A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice
Pascal Bernatchez, … , Ethan Marin, William C. Sessa
Pascal Bernatchez, … , Ethan Marin, William C. Sessa
Published August 1, 2011
Citation Information: J Clin Invest. 2011;121(9):3747-3755. https://doi.org/10.1172/JCI44778.
View: Text | PDF | Corrigendum
Research Article Cell biology

A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice

Abstract

Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical for the inhibitory actions of Cav-1 toward eNOS. Herein, we show that F92A–Cav-1 and a mutant cell–permeable scaffolding domain peptide called Cavnoxin can increase basal NO release in eNOS-expressing cells. Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed with eNOS- or Cav-1–deficient mice showed that the vasodilatory effect of Cavnoxin is abolished in the absence of these gene products, which indicates a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that noninhibitory F92A–Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with the other actions of endogenous Cav-1. They also suggest a therapeutic application for regulating the eNOS/Cav-1 interaction in diseases characterized by decreased NO release.

Authors

Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa

×

Figure 1

F92A–Cav-1 increases NO release.

Options: View larger image (or click on image) Download as PowerPoint
F92A–Cav-1 increases NO release. (A) Left panel shows confluent BAECs we...
(A) Left panel shows confluent BAECs were infected with low (5 MOI) or high (50 MOI) of purified adenoviruses, and the accumulation of nitrite in medium was quantified after 16 hours by NO-specific chemiluminescence. Expression of WT (myc tagged) and F92A–Cav-1 (HA tagged) was confirmed by Western blotting. Data are individual values of nitrite per 1 × 106 cells and correlate with the Western blotting data below, performed in triplicate. *P < 0.001 compared with β-gal–infected ECs. Right panel shows correlation depicting enhanced nitrite accumulation in cells expressing F92A–Cav-1 versus WT Cav-1 as a function of viral Cav-1 expression (nitrite accumulation per 1 × 106 cells/AU of Cav-1 expressed). (B) Left panel shows cotransfection of F92A–Cav-1 cDNA with eNOS cDNA increases nitrite accumulation in HEK293 cells, whereas cotransfection with WT Cav-1 cDNA decreases nitrite accumulation. Cells were transfected with fixed amounts of eNOS cDNA and increasing amount of HA-tagged Cav-1 constructs. Individual nitrite values and correlating Western blot data are shown. *P < 0.001 compared with eNOS-expressing cells. Right panel shows correlation depicting increased and decreased nitrite release from cells transfected with F92A or WT Cav-1 cDNAs, respectively. Similar experiments were repeated 2 additional times.