Faculty Opinions recommendation of Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes.

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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LOW-INTENSITY ULTRASOUND INCREASES THE ENDOTHELIAL NITRIC OXIDE SYNTHASE (eNOS) EXPRESSION OF ENDOTHELIAL CELLS POSSIBLY VIA THE PHOSPHATIDYLINOSITOL 3-KINASE/AKT/PROTEIN KINASE A SIGNALING PATHWAY

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s1016237210002122 ◽

2010 ◽

Vol 22 (05) ◽

pp. 367-376

Author(s):

Shan-hui Hsu ◽

Tsung-Bin Huang ◽

Kuan-Pu Wang

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Protein Kinase ◽

Protein Kinase A ◽

Ultrasound Exposure ◽

Low Intensity ◽

Low Intensity Ultrasound ◽

Oxide Synthase ◽

Endothelial Nitric Oxide ◽

Kinase A

The stimulative effects and the associated mechanism of low-intensity ultrasound on endothelial cells were investigated in this study. Human umbilical cord endothelial cells (HUVECs) were exposed to 1 MHz pulsed ultrasound at 34 mW/cm2 for 10 min daily. The cell proliferation, nitric oxide release, intracellular calcium concentration, and endothelial nitric oxide synthase (eNOS) gene and protein expressions after ultrasound exposure were analyzed. It was found that ultrasound exposure did not change the cell proliferation, but it enhanced the release of nitric oxide and Ca2+ ions, and increased the eNOS activity. The induced phosphorylation of eNOS (Ser1177) by the ultrasound exposure was inhibited by the addition of N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H89, a protein kinase A inhibitor), but not 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, a phosphoatidylinositol 3-kinase inhibitor). The induced phosphorylation of Akt (protein kinase B, Ser473) by the ultrasound exposure was inhibited by the addition of LY294002, but not by the presence of H89. The results also applied to bovine carotid artery endothelial cells (BAECs) exposed to ultrasound at a larger intensity (68 mW/cm2). The above experiments suggested that low-intensity ultrasound may promote eNOS activity of endothelial cells via the phosphatidylinositol 3-kinase/Akt/protein kinase A signaling pathway. This may have implications on the therapeutic effect of ultrasound in tissue repair.

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