Flow Shear Stress Stimulates Gab1 Tyrosine Phosphorylation to Mediate Protein Kinase B and Endothelial Nitric-oxide Synthase Activation in Endothelial Cells

ABSTRACT Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.

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Nitrite Activates 5′AMP-Activated Protein Kinase-Endothelial Nitric Oxide Synthase Pathway in Human Glomerular Endothelial Cells

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b17-00316 ◽

2017 ◽

Vol 40 (11) ◽

pp. 1866-1872 ◽

Cited By ~ 2

Author(s):

Licht Miyamoto ◽

Megumi Yamane ◽

Yosuke Tomida ◽

Mai Kono ◽

Tomomi Yamaoka ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Glomerular Endothelial Cells ◽

Human Glomerular Endothelial Cells ◽

Amp Activated Protein Kinase ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

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Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation

Journal of Biological Chemistry ◽

10.1074/jbc.m111.220236 ◽

2011 ◽

Vol 286 (22) ◽

pp. 20100-20108 ◽

Cited By ~ 26

Author(s):

Zhihong Xiao ◽

Tingting Wang ◽

Honghua Qin ◽

Chao Huang ◽

Youmei Feng ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Protein Phosphorylation ◽

Endothelial Nitric Oxide Synthase ◽

No Production ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca2+ and protein phosphorylation, but the interrelationship between intracellular Ca2+ mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca2+ release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca2+ release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca2+ release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca2+ and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca2+ release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca2+/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca2+ release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca2+ release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca2+ is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca2+-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.

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Propofol Depresses Angiotensin II–induced Cell Proliferation in Rat Cardiac Fibroblasts

Anesthesiology ◽

10.1097/01.anes.0000365960.74268.21 ◽

2010 ◽

Vol 112 (1) ◽

pp. 108-118 ◽

Cited By ~ 15

Author(s):

Tzu-Hurng Cheng ◽

Yuk-Man Leung ◽

Chi-Wai Cheung ◽

Cheng-Hsien Chen ◽

Yen-Ling Chen ◽

...

Keyword(s):

Nitric Oxide ◽

Cell Proliferation ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Protein Kinase B ◽

Cardiac Fibroblasts ◽

Ang Ii ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Background Propofol may have beneficial effects on the prevention of angiotensin II (Ang II)-induced cardiac fibroblast proliferation via its antioxidative properties. The authors hypothesized that propofol may alter Ang II-induced cell proliferation and aimed to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Methods Cultured rat cardiac fibroblasts were pretreated with propofol then stimulated with Ang II; cell proliferation and endothelin-1 gene expression were examined. The effect of propofol on Ang II-induced nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity were also examined. The effect of propofol on nitric oxide production and protein kinase B and endothelial nitric oxide synthase phosphorylations were also tested to elucidate the intracellular mechanism of propofol in proliferation. Results Ang II (100 nm) increased cell proliferation and endothelin-1 expression, which were partially inhibited by propofol (10 or 30 microm). Propofol also inhibited Ang II-increased nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity. Propofol was also found to increase nitric oxide generation and protein kinase B and nitric oxide synthase phosphorylations. Nitric oxide synthase inhibitor (N-nitro-L-arginine methylester) and the short interfering RNA transfection for protein kinase B or endothelial nitric oxide synthase markedly attenuated the inhibitory effect of propofol on Ang II-induced cell proliferation. Conclusions The authors' results suggest that propofol prevents cardiac fibroblast proliferation by interfering with the generation of reactive oxygen species and involves the activation of the protein kinase B-endothelial nitric oxide synthase-nitric oxide pathway.

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