1 8 h with pure egg phosphatidylcholine (PC), or cholesterol-enriched liposom es (4 mg PC;ml). EPA and DHA alpha-tocopherol-acetate were emulsified with PC and incubated with HUVEC (10 mM). Na, K-ATPase and 5'-nucleotidase activ ities were determined using the coupled assay method on microsomal fraction s obtained from cultured cells using non treated cells as control. Choleste rol enrichment significantly reduced both Na, K-ATPase and 5'-nucleotidase activities by a similar level (-40%), whereas pure phospholipid liposomes i nhibited this activity only by 22%. The dose-response curves of Na, K-ATPas e activity were all biphasic assuming the presence of two independent sites exhibiting different affinities for ouabain of nM and mu M respectively. T he cholesterol induced inhibitory effect was greater for low affinity sites (-54%) as compared to that of the high affinity sites (-24%) whereas omega -3 fatty acids reduced the activity of both sites by 22%. Short term effect s of EPA and DHA on Na, K-ATPase activity were determined by incubating mic rosomal fractions from untreated cells with various concentrations of free fatty acids (from 1 to 200 mu M) for 20 min. Both EPA and DHA significantly reduced Na, K-ATPase activity but inhibition by EPA seems to be more effec tive than DHA. These results suggest that cholesterol and omega-3 fatty aci ds reduce Na, K-ATPase activity in HUVEC. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.