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

1. Vasorelaxation to β2-adrenoceptor stimulation occurs through both endothelium-dependent and endothelium-independent mechanisms, and the former is mediated through Ca2+-independent activation of endothelial-type nitric oxide synthase (NOS-3). Since Ca2+-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 β2-adrenergic relaxation of rat aorta. 2. Rat aortic rings were pre-incubated with the PKA inhibitor H-89 (10-7 M), th ephosphatidylinositol 3-kinase (P13K) inhibitor wortmannin (5 x 10-7 M), Akt inhibitor (10-3M), or vehicle, in the absence or presence of the NOS inhibitor N6-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 β2-andrenoceptor agonist albuterol. 3. Rings exhibited a concentraion-dependent relaxation to albuterol pEC50 6.9 %±0.2, Emax 88.2 ± 4.0%, L-NAME attenuated Emax to 60.2 ± 3.5% (P<0.001). 4. In the presence of L-NAME, wortmannin or Akt inhibitor did not influence albuterol responses, whereas H-89 reduced Emax further, to 27.5 ± 2.2 % (P<0.0001). 5. In the absence of L-NAME, Emax 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 Emax further still. 6. 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. 7. Our results indicate that β2-adrenoceptor stimulaion relaxes rat aorta through both NO-dependent and independent mechanisms. The latter is predominantly PKA-mediated, whereas the former occurs through both PKA and P13K/Akt activation. [ABSTRACT FROM AUTHOR]