Objective: To determine whether DHA suppresses oxidative stress induced endothelial cell calcium influx by altering lipid composition and TRPC1 distribution in membrane rafts.
Methods: Endothelial cells (EC) were pretreated with DHA or stearic acid, then incubated for another 3h with media containing H(2)O(2). Membrane lipid rafts were isolated using the discontinuous sucrose density gradient ultracentrifugation method. Intracellular calcium was detected with laser scanning confocal microscope. TRPC1 protein in membrane fractions was detected by immunoblotting. Membrane fatty acids compositions were analyzed by gas chromatography; raft cholesterol level was assayed by an Amplex Red Cholesterol Assay kit, and DAG concentration was quantified by a DAG kinase assay.
Results: DHA significantly reduced oxidative stress induced calcium influx; pretreated with DHA the n-3 PUFAs were significantly increased in raft fractions, as well as saturated myristic acid, palmitic acid content of membrane rafts in EC; while the stearic acid, monounsaturated oleic acid and cis-oleic acid were decreased. Incubation with DHA also significantly reduced the amount of SM and cholesterol levels in the raft. Interestingly, we fractioned plasma membrane subcellular compartments and discovered that certain amounts of TRPC1 existed in detergent-resistant plasma membrane fractions of EC. After DHA treatment, TRPC1 was partly displaced from lipid raft to detergent-soluble membrane fractions.
Conclusions: DHA significantly reduces oxidative stress induced endothelial calcium influx, this effect might be associated with, at least in part, altered raft lipid environment, and suppresses TRPC1-mediated calcium signaling pathway by partially displacing TRPC1 from membrane caveolar lipid rafts.
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