Mechanisms underlying beta2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells.

Endothelial beta(2)-adrenoceptor (beta(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 beta(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 betaAR agonists or cyclic AMP-elevating agents. beta(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(omega)-nitro-l-arginine methyl ester (l-NAME) inhibited l-arginine transport. beta(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. beta(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 beta(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.

Aspirin modifies nitric oxide synthase activity in platelets: effects of acute versus chronic aspirin treatment.

OBJECTIVE: We examined the effects of aspirin on basal and beta-adrenoceptor (beta-AR)-mediated nitric oxide synthase (NOS) activity in normal platelets. METHODS: NOS activity was determined from the conversion of L-[3H]arginine to L-[3H]citrulline, both basally and following beta-AR stimulation, in platelets from healthy human subjects following both short- and long-term aspirin administration. RESULTS: Basal L-[3H]citrulline increased following aspirin 800 mg administered intravenously in vivo, from 0.31+/-0.12 to 0.76+/-0.14 pmol/10(8) platelets (P<0.01). Isoproterenol at 1 micromol/l increased platelet NOS activity before but not following intravenous aspirin. After short-term in vitro treatment with aspirin 10 micromol/l, 400 micromol/l or 4 mmol/l, basal platelet L-[3H]citrulline increased similarly, an effect not seen with indomethacin 100 micromol/l or ibuprofen 10 micromol/l. Platelet NOS activity was not increased by albuterol 1 micromol/l, in the presence of indomethacin, ibuprofen or aspirin in vitro. By contrast, oral aspirin 75 mg daily for 14 days did not affect basal platelet NOS activity, but abolished beta-adrenergic NOS activation. CONCLUSIONS: Aspirin activates basal platelet NOS acutely, but not chronically, through a mechanism independent of cyclooxygenase (COX) inhibition. By contrast, both short- and long-term aspirin treatment inhibit platelet beta-adrenergic NOS activation by a COX-dependent mechanism. This indicates that aspirin exerts divergent effects on basal and beta-AR-stimulated platelet NOS activity, which are likely to be of clinical relevance.