Molecular cloning and characterization of human endothelial nitric oxide synthase

Marsden, Philip A.; Schappert, Keith; Chen, Hai Sheine; Flowers, Michele; Sundell, Cynthia L.; Wilcox, Josiah N.; Lamas, Santiago; Michel, Thomas

doi:10.1016/0014-5793(92)80697-f

Cited by 455 publications

(194 citation statements)

References 19 publications

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“…Cell Culture-Human umbilical vein endothelial cells (HUVEC) were cultured in M199 (Invitrogen) containing 20% fetal bovine serum (Hyclone, Logan, UT), 17 units/ml heparin, and 0.05 mg/ml endothelial mitogen (Biomedical Technologies Inc., Stoughton, MA), as described previously (17). VSMC and human dermal microvascular endothelial cells (MVEC), purchased from Clonetics (Cambrex, East Rutherford, NJ), were cultured as recommended by the supplier.…”

Section: Methodsmentioning

confidence: 99%

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Hypoxia-inducible Expression of a Natural cis-Antisense Transcript Inhibits Endothelial Nitric-oxide Synthase

Fish

Matouk

Yeboah

et al. 2007

Journal of Biological Chemistry

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133

120

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The destabilization of endothelial nitric-oxide synthase (eNOS) mRNA in hypoxic endothelial cells may be important in the etiology of vascular diseases, such as pulmonary hypertension. Recently, an overlapping antisense transcript to eNOS/NOS3 was implicated in the post-transcriptional regulation of eNOS. We demonstrate here that expression of sONE, also known as eNOS antisense (NOS3AS) or autophagy 9-like 2 (APG9L2), is robustly induced by hypoxia or functional deficiency of von Hippel-Lindau protein. sONE is also up-regulated in the aortas of hypoxic rats. In hypoxic endothelial cells, sONE expression negatively correlates with eNOS expression. Blocking the hypoxic induction of sONE by RNA interference attenuates the fall in both eNOS RNA and protein. We provide evidence that the induction of sONE primarily involves transcript stabilization rather than increased transcriptional activity and is von Hippel-Lindau-but not hypoxia-inducible factor 2␣-dependent. We also demonstrate that sONE transcripts are enriched in the nucleus of normoxic cells and that hypoxia promotes an increase in the level of cytoplasmic and polyribosome-associated, sONE mRNA. The finding that eNOS expression can be regulated by an overlapping cis-antisense transcript in a stimulus-dependent fashion provides evidence that sense/antisense interactions may play a previously unappreciated role in vascular disease pathogenesis.

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Section: Methodsmentioning

confidence: 99%

“…To mimic hypoxia, cells were treated with a 140 M concentration of the iron-chelator desferrioxamine (DFO) (Sigma). After treatment, RNA was extracted as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

Hypoxia-inducible Expression of a Natural cis-Antisense Transcript Inhibits Endothelial Nitric-oxide Synthase

Fish

Matouk

Yeboah

et al. 2007

Journal of Biological Chemistry

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133

120

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“…All the surface cysteines (Cys 93, Cys 98, Cys 211, Cys 234, and Cys 381) were identified as sites of S-nitrosylation in our studies, except for Cys 200. In the reductase domain, we have found that regions containing cysteine-660, À801, and À1113 are known to bind with FMN, FAD, and NADPH (Mardsen et al, 1992). Other sites found to undergo S-nitrosylation (cysteines 852, 975=990, and 1047=1049) have not been implicated in any known biochemical functions.…”

Section: Determination Of S-nitrosylation Sites In Enos Treated With mentioning

confidence: 99%

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Tummala

Ryzhov

Ravi

et al. 2008

DNA and Cell Biology

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S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a -NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that Snitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975=990, and 1047=1049 as being susceptible to Snitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse.

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“…It is synthesized from Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3) or constitutive NOS (cNOS), is an enzyme that in humans is encoded by the NOS3 gene located in the 7q35-7q36 region of chromosome 7 [13]. Nitric oxide (NO) is an important protective molecule in the vasculature, and endothelial NO synthase (eNOS) is responsible for most of the vascular NO produced.…”

Section: Nitric Oxidementioning

confidence: 99%

Association of HLA Class I and II Antigens with Vitiligo in Egyptian Population

Elgendy¹,

Alshawadfy²,

Ali³

et al. 2016

Mol Enzyme Drug Targ

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Endothelial dysfunction is regarded as an important factor in the pathogenesis of diabetes. Endothelial dysfunction has been posited to play an important role in the pathogenesis of diabetic nephropathy (DN).The vascular complications of diabetes impose a huge burden on the management of this disease. The higher incidence of cardiovascular complications and the unfavorable prognosis among diabetic individuals who develop such complications have been correlated to the hyperglycemia-induced oxidative stress and associated endothelial dysfunction. Both hyperglycemia, as well as the metabolic consequences of glucose dysregulation, are thought to lead to endothelial cell dysfunction. In this regard, endothelial nitric oxide synthase (eNOS) plays a central role in dysfunction. This review will focus on the mechanisms and therapeutics that specifically target endothelial dysfunction in the context of a diabetic setting.

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Molecular cloning and characterization of human endothelial nitric oxide synthase

Cited by 455 publications

References 19 publications

Hypoxia-inducible Expression of a Natural cis-Antisense Transcript Inhibits Endothelial Nitric-oxide Synthase

Hypoxia-inducible Expression of a Natural cis-Antisense Transcript Inhibits Endothelial Nitric-oxide Synthase

Identification of the Cysteine Nitrosylation Sites in Human Endothelial Nitric Oxide Synthase

Association of HLA Class I and II Antigens with Vitiligo in Egyptian Population

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