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

Endothelial β2‐adrenoceptor (β2AR) 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 β2AR‐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 βAR agonists or cyclic AMP‐elevating agents. β2AR 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 Nω‐nitro‐l‐arginine methyl ester (l‐NAME) inhibited l‐arginine transport. β2AR 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. 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 in l‐arginine uptake resulting from NO‐mediated membrane hyperpolarization.