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.

Figure 1

(a) Concentration–relaxation curves to isoproterenol in rat aortic rings, in the absence or presence of the β₁AR-selective antagonist CGP 20712A (3 × 10⁻⁷ M) or the β₂AR-selective antagonist ICI 118551 (10⁻⁷ M). (b) Concentration–relaxation curves to albuterol in rat aortic rings, in the absence or presence of the NOS inhibitor L-NAME (10⁻⁴ M). All data are shown as mean±s.e.m. of experiments on aortic rings derived from six different rats. ^*P<0.05, ^**P<0.01 and ^***P<0.001 as compared with control.

Figure 2

Concentration–relaxation curves to albuterol in rat aortic rings, in the presence (a) or absence (b) of the NOS inhibitor L-NAME (10⁻⁴ M). Responses are shown in the concomitant absence or presence of the PKA inhibitor H-89 (10⁻⁷ M), the PI3K inhibitor wortmannin (5 × 10⁻⁷ M), or both. All data are shown as mean±s.e.m. of experiments on aortic rings derived from six different rats. ^**P<0.01 and ^***P<0.001 as compared with control. ^#P<0.05 and ^###P<0.001 as compared with H-89 alone.

Figure 3

Concentration–relaxation curves to albuterol in rat aortic rings, in the presence (a) or absence (b) of the NOS inhibitor L-NAME (10⁻⁴ M). Responses are shown in the concomitant absence or presence of Akt inhibitor (10⁻⁵ M) or the combination of Akt inhibitor (10⁻⁵ M) and the PKA inhibitor H-89 (10⁻⁷ M). All data are shown as mean±s.e.m. of experiments on aortic rings derived from six different rats. ^***P<0.001 as compared with control. ^#P<0.05 and ^###P<0.001 as compared with Akt inhibitor alone.

Figure 4

Western blots depicting the presence of a 135 kDa band (the known molecular mass of NOS-3) in NOS-3 immunoprecipitates prepared from rat aortic homogenates, the blots being probed with (a) anti-NOS-3 antibody or (b) anti-phosphoserine IgG. Lanes: 1=basal (vehicle treatment); 2=albuterol (10⁻⁶ M); 3=albuterol (10⁻⁶ M)+H-89 (10⁻⁷ M); 4=albuterol (10⁻⁶ M)+Akt inhibitor (10⁻⁵ M); 5=albuterol (10⁻⁶ M)+H-89 (10⁻⁷ M)+Akt inhibitor (10⁻⁵ M). (c) Densitometric ratio of 135 kDa phosphoserine/NOS-3 bands, in rat aortas treated with each of these combinations, expressed as mean±s.e.m. of experiments on aortic rings derived from six different rats. ^*P<0.05 and ^**P<0.01 as compared with basal; ^#P<0.05 and ^##P<0.01 as compared with albuterol alone.