Mechanisms underlying β 2 ‐adrenoceptor‐mediated nitric oxide generation by human umbilical vein endothelial cells

Endothelial β 2 ‐adrenoceptor (β 2 AR) 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 β 2 AR‐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 [ 3 H]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. β 2 AR 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 ω ‐nitro‐ l ‐arginine methyl ester ( l ‐NAME) inhibited l ‐arginine transport. β 2 AR 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. β 2 AR 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 β 2 AR‐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.