Acute exercise induce endothelial nitric oxide synthase phosphorylation via Akt and AMP-activated protein kinase in aorta of rats: Role of reactive oxygen species

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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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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Isoform-selective 5′-AMP-activated protein kinase-dependent preconditioning mechanisms to prevent postischemic leukocyte-endothelial cell adhesive interactions

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00944.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. H1352-H1360 ◽

Cited By ~ 19

Author(s):

F. Spencer Gaskin ◽

Kazuhiro Kamada ◽

Mozow (Yusof) Zuidema ◽

Allan W. Jones ◽

Leona J. Rubin ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cell ◽

Protein Kinase ◽

Endothelial Nitric Oxide Synthase ◽

Ischemia Reperfusion ◽

Cell Adhesive ◽

Adhesive Interactions ◽

Amp Activated Protein Kinase ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

We previously demonstrated that preconditioning induced by ethanol consumption at low levels [ethanol preconditioning (EPC)] or with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR-PC) 24 h before ischemia-reperfusion prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism that is initiated by nitric oxide formed by endothelial nitric oxide synthase. Recent work indicates that 1) ethanol increases the activity of AMP-activated protein kinase (AMPK) and 2) AMPK phosphorylates endothelial nitric oxide synthase at the same activation site seen following EPC (Ser1177). In light of these observations, we postulated that the heterotrimeric serine/threonine kinase, AMPK, may play a role in triggering the development of the anti-inflammatory phenotype induced by EPC. Ethanol was administered to C57BL/6J mice by gavage in the presence or absence of AMPK inhibition. Twenty-four hours later, the numbers of rolling and adherent leukocytes in postcapillary venules of the small intestine were recorded using an intravital microscopic approach. Following 45 min of ischemia, LEI were recorded after 30 and 60 min of reperfusion or at equivalent time points in control animals. Ischemia-reperfusion induced a marked increase in LEI relative to sham-operated control mice. The increase in LEI was prevented by EPC, an effect that was lost with AMPK inhibition during the period of ethanol exposure. Studies conducted in AMPK α1- and α2-knockout mice suggest that the anti-inflammatory effects of AICAR are not dependent on which isoform of the catalytic α-subunit is present because a deficiency of either isoform results in a loss of protection. In sharp contrast, EPC appears to be triggered by an AMPK α2-isoform-dependent mechanism.

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