Direct Interaction of Endothelial Nitric-oxide Synthase and Caveolin-1 Inhibits Synthase Activity

Ju, Hong; Zou, Rong; Venema, Virginia J.; Venema, Richard C.

doi:10.1074/jbc.272.30.18522

Cited by 576 publications

(496 citation statements)

References 27 publications

(29 reference statements)

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“…A recent report suggests that sequences both within the N-and C-terminal halves of caveolin appear to interact with eNOS in addition to the caveolin scaffolding domain (21). However, our data indicate that the caveolin scaffolding domain is both necessary and sufficient for caveolin to interact with eNOS, and thereby leads to enzyme inhibition.…”

Section: Discussioncontrasting

confidence: 63%

Caveolin versus Calmodulin

Michel¹,

Féron²,

Sase

et al. 1997

Journal of Biological Chemistry

277

105

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Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca 2؉ -calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca 2؉ -calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.The mammalian nitric oxide (NO) 1 synthases comprise a family of three related proteins and modulate diverse biological processes ranging from neurotransmission to vascular homeostasis to immunological surveillance (1, 2). These homodimeric proteins share similar overall catalytic schemes to produce NO by the NADPH-, heme-, and O 2 -dependent oxidation of L-arginine in a complex reaction involving numerous redox cofactors, and are absolutely dependent on the ability of the enzymes to be allosterically activated by Ca 2ϩ -calmodulin. The roles of calmodulin in NOS catalysis have been extensively studied, and the binding of calmodulin appears to facilitate interdomain electron transfer for all three enzyme isoforms. However, the NOS isoforms differ in important aspects of their Ca 2ϩ -dependence for enzyme activation by calmodulin. For the endothelial and neuronal nitric oxide synthase isoforms (termed eNOS and nNOS, respectively), transient changes in intracellular Ca 2ϩ promote calmodulin binding and enzyme activation; eNOS and nNOS are characteristically activated as a short-term response to receptor-dependent calcium transients. By contrast, the inflammation-related NOS (iNOS) binds calmodulin avidly and appears to be fully active even at low ambient intracellular calcium levels in immunostimulated cells.The different NOS isoforms may also be distinguished by their subcellular distribution. eNOS is unique among the NOS isoforms in being targeted to the signal-transducing membrane microdomains termed plasmalemmal caveolae (3). Plasmalemmal caveola...

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Section: Discussioncontrasting

confidence: 63%

Caveolin versus Calmodulin

Michel¹,

Féron²,

Sase

et al. 1997

Journal of Biological Chemistry

277

105

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“…Surprisingly, AKAP150 staining was not observed at the MEPs in PAs (Figure 1E), and only a small fraction of TRPV4 sparklets occurred at the MEPs (Figure 1D). Caveolin, a major structural protein of caveolae, directly interacts with and inhibits eNOS activity 57. Endothelial hemoglobin‐α and cytochrome b5 reductase 3 (CytB5R3) have also been shown to regulate the effects of NO on vascular reactivity 58.…”

Section: Discussionmentioning

confidence: 99%

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

Marziano

Hong

Cope

et al. 2017

JAHA

132

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BackgroundRecent studies demonstrate that spatially restricted, local Ca2+ signals are key regulators of endothelium‐dependent vasodilation in systemic circulation. There are drastic functional differences between pulmonary arteries (PAs) and systemic arteries, but the local Ca2+ signals that control endothelium‐dependent vasodilation of PAs are not known. Localized, unitary Ca2+ influx events through transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, regulate endothelium‐dependent vasodilation in resistance‐sized mesenteric arteries via activation of Ca2+‐dependent K+ channels. The objective of this study was to determine the unique functional roles, signaling targets, and endogenous regulators of TRPV4 sparklets in resistance‐sized PAs.Methods and ResultsUsing confocal imaging, custom image analysis, and pressure myography in fourth‐order PAs in conjunction with knockout mouse models, we report a novel Ca2+ signaling mechanism that regulates endothelium‐dependent vasodilation in resistance‐sized PAs. TRPV4 sparklets exhibit distinct spatial localization in PAs when compared with mesenteric arteries, and preferentially activate endothelial nitric oxide synthase (eNOS). Nitric oxide released by TRPV4‐endothelial nitric oxide synthase signaling not only promotes vasodilation, but also initiates a guanylyl cyclase‐protein kinase G‐dependent negative feedback loop that inhibits cooperative openings of TRPV4 channels, thus limiting sparklet activity. Moreover, we discovered that adenosine triphosphate dilates PAs through a P2 purinergic receptor‐dependent activation of TRPV4 sparklets.ConclusionsOur results reveal a spatially distinct TRPV4‐endothelial nitric oxide synthase signaling mechanism and its novel endogenous regulators in resistance‐sized PAs.

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“…eNOS is regulated by caveolin-1. By its interaction with caveolin-1, eNOS is maintained in an inactive state (Garcia-Cardena et al, 1996;Ju et al, 1997). Upon stimulation followed by an increase in intracellular calcium concentration, eNOS dissociates from caveolin-1, a necessary step for its activation and NO production (Feron et al, 1996;Gratton et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Linder¹,

McCluskey²,

Cole³

et al. 2005

J Pharmacol Exp Ther

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Classically, nitric oxide (NO) formed by endothelial NO synthase (eNOS) freely diffuses from its generation site to smooth muscle cells where it activates soluble guanylyl cyclase (sGC), producing cGMP. Subsequently, cGMP activates both cGMPand cAMP-dependent protein kinases [cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively], leading to smooth muscle relaxation. In endothelial cells, eNOS has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine (ACh)-induced relaxation due to alteration in caveolar structure. Given the nature of NO to be more soluble in a hydrophobic environment than in water, and assuming that colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation, we hypothesize that sGC, PKA, and PKG activation may occur at the plasma membrane caveolae. In endothelium-intact rat aortic rings, the relaxation induced by ACh, by the sGC activator 3-(5Ј-hydroxymethyl-2Јfuryl)-1-benzyl indazole (YC-1), and by 8-bromocGMP was impaired in the presence of methyl-␤-cyclodextrin, a drug that disassembles caveolae by sequestering cholesterol from the membrane. sGC, PKG, and PKA were colocalized with caveolin-1 in aortic endothelium, and this colocalization was abolished by methyl-␤-cyclodextrin. Methyl-␤-cyclodextrin efficiently disassembled caveolae in endothelium. In summary, our results provide evidence of compartmentalization of sGC, PKG, and PKA in endothelial caveolae contributing to NO signaling cascade, giving new insights by which the endothelium mediates vascular smooth muscle relaxation.

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Direct Interaction of Endothelial Nitric-oxide Synthase and Caveolin-1 Inhibits Synthase Activity

Cited by 576 publications

References 27 publications

Caveolin versus Calmodulin

Caveolin versus Calmodulin

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

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