Reductase Domain Cysteines 1048 and 1114 Are Critical for Catalytic Activity of Human Endothelial Cell Nitric Oxide Synthase as Probed by Site-Directed Mutagenesis

Zhang, J.L.; Patel, Jay; Li, Y.D.; Block, Edward R.

doi:10.1006/bbrc.1996.1348

Cited by 17 publications

(8 citation statements)

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“…A point mutation of this amino acid leads to a 90% decrease in enzymatic activity. 19 Exon 25 encodes a conserved sequence motif, which binds to the adenine moiety of NADPH. Within this sequence is located another conserved cysteine (Cys 1114), which also leads to a drastic decrease of eNOS activity when mutated.…”

Section: Discussionmentioning

confidence: 99%

“…Within this sequence is located another conserved cysteine (Cys 1114), which also leads to a drastic decrease of eNOS activity when mutated. 19 DNA sequences encoding these elements were deleted from the mouse genome. In addition, because of the insertion of the neomycin resistance gene, the C-terminal end of eNOS encoded by exon 26 can be assumed to be absent from a truncated protein synthesized from exons 1 to 23.…”

Section: Discussionmentioning

confidence: 99%

“…Expression of the disrupted gene from exons 1 to 23 can be expected to result in the formation of an inactive enzyme because the NADPH-adenine-nucleotide binding site as well as other sequences implicated in NADPH binding are encoded by exon 24,19 as well as by exon 26. 24 Exons 24 and 25 are located on a 0.8-kb EcoRI fragment, which was replaced by the neomycin resistance gene in the targeting vector pKO4 (Fig 1A).…”

Section: Disruption Of the Enos Gene By Gene Targetingmentioning

confidence: 99%

“…This results in deletion of the adenine nucleotide binding site of the NADPH binding site and of other sequences implicated in NADPH binding located in exon 24. 19 The targeting vector was constructed by placing a 0.8-kb EcoRI-Sca I fragment (short arm) on the 5Ј end and an 8-kb EcoRI fragment (long arm) 3Ј to the neoR gene. 20 The long arm was flanked by a herpes simplex thymidine kinase gene.…”

Section: Cloning Of the Murine Enos Gene And Construction Of The Targmentioning

confidence: 99%

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Coronary Hemodynamics in Endothelial NO Synthase Knockout Mice

Gödecke

Decking

Ding

et al. 1998

Circulation Research

247

181

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Abstract-For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOSϪ/Ϫ). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOSϪ/Ϫ mice. Aortic endothelial cells derived from eNOSϪ/Ϫ mice displayed only background levels of NO x formation compared with wild-type (WT) cells (88 versus 1990 pmol). eNOSϪ/Ϫ mice were hypertensive (mean arterial pressure, 135Ϯ15 versus 107Ϯ8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with N -nitro-L-arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, Ϫ35%) in WT hearts was followed by sustained vasodilation (ϩ190%). L-NAME significantly reduced vasodilation in WT hearts (ϩ125%) but did not alter the initial vasoconstriction. In eNOSϪ/Ϫ hearts, the initial vasoconstriction was augmented (Ϫ70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (Ϫ49% decrease of basal flow). This effect was even more pronounced in eNOSϪ/Ϫ hearts (Ϫ71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOSϪ/Ϫ mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) Key Words: heart Ⅲ gene targeting Ⅲ reactive hyperemia Ⅲ coronary flow Ⅲ blood pressure E ndothelial NO synthase, also called type III NO synthase, is the major NOS isoenzyme that is widely expressed in endothelial cells throughout the vascular bed. It is generally accepted that endothelium-derived NO is an important factor in the control of basal vascular tone.1 NO is also involved in receptor-mediated vasodilation in response to various agonists such as ACh, ATP, thrombin, bradykinin, and others. Through experiments using NOS inhibitors 2,3 but also by use of genetically modified animals, 4,5 it has been demonstrated that functional inactivation of eNOS activity results in hypertension. In addition to the control of vascular tone, NO inhibits platelet aggregation and leukocyte adhesion to the vessel wall as well as proliferation and migration of smooth muscle cells. Thus, eNOS is considered to play an important role in maintaining the antiatherogenic surface of the vessel wall. 6In the heart, eNOS is expressed primarily in the coronary and endocardial endothelia. In addition, eNOS has been localized to cardiac myocytes and the specialized cells of s...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Disruption Of the Enos Gene By Gene Targetingmentioning

confidence: 99%

Section: Cloning Of the Murine Enos Gene And Construction Of The Targmentioning

confidence: 99%

See 2 more Smart Citations

Coronary Hemodynamics in Endothelial NO Synthase Knockout Mice

Gödecke

Decking

Ding

et al. 1998

Circulation Research

247

181

View full text Add to dashboard Cite

show abstract

“…The catalytic activity of eNOS is regulated by multiple post-transcriptional mechanisms involving a variety of factors including phosphorylation state [2,3], active site cysteine modulation in oxygenase and reductase domains of eNOS [4][5][6][7], myristoylation/palmitoylation [8][9][10], and by protein:protein interaction including caveolin:eNOS association-dissociation [3,5,11,12]. A family of three proteins, Caveolin-1,-2,-3, serve as marker proteins of caveolae, flask-shaped invaginations of plasma membranes with distinct lipid rafts enriched in cholesterol in various cell types [13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Peptide-Stimulation Enhances Compartmentalization and the Catalytic Activity of Lung Endothelial NOS

Hutchinson

Kuchibhotla

Block

et al. 2009

Cell Physiol Biochem

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We reported that an 11 amino acid synthetic peptide (P1) activates lung endothelial cell nitric oxide synthase (eNOS) independent of its change in expression and/or phosphorylation. Since caveolae/eNOS dissociation is known to enhance the catalytic activity of eNOS, we examined whether P1-mediated increase of eNOS activity is associated with caveolae/cholesterol modulation, increased caveolin-1 phosphorylation, and intracellular compartmentalization of eNOS in pulmonary artery endothelial cells (PAEC). PAEC were incubated with or without (control) P1 or cholesterol modulators/caveolae disruptors, cholesterol oxidase (CHOX) and methyl-β-cyclodextrin (CD), for 1 h at 37°C. After incubation cells were used for: i) immunoprecipitation, ii) isolation of plasma membrane (PM)-, Golgi complex (GC)-, and non-Golgi complex (NGC)-enriched fractions, iii) immunofluorescence confocal imaging, and iv) electron microscopy for localization and/or eNOS activity. P1, CHOX, and CD-stimulation caused dissociation of eNOS from PM with increased localization to GC and/or NGC. P1 and CHOX significantly increased eNOS activity in PM and GC and CD-stimulation increased eNOS activity localized only in GC. P1 increased phosphorylation of caveolin-1 in intact cells and GC fraction. Immunofluorescence and/or immunogold labeled imaging/electron microscopy analysis of P1-, CHOX-, and CD-stimulated intact cells confirmed eNOS/caveolae dissociation and translocation of eNOS to GC. These results suggest that: i) P1-stimulation translocates eNOS to GC and enhances the catalytic activity of eNOS in both the PM and GC fractions of PAEC, ii) CHOX- but not CD-mediated caveolae and/or cholesterol modulation mimics the effect of P1-stimulated compartmentalization and activation of eNOS in PAEC, and iii) P1-stimulated caveolae/cholesterol modulation, phosphorylation of caveolin-1, and activation of eNOS is physiologically relevant since P1 is known to enhance NO/cGMP-dependent vasorelaxation in the pulmonary circulation.

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