Polyunsaturated docosahexaenoic acid suppresses oxidative stress induced endothelial cell calcium influx by altering lipid composition in membrane caveolar rafts

doi:10.1016/j.plefa.2010.02.002

Prostaglandins, Leukotrienes and Essential Fatty Acids

Volume 83, Issue 1, July 2010, Pages 37-43

https://doi.org/10.1016/j.plefa.2010.02.002 Get rights and content

Abstract

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 3 h with media containing H₂O₂. 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.

Introduction

Endothelial dysfunction is critically involved in activated leukocytes transendothelial infiltration, rejection reaction, neointimal and media thickening, vessels remodeling, transplant associated arteriosclerosis or irreversible chronic graft loss [1], [2]. One of the major pathophysiological change in activated or injured EC is the [Ca²⁺]_i overload. Calcium entry in EC occurs in different pathways by various calcium channels. One major route for Ca²⁺ influx is through store-operated Ca²⁺ entry (SOCE) channels in the plasma membrane. However, the mechanism involved in activation or inactivation of SOCE is still elusive. The transient receptor potential (TRP) family ion channels are permeant to cations, most resulting in changes of [Ca²⁺]_i. They are largely cation-nonselective, passing monovalent and divalent cations with little discrimination, at least 19 TRP channels isoforms are expressed in EC [3]. Members of the TRPC protein family have been suggested as molecular components of the SOCE channel [4], and TRPC1 has been reported to be required for SOCE in several cell types [5], [6]. However, neither the molecular mechanism of SOCE nor the role of TRPC in this process is yet understood. In recent years, much evidence has emerged which demonstrates that key molecules involved in Ca²⁺ signaling are associated with caveolar lipid rafts [7]. Lipid rafts are detergent-insoluble special membrane microdomains formed by the dynamic clustering of sphingolipids and cholesterol which have been suggested to function as platform for protein attachment, trafficking and signaling. Several studies have shown that TRPC1 are associated with caveolar lipid raft, and the molecular architecture of caveolae can facilitate intra-molecular interactions between TRPC channels and associated proteins that are involved in activation and/or inactivation of SOCE, moreover, the integrity of the caveolar lipid domain is critical for TRPC1 function [8], [9].

n-3 PUFAs, such as EPA (C20:5) and DHA (C22:6), which refers to 20-carbon (or 22-carbon) fatty acid with 5(or 6) double bonds, the first of which is on carbon atom 3 from the methyl end. Recently, many studies in experimental animals and human subjects have demonstrated that n-3 PUFAs protect against arrhythmia, atherosclerosis or hypertension by blocking Ca²⁺ channel and reducing Ca²⁺ influx [10], [11]. Moreover, n-3 PUFAs can displace proteins from membrane rafts by altering raft lipid composition in several human cell types [12], [13].

In the present study, we hypothesize that polyunsaturated DHA inhibits oxidative stress induced calcium influx which is correlated with its property of changing raft lipid composition, and the alteration of lipid environment affects the location of TRPC1 protein in lipid rafts. So, we isolated the detergent-insoluble and soluble membrane fractions of EC, and then determined the lipid compositions. Meanwhile, we investigated the possibility of DHA regulating TRPC1 protein function by altering membrane raft phospholipids compositions. We have shown here our preliminary results that DHA can alter lipid components of rafts and suppresses calcium influx partially by displacing TRPC1 from lipid rafts. Thus, our data provided some evidences for a functional modification in lipid rafts by DHA treatment and explained DHA-mediated EC protective effects.

Section snippets

Materials and reagents

Cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA, 22:6 n-3) and stearic acid (18:0) were purchased from Sigma-Aldrich Inc. (St, Louis, Mo). Propidium iodide (minimum 95%, HPLC) was from Sigma, and all other chemicals were also from Sigma unless stated otherwise. DMEM medium, bovine calf serum, and serum-free Iscove's modified Dulbecco's medium were from Invitrogen Inc. (Grand Island, NY). Rabbit polyclonal TRPC1 antibody from Sigma was used for western blotting. The rabbit anti-mouse IgG

Oxidative stress induced EC intracellular calcium changes with DHA pretreatment

The cells were incubated with media containing an additional 4,2,1 mmol/L H₂O₂ (Sigma) for 3 h, respectively. The results indicated that the disparity is significant when comparisons were made between groups (Fig. 1). In order to further investigate the effect of DHA on H₂O₂ induced [Ca²⁺]_i changes, the cells were preincubated with 20 μmol/L of DHA for 36 h, and then incubated for another 3 h with media containing an additional 4,2,1 mmol/L H₂O₂, respectively. Our data showed that DHA significantly

Discussion

It is widely accepted that n-3 PUFAs rich in fish oils protect against several types of inflammatory and cardiovascular diseases. DHA and EPA may be the active biological components of these effects. There is considerable evidence to suggest that DHA acts as cardiovascular protection agents principally because it reduced Ca²⁺ entry by blocking Ca²⁺ channel [4]. One major route for Ca²⁺ influx is through SOCE channels in the plasma membrane. However, the mechanism involved in activation or

Acknowledgements

This study was supported by The National Natural Science Foundation of China (30772136), and China Postdoctoral Science Foundation (2004035665).

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Polyunsaturated docosahexaenoic acid suppresses oxidative stress induced endothelial cell calcium influx by altering lipid composition in membrane caveolar rafts

Abstract

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Materials and reagents

Oxidative stress induced EC intracellular calcium changes with DHA pretreatment

Discussion

Acknowledgements

Curr. Opin. Immunol.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Nutr.

J. Biol. Chem.

Anal. Biochem.

Biochem. Pharmacol.

Eur. J. Pharmacol.

J. Thromb. Haemostasis

Prog. Lipid Res.

Biochim. Biophys. Acta

J. Biol. Chem.

Tissue injury and repair in allografts: novel perspectives

Curr. Opin. Nephrol. Hypertension