A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice
Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa
Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa
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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

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Figure 3

F92A–Cav-1 interacts with eNOS and prevents Cav-1 from inhibiting eNOS.

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F92A–Cav-1 interacts with eNOS and prevents Cav-1 from inhibiting eNOS. ...
(A) BAEC lysates were incubated with GSH-coupled beads coated with GST alone, GST–Cav-1 (aa 62–101), or GST–F92A–Cav-1 (aa 62–101), washed, and eNOS binding determined by immunoblotting. Top panels show interaction of eNOS with Cav-1 domain and the bottom panel documents a similar amount of GST protein input. Performed in triplicate. (B) Recombinant eNOS activity assay. Purified eNOS was incubated with limiting amounts of GST–Cav-1–coated beads (+) or supplemented with excess soluble GST–Cav-1 (++) or excess soluble GST–F92A–Cav-1 (++) and NOS activity quantified. n = 5 in triplicate. *P < 0.05. (C) CaM/HSP90-dependent displacement of eNOS binding to GST–Cav-1 and GST–F92A–Cav-1. eNOS (2 μg) and HSP90 (4 μg) were incubated with beads in the absence (–) or presence of 0.01 (+) or 1 (++) μM of CaM. Experiments were performed in triplicate; typical data shown. (D) F92A–Cav-1 does not affect its cosedimentation or trafficking (E) with eNOS. WT Cav-1 (myc, top) or F92A–Cav-1 (HA, bottom) localization was examined by confocal microscopy by using anti-myc/HA (red) or anti-eNOS (green). Nuclei were visualized with DAPI (blue). Inset depicts lack of myc or HA staining in noninfected cells. n = 5 individual cells. Scale bar: 7 μm. (F) Decreased superoxide release in BAECs infected with F92A–Cav-1. BAECs were infected with Ad-WT and Ad-F92A–Cav-1 for 48 hours, and superoxide formation was evaluated by cytochrome c reduction. Western blots show protein expression for each condition. (n = 7–10; *P < 0.05).