Cholesterol- and caveolin-rich membrane domains are essential for phospholipase A2-dependent EDHF formation.
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-beta-cyclodextrin (MbetaCD 10 mM). MbetaCD-induced modulation of non-nitric oxide-/non-prostanoid-dependent (EDHF)-mediated vasorelaxation was studied in pig coronary arteries. Effects of MbetaCD on endothelial Ca(2+) signaling and phospholipase A(2) (cPLA(2)) activity were determined using fura-2 imaging and measurement of [(3)H]-arachidonate mobilization in cultured pig aortic endothelial cells (PAEC). Cellular localization of caveolin-1 and phospholipase A(2) was investigated by cell fractionation, and interaction of cPLA(2) with caveolin-1 was tested by immunoprecipitation experiments.


RESULTS
MbetaCD 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 microM). Exposure of arteries to cholesterol-saturated MbetaCD failed to affect EDHF-mediated relaxations. Cholesterol depletion with MbetaCD did not affect bradykinin or ionomycin-induced Ca(2+) signaling in pig aortic endothelial cells, but was associated with enhanced basal and reduced Ca(2+)-dependent release of arachidonic acid (AA). Cell fractionation experiments indicated targeting of cPLA(2) to low density, caveolin-1 rich membranes and immunoprecipitation experiments demonstrated association of phospholipase A(2) with the scaffold protein of caveolae, caveolin-1. Cholesterol depletion with MbetaCD did not disrupt the interaction between cPLA(2) and caveolin-1 but prevented targeting of cPLA(2) to low density membranes. Exogenous supplementation of arachidonic acid after cholesterol depletion partially restored EHDF responses in pig coronary arteries.


CONCLUSIONS
The integrity of caveolin-1-containing membrane microdomains is prerequisite for arachidonic acid recruitment and EDHF signaling in porcine arteries.
Department of Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria.
Annarita
A
Graziani
Verena
V
Bricko
Marco
M
Carmignani
Wolfgang F
WF
Graier
Klaus
K
Groschner
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D001685:Biological Factors / Q000096:biosynthesis*; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D003331:Coronary Vessels; D046508:Culture Techniques; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D015398:Signal Transduction / Q000502:physiology*; D013552:Swine
D001685:Biological Factors; D051242:Caveolin 1; D022461:Caveolins; C067410:endothelium-dependent hyperpolarization factor; D016718:Arachidonic Acid; D002784:Cholesterol; D010741:Phospholipases A; D054467:Phospholipases A2; D001920:Bradykinin; D002118:Calcium