Objective: We analyzed the role of caveolin-1 (Cav-1) in the cross-talk between NADPH oxidase and endothelial nitric oxide synthase (eNOS) signaling in endothelial caveolae.
Methods and results: In intact endothelial cells, angiotensin II (AII) concurrently increased NO and O(2)(-·) production (to 158±12% and 209±5% of control). NO production was sensitive to inhibition of NADPH oxidase and small interfering RNA downregulation of nonreceptor tyrosine kinase cAbl. Reciprocally, N-nitro-l-arginine methyl ester, a NOS inhibitor, partly inhibited O(2)(-·) stimulated by AII (by 47±11%), indicating eNOS uncoupling, as confirmed by increased eNOS monomer/dimer ratio (by 35%). In endothelial cell fractions separated by isopycnic ultracentrifugation, AII promoted colocalization of cAbl and the NADPH oxidase subunit p47phox with eNOS to Cav-1-enriched fractions, as confirmed by proximity ligation assay. Downregulation of Cav-1 by small interfering RNA (to 50%), although it preserved eNOS confinement, inhibited AII-stimulated p47phox translocation and NADPH oxidase activity in Cav-1-enriched fractions and reversed eNOS uncoupling. AII infusion produced hypertension and decreased blood hemoglobin-NO in Cav-1(+/+) mice but not in heterozygote Cav-1(+/-) mice with similar Cav-1 reduction.
Conclusions: Cav-1 critically regulates reactive oxygen species-dependent eNOS activation but also eNOS uncoupling in response to AII, underlining the possibility to treat endothelial dysfunction by modulating Cav-1 abundance.