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CARDIOVASCULAR |
1 Cardiovascular Division, King's College London, London, UK
2 Cardiovascular Department, Affiliated Gulou Hospital, Medical School of Nanjing University, Nanjing, China
3 Department of Cardiology, Zhongda Hospital of Southeast University, Nanjing, China
Endothelial ß2-adrenoceptor (ß2AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of L-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in ß2AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed L-arginine uptake, NOS activity (from L-arginine to L-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 ßAR agonists or cyclic AMP-elevating agents. ß2AR stimulation increased L-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade of L-arginine uptake by L-lysine inhibited NOS activity and, conversely, blockade of NOS using N
-nitro-L-arginine methyl ester (L-NAME) inhibited L-arginine transport. ß2AR stimulation also caused a membrane hyperpolarization inhibitable by L-NAME, suggesting that the increase in L-arginine uptake occurred in response to NO-mediated hyperpolarization. ß2AR 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 ß2AR-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 in L-arginine uptake resulting from NO-mediated membrane hyperpolarization.
(Received 26 June 2006;
accepted after revision 26 July 2006;
first published online 27 July 2006)
Corresponding author A. Ferro: 2.38A, New Hunts Hose, King's College London, Guy's Hospital Campus, London Bridge, London SE1 1UL, UK. Email: albert.ferro{at}kcl.ac.uk
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