beta(2)-adrenoceptors activate nitric oxide synthase in human platelets.
Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet beta-adrenoceptors (betaARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, after betaAR stimulation or cAMP elevation. Basal NOS activity was 0.11+/-0.03 pmol L-citrulline/10(8) platelets. The betaAR agonist isoproterenol 1 micromol/L and the adenylyl cyclase activator forskolin 1 micromol/L each increased NOS activity, to 0.26+/-0.04 and 0.23+/-0.03 pmol L-citrulline/10(8) platelets, respectively (P<0.01 for each). Both responses were abolished by the adenylyl cyclase inhibitor SQ22536 50 micromol/L. NOS activation by isoproterenol or forskolin was not associated with a change in intracellular Ca(2+). In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3+/-0.2% versus 2.6+/-0.2% respectively; P<0.001). Effects of isoproterenol on NOS activity, platelet aggregation, and adhesion were mediated exclusively through beta(2)ARs, as determined by coincubation with betaAR subtype-selective antagonists. We conclude that beta(2)ARs activate platelet NOS by increasing cAMP, and that this activation is Ca(2+)-independent. beta(2)ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the L-arginine/NO system mediates the effects of beta(2)ARs on adhesion but not aggregation.
Queen
L R
LR
Department of Clinical Pharmacology, Center for Cardiovascular Biology & Medicine, King's College London, St Thomas' Hospital, London, UK.
Xu
B
B
Horinouchi
K
K
Fisher
I
I
Ferro
A
A
D001792:Blood Platelets / Q000201:enzymology* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002956:Citrulline / Q000096:biosynthesis; D000242:Cyclic AMP / Q000096:biosynthesis; D004789:Enzyme Activation; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000097:blood*; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology*
D018343:Receptors, Adrenergic, beta-2; D002956:Citrulline; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D007545:Isoproterenol; D002118:Calcium