Beta-2-adrenoceptor (ß₂AR)-mediated vasorelaxation is mediated through both nitric oxide (NO)-dependent and independent mechanisms in many blood vessels. In this work, we investigated the relative contributions of NO to ß₂AR relaxation in rat aorta in vitro, and the roles of protein kinase A (PKA) and protein kinase B (PKB, Akt) in these responses.

Male Wistar rats (175-200g) were killed by cervical dislocation, thoracic aorta was dissected out quickly and 2mm-long rings were placed in 25mL organ baths containing Krebs buffer bubbled constantly with 95% O₂ / 5% CO₂ at 37°C. Preliminary length-tension curves suggested that a resting tension of 2g gave optimal contractile responses to KCl 45 mM, therefore this level of resting tension was used in all experiments. Once stable and reproducible contractions were obtained with KCl 45 mM, rings were washed extensively and subsequently contracted with phenylephrine 10^-7M. Following attainment of plateau constriction, acetylcholine 1 µM was added; only rings which demonstrated 60% relaxation to acetylcholine were considered to have functional endothelium, and were used for further experiments. Following further extensive washing, rings were incubated with the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) 10^-4M or corresponding vehicle. Additionally, incubations (both in the absence and presence of L-NAME) were performed with the PKA inhibitor H-89 10^-7M, the phosphatidylinositol 3-kinase inhibitor wortmannin 500nM, Akt inhibitor 10^-5M, or corresponding vehicle. All these incubations were for 15 min, following which rings were once again contracted with phenylephrine 10^-7M and, after re-attainment of plateau, concentration-relaxation responses were determined to the ß₂AR agonist albuterol (10⁹-10^-4M). Data are expressed as mean ± s.e.m. of 6 separate experiments for each condition, and statistical analysis was by repeated measures two-way ANOVA, with P<0.05 (two-tailed) taken as significant.

Aortic rings exhibited a concentration-dependent relaxation to albuterol: pEC₅₀ (M) 6.8±0.1, E_max 89.5±1.6%. L-NAME did not significantly affect pEC₅₀, but attenuated E_max to 56.2±2.2% (P<0.001). In the presence of L-NAME, co-incubation with wortmannin or Akt inhibitor did not further influence albuterol responses, whereas co-incubation with 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 55.9±1.0% respectively (P<0.001 for each); in the combined presence of H-89 and wortmannin, E_max was decreased to 33.5±3.5% (P<0.001 as compared with either antagonist alone), and in the combined presence of H-89 and Akt inhibitor, E_max was reduced to 28.3±1.1% (P<0.001 as compared with either antagonist alone). pEC₅₀ was not significantly affected by H-89, wortmannin or Akt inhibitor.

In conclusion, ß₂AR stimulation relaxes rat aorta through both NO-dependent and independent mechanisms. Inhibition of the PKA or PKB systems alone partially inhibits, whilst inhibition of both almost entirely abolishes, ß₂AR- mediated relaxation. The NO-independent component of this relaxation response appears to be purely PKA-mediated.