Aortic smooth muscle cell proliferation and endothelial nitric oxide synthase activity in fructose-fed rats

We have produced specific monoclonal antibodies (MAb) against particulate bovine aortic endothelial nitric oxide synthase. In Western blots, native and cultured bovine aortic endothelial cells as well as cultured bovine microvascular endothelial cells possess immunoreactive NO synthase. In dot blots, MAb H210 and H32 detect 1 ng and 100 pg of purified endothelial NO synthase, respectively. Both antibodies are specific to the endothelial NO synthase and do not cross-react with other known isoforms of NO synthase, namely from the brain, from cytokine/endotoxin-induced macrophages, or from cytokine/endotoxin-induced vascular smooth muscle cells. Immunohistochemical studies demonstrated the specificity of endothelial NO synthase for endothelial cells in various bovine and human tissues. Many types of endothelial cells, macrovascular, microvascular, arterial, and venous were found to possess this specific isoform of NO synthase. Electron microscopy showed the enzyme to be associated with the plasma membrane, membranes of cytoplasmic vesicles, and in the cytoplasm in human umbilical vein endothelial cells. The results demonstrate that particulate endothelial NO synthase is present in a site to act rapidly to produce NO for release into the blood or toward the smooth muscle in many vascular beds.

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eNOS activation and NO function: Structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity

Journal of Endocrinology ◽

10.1530/joe-11-0083 ◽

2011 ◽

Vol 210 (3) ◽

pp. 271-284 ◽

Cited By ~ 135

Author(s):

Ruslan Rafikov ◽

Fabio V Fonseca ◽

Sanjiv Kumar ◽

Daniel Pardo ◽

Charles Darragh ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Posttranslational Modifications ◽

Endothelial Nitric Oxide Synthase ◽

Regulatory Region ◽

Enos Uncoupling ◽

Oxide Synthase ◽

Endothelial Nitric Oxide ◽

Nitric Oxide Synthase Activity

Rather than being a constitutive enzyme as was first suggested, endothelial nitric oxide synthase (eNOS) is dynamically regulated at the transcriptional, posttranscriptional, and posttranslational levels. This review will focus on how changes in eNOS function are conferred by various posttranslational modifications. The latest knowledge regarding eNOS targeting to the plasma membrane will be discussed as the role of protein phosphorylation as a modulator of catalytic activity. Furthermore, new data are presented that provide novel insights into how disruption of the eNOS dimer prevents eNOS uncoupling and the production of superoxide under conditions of elevated oxidative stress and identifies a novel regulatory region we have termed the ‘flexible arm’.

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Inhibition of MEK/ERK1/2 signalling alters endothelial nitric oxide synthase activity in an agonist-dependent manner

Biochemical Journal ◽

10.1042/bj20060371 ◽

2006 ◽

Vol 398 (2) ◽

pp. 279-288 ◽

Cited By ~ 21

Author(s):

Jacqueline M. Cale ◽

Ian M. Bird

Keyword(s):

Nitric Oxide ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Intracellular Trafficking ◽

Enos Activity ◽

Oxide Synthase ◽

Endothelial Nitric Oxide ◽

Nitric Oxide Synthase Activity

eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein–protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+]i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+]i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+]i, phosphorylation and, possibly, intracellular trafficking.

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