Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

Motley, Evangeline D.; Eguchi, Kunie; Patterson, Myla M.; Palmer, Phillip D.; Suzuki, Hiroyuki; Eguchi, Satoru

doi:10.1161/01.hyp.0000255954.80025.34

Cited by 76 publications

(84 citation statements)

References 38 publications

(35 reference statements)

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“…However, calcium, PKA, and PKC have also been reported to phosphorylate eNOS on S1177 in response to various stimuli (12,20,39). Similarly, we have found that a PKC inhibitor, rottlerin, leads to the inhibi- The role of alternate pathways in allowing the phosphorylation of eNOS may therefore explain how VEGF and hyperactivated VEGFR2 can induce increased phosphorylation of eNOS in DEP-1-depleted cells, in which Akt activation is suboptimal.…”

Section: Discussionmentioning

confidence: 53%

New Role for the Protein Tyrosine Phosphatase DEP-1 in Akt Activation and Endothelial Cell Survival

Chabot

Spring

Gratton

et al. 2009

Molecular and Cellular Biology

126

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Functional inactivation of the protein tyrosine phosphatase DEP-1 leads to increased endothelial cell proliferation and failure of vessels to remodel and branch. DEP-1 has also been proposed to contribute to the contact inhibition of endothelial cell growth via dephosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), a mediator of vascular development. However, how DEP-1 regulates VEGF-dependent signaling and biological responses remains ill-defined. We show here that DEP-1 targets tyrosine residues in the VEGFR2 kinase activation loop. Consequently, depletion of DEP-1 results in the increased phosphorylation of all major VEGFR2 autophosphorylation sites, but surprisingly, not in the overall stimulation of VEGFdependent signaling. The increased phosphorylation of Src on Y529 under these conditions results in impaired Src and Akt activation. This inhibition is similarly observed upon expression of catalytically inactive DEP-1, and coexpression of an active Src-Y529F mutant rescues Akt activation. Reduced Src activity correlates with decreased phosphorylation of Gab1, an adapter protein involved in VEGF-dependent Akt activation. Hypophosphorylated Gab1 is unable to fully associate with phosphatidylinositol 3-kinase, VEGFR2, and VEcadherin complexes, leading to suboptimal Akt activation and increased cell death. Overall, our results reveal that despite its negative role on global VEGFR2 phosphorylation, DEP-1 is a positive regulator of VEGFmediated Src and Akt activation and endothelial cell survival.

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Section: Discussionmentioning

confidence: 53%

New Role for the Protein Tyrosine Phosphatase DEP-1 in Akt Activation and Endothelial Cell Survival

Chabot

Spring

Gratton

et al. 2009

Molecular and Cellular Biology

126

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“…47 However, the eNOS protein expression does not directly correlate with NO production since the protein must be activated to produce NO. 48 The amount of NO produced by CB-EPCs and HAECs increased significantly after exposure to fluid shear stress for both substrates, and there was no significant difference in the amount of NO produced by the two cell types. While cultured SMCs alone do produce NO in response to fluid shear stress by neuronal NOS, 49 SMCs in coculture are not exposed to shear stress directly and the similarity in the levels of NO produced with and without SMC coculture suggests that SMC NO production was not significant.…”

Section: Cord Blood Endothelial Progenitor Cellsmentioning

confidence: 86%

Characterization of Umbilical Cord Blood–Derived Late Outgrowth Endothelial Progenitor Cells Exposed to Laminar Shear Stress

Brown

Wallace

Angelos

et al. 2009

Tissue Engineering Part A

114

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Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising source of endothelial cells for synthetic vascular grafts and tissue-engineered blood vessels since they are readily attainable, can be easily isolated, and possess a high proliferation potential. The objective of this study was to compare the functional behavior of late outgrowth CB-EPCs with human aortic endothelial cells (HAECs). CB-EPCs and HAECs were cultured on either smooth muscle cells in a coculture model of a tissue-engineered blood vessels or fibronectin adsorbed to Teflon-AFÔ-coated glass slides. Late outgrowth CB-EPCs expressed endothelial cellspecific markers and were negative for the monocytic marker CD14. CB-EPCs have higher proliferation rates than HAECs, but are slightly smaller in size. CB-EPCs remained adherent under supraphysiological shear stresses, oriented and elongated in the direction of flow, and expressed similar numbers of a 5 b 1 and a v b 3 integrins and antithrombotic genes compared to HAECs. There were some differences in mRNA levels of Eselectin and vascular cell adhesion molecule 1 between CB-EPCs and HAECs; however, protein levels were similar on the two cell types, and CB-EPCs did not support adhesion of monocytes in the absence of tumor necrosis factor-a stimulation. Although CB-EPCs expressed significantly less endothelial nitric oxide synthase protein after exposure to flow than HAECs, nitric oxide levels induced by flow were not significantly different. These results suggest that late outgrowth CB-EPCs are functionally similar to HAECs under flow conditions and are a promising cell source for cardiovascular therapies.

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“…5 Previously, others have shown that PKCd phosphorylates serine (1177/1179) resulting in eNOS activation in bovine aortic endothelial cells. 38 Attenuation of PKCd (via small interfering RNA) decreased NO production in fibroblasts. 39 In both of these studies, the authors used Rottlerin, which at the time was thought to be the only specific inhibitor of PKCd but others have shown that Rottlerin is not specific for PKCd.…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase C Delta Modulates Endothelial Nitric Oxide Synthase after Cardiac Arrest

Lin

Gresia

Stradecki

et al. 2014

J Cereb Blood Flow Metab

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We previously showed that inhibition of protein kinase C delta (PKCd) improves brain perfusion 24 hours after asphyxial cardiac arrest (ACA) and confers neuroprotection in the cortex and CA1 region of the hippocampus 7 days after arrest. Therefore, in this study, we investigate the mechanism of action of PKCd-mediated hypoperfusion after ACA in the rat by using the two-photon laser scanning microscopy (TPLSM) to observe cortical cerebral blood flow (CBF) and laser Doppler flowmetry (LDF) detecting regional CBF in the presence/absence of dV1-1 (specific PKCd inhibitor), nitric oxide synthase (NOS) substrate (L-arginine, L-arg) and inhibitor (N o -Nitro-L-arginine, NLA), and nitric oxide (NO) donor (sodium nitroprusside, SNP). There was an increase in regional LDF and local (TPLSM) CBF in the presence of dV1-1 þ L-arg, but only an increase in regional CBF under dV1-1 þ SNP treatments. Systemic blood nitrite levels were measured 15 minutes and 24 hours after ACA. Nitrite levels were enhanced by pretreatment with dV1-1 30 minutes before ACA possibly attributable to enhanced endothelial NOS protein levels. Our results suggest that PKCd can modulate NO machinery in cerebral vasculature. Protein kinase C delta can depress endothelial NOS blunting CBF resulting in hypoperfusion, but can be reversed with dV1-1 improving brain perfusion, thus providing subsequent neuroprotection after ACA. Keywords: asphyxial cardiac arrest; middle cerebral artery occlusion; neuroprotection; palmitic acid methyl ester; stearic acid methyl ester INTRODUCTIONWe previously showed that inhibition of protein kinase C delta (PKCd) via dV1-1 can increase perfusion 24 hours after asphyxial cardiac arrest (ACA). Global cerebral ischemia (via ACA) causes derangement of cerebral blood flow (CBF) responsible for neuronal cell death in the CA1 region of the hippocampus as well as the cortex.1 Owing to the overall decrease in CBF after ischemia, neuronal cell death 1 can occur in major regions of the brain responsible for learning, memory, and cognitive function.2 It is thought that during global ischemia, PKCd (a novel PKC) levels are elevated causing PKCd to translocate to the nucleus (activation) resulting in cellular damage. In the normal brain, PKCd levels are nominal whereas global ischemia can cause activation/translocation of PKCd.3 Inhibition of PKCd (via specific inhibitor of PKCd, dV1-1) can cause a revival of CBF 24 hours after ischemia to counteract hypoperfusion or low CBF found to be suppressed after cardiac arrest from 38% to 65%. 1,4 We previously showed that pretreatment of dV1-1 before ACA can enhance perfusion 24 hours after ischemia, resulting in improved neuronal survival in the hippocampal CA1 and cortex regions in our rat model of ACA.1 Here, we sought out to define the specific mechanism(s) of how inhibition of PKCd can alleviate these pathologies. The possible endothelium and endothelial-mediated nitric oxide synthase (eNOS) involvement as a target for PKCd relating to general circulation was first reported by Monti et al.

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Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

Cited by 76 publications

References 38 publications

New Role for the Protein Tyrosine Phosphatase DEP-1 in Akt Activation and Endothelial Cell Survival

New Role for the Protein Tyrosine Phosphatase DEP-1 in Akt Activation and Endothelial Cell Survival

Characterization of Umbilical Cord Blood–Derived Late Outgrowth Endothelial Progenitor Cells Exposed to Laminar Shear Stress

Protein Kinase C Delta Modulates Endothelial Nitric Oxide Synthase after Cardiac Arrest

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