Nitric oxide-dependent beta2-adrenergic dilatation of rat aorta is mediated through activation of both protein kinase A and Akt

Abstract

Vasorelaxation to beta(2)-adrenoceptor stimulation occurs through both endothelium-dependent and endothelium-independent mechanisms, and the former is mediated through Ca(2+)-independent activation of endothelial-type nitric oxide synthase (NOS-3). Since Ca(2+)-independent NOS-3 activation may occur through its serine phosphorylation via protein kinase A (PKA) or Akt, we determined the PKA and Akt dependency of beta(2)-adrenergic relaxation of rat aorta. Rat aortic rings were pre-incubated with the PKA inhibitor H-89 (10(-7) m), the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (5 x 10(-7) m), Akt inhibitor (10(-5) m), or vehicle, in the absence or presence of the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 10(-4) m). Rings were then contracted with phenylephrine (10(-7) m), and concentration-relaxation responses determined to the beta(2)-adrenoceptor agonist albuterol. Rings exhibited a concentration-dependent relaxation to albuterol: pEC(50) 6.9+/-0.2, E(max) 88.2+/-4.0%. l-NAME attenuated E(max) to 60.2+/-3.5% (P<0.001). In the presence of l-NAME, wortmannin or Akt inhibitor did not influence albuterol responses, whereas H-89 reduced E(max) further, to 27.5+/-2.2% (P<0.001). In the absence of l-NAME, E(max) to albuterol was reduced by H-89, wortmannin or Akt inhibitor, to 56.2+/-2.2, 56.0+/-1.6 and 55.4+/-1.8%, respectively (P<0.001 for each); the combinations H-89 plus wortmannin or H-89 plus Akt inhibitor reduced E(max) further still. Western blotting of NOS-3 immunoprecipitates from rat aortas confirmed that albuterol increased serine phosphorylation of NOS-3, and this increase was attenuated by H-89 or Akt inhibitor. Our results indicate that beta(2)-adrenoceptor stimulation relaxes rat aorta through both NO-dependent and independent mechanisms. The latter is predominantly PKA-mediated, whereas the former occurs through both PKA and PI3K/Akt activation.