Mechanisms underlying β2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells.

Autor: Queen, Lindsay R., Ji, Yong, Xu, Biao, Young, Lora, Yao, Kang, Wyatt, Amanda W., Rowlands, David J., Siow, Richard C. M., Mann, Giovanni E., Ferro, Albert
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Zdroj: Journal of Physiology; Oct2006, Vol. 576 Issue 2, p585-594, 10p, 1 Diagram, 7 Graphs
Abstrakt: Endothelial β2-adrenoceptor (β2AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role ofl-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in β2AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessedl-arginine uptake, NOS activity (froml-arginine tol-citrulline conversion), membrane potential (using [3H]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with βAR agonists or cyclic AMP-elevating agents. β2AR stimulation increasedl-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade ofl-arginine uptake byl-lysine inhibited NOS activity and, conversely, blockade of NOS using Nω-nitro-l-arginine methyl ester (l-NAME) inhibitedl-arginine transport. β2AR stimulation also caused a membrane hyperpolarization inhibitable byl-NAME, suggesting that the increase inl-arginine uptake occurred in response to NO-mediated hyperpolarization. β2AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that β2AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase inl-arginine uptake resulting from NO-mediated membrane hyperpolarization. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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