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Cholesterol- and caveolin-rich membrane domains are essential for phospholipase A2-dependent EDHF formation

Annarita Graziani, Verena Bricko, Marco Carmignani, Wolfgang F. Graier, Klaus Groschner
DOI: http://dx.doi.org/10.1016/j.cardiores.2004.06.026 234-242 First published online: 1 November 2004

Abstract

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-11α, 9α-epoxymethano-prostaglandin F
    l-NNA
    Nω-nitro-l-arginine
    MβCD
    methyl-beta-cyclodextrin
    cPLA2
    cytosolic calcium-dependent phospholipase A2
    dpm
    disintegrations per minute
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