© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Cholesterol- and caveolin-rich membrane domains are essential for phospholipase A2-dependent EDHF formation
aDepartment of Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria
bDepartment of Basic and Applied Biology, University of L'Aquila, Coppito, Italy
cDepartment of Medical Biochemistry and Medical Molecular Biology, Karl-Franzens-University, Graz, Austria
* Corresponding author. Department of Pharmacology and Toxicology, University of Graz, Universitaetsplatz 2, A-8010 Graz, Austria. Tel.: +43 316 380 5570; fax: +43 316 380 9890. Email address: klaus.groschner{at}uni-graz.at
Objective: Cholesterol-rich membrane domains, which contain the scaffold protein caveolin-1 (Cav-1) (caveolae), represent an important structural element involved in endothelial signal transduction. The present study was designed to investigate the role of these signaling platforms in the generation of endothelial-derived hyperpolarizing factor (EDHF).
Methods: Caveolae were disrupted by cholesterol depletion with methyl-β-cyclodextrin (MβCD 10 mM). MβCD-induced modulation of non-nitric oxide-/non-prostanoid-dependent (EDHF)-mediated vasorelaxation was studied in pig coronary arteries. Effects of MβCD on endothelial Ca2+ signaling and phospholipase A2 (cPLA2) activity were determined using fura-2 imaging and measurement of [3H]-arachidonate mobilization in cultured pig aortic endothelial cells (PAEC). Cellular localization of caveolin-1 and phospholipase A2 was investigated by cell fractionation, and interaction of cPLA2 with caveolin-1 was tested by immunoprecipitation experiments.
Results: MβCD inhibited EDHF-mediated relaxations of pig coronary arteries induced by bradykinin (100 nM) or ionomycin (300 nM) but not relaxations induced by the NO donor DEA/NO (1 µM). Exposure of arteries to cholesterol-saturated MβCD failed to affect EDHF-mediated relaxations. Cholesterol depletion with MβCD did not affect bradykinin or ionomycin-induced Ca2+ signaling in pig aortic endothelial cells, but was associated with enhanced basal and reduced Ca2+-dependent release of arachidonic acid (AA). Cell fractionation experiments indicated targeting of cPLA2 to low density, caveolin-1 rich membranes and immunoprecipitation experiments demonstrated association of phospholipase A2 with the scaffold protein of caveolae, caveolin-1. Cholesterol depletion with MβCD did not disrupt the interaction between cPLA2 and caveolin-1 but prevented targeting of cPLA2 to low density membranes. Exogenous supplementation of arachidonic acid after cholesterol depletion partially restored EHDF responses in pig coronary arteries.
Conclusion: The integrity of caveolin-1-containing membrane microdomains is prerequisite for arachidonic acid recruitment and EDHF signaling in porcine arteries.
KEYWORDS Endothelial-derived hyperpolarizing factor; Lipid rafts; Caveolin; Phospholipase A2; Arachidonic acid
Abbreviations: PAEC, pig aortic endothelial cells • DEA/NO, 2-(N, N-diethylamino)-diazenolate-2-oxide.diethylammonium salt • EDHF, endothelium-derived hyperpolarizing factor • PBS, phosphate buffered saline • U46619, 9, 11-dideoxy-11
, 9-epoxymethano-prostaglandin F2 • L-NNA, N
-nitro-L-arginine • MβCD, methyl-beta-cyclodextrin • cPLA2, cytosolic calcium-dependent phospholipase A2 • dpm, disintegrations per minute
Time for primary review 25 days
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