Loss of basal endothelial nitric oxide synthase activity but not agonist-induced relaxation after isolation of the mouse aorta

Johanna van Langen, Cor E. Van Hove, Paul Fransen, Hidde Bult. Div. Pharmacology, University of Antwerp, Antwerp-Wilrijk, 2610, Belgium

Aim. Endothelial nitric oxide (NO) synthase (eNOS) is a key regulator of vascular tone and arterial stiffness. This study addresses changes in eNOS activity after isolation of the mouse aorta by investigating basal and agonist-induced vasodilator effects of NO.

Methods. Segments (2mm) of the thoracic aorta of C57/Bl6 mice were mounted under 16 mN resting tension in organ baths containing Krebs solution at 37°C and gassed with 95% O2 and 5% CO2 to measure isometric forces. At 1h intervals basal NO was assessed by its ability to suppress phenylephrine (PE, 10-6 M) or prostaglandin F2α (PGF2α 10-5 M)-induced tension. After stabilisation of the fifth contraction, rings were treated with 300µM NΩ -nitro-L-arginine methyl ester (L-NAME) and 300µM NΩ -nitro-L-arginine to assess maximum force. This was followed by a concentration response curve (CRC) with diethylamine NO (DEANO) to determine sensitivity to exogenous NO. Parallel rings were also constricted at 1h intervals with PE or PGF2α, but when tone had stabilised this was followed by CRCs with acetylcholine (ACh) or the alpha-2 adrenoceptor agonist UK 14,304 to investigate agonist-induced NO release. Immunoblotting was used to investigate Akt phosphorylation. Results are shown as mean ± s.e., n refers to the number of mice.

Results. Exactly 1h after dissection of the aorta, PE or PGF2α induced very small contractions, resp. 17±4% (n=5) or 31±7% (n=5) of maximum tension in the presence L-NAME. Forces evoked by both agonists increased gradually every hour. In the presence of 100µM L-NAME, PE-induced contractions were much larger and remained stable between 2 and 5 h. In contrast, PE contractions were unaffected by selective inhibitors of neuronal or inducible NOS. PE-induced forces were interpolated on DEANO CRCs to estimate basal NO equivalents. This bioassay pointed to an exponential decline of basal NO with a half-life of 1.3 h. ACh and UK 14,304 induced resp. 86 ± 5% (n=4) and 47±4 % (n=4) relaxation of pre-constricted segments at 1h. Both relaxation curves remained largely unaltered between 1h and 5h. Addition of the phosphoinositide kinase-3 (PI3K) inhibitor wortmannin (30nM) at 1 h raised PE-induced force from 32±2 % to 70±2 % of maximum force in the presence L-NAME (P<0.01, paired t test, n=4). In contrast, wortmannin did not influence amplitude or the concentration giving half maximal relaxation (EC50) of ACh- or DEANO, or PE-induced force in the presence of L-NAME. Western blotting documented unaltered Akt expression, but Akt phosphorylation declined by 47% between 1 and 4 h (normalised ratio phosphoAkt/Akt 1.01±0.03 at 1h, 0.53±0.11 at 4h, n=4, P=0.005, one-way ANOVA and Dunnett’s test).

Conclusions. These results indicate that basal NO production in the mouse aorta is initially very high. However, it rapidly declines in the organ bath via an exponential time-course. The selective NOS inhibitors showed that basal NO is exclusively produced by eNOS. In contrast to basal NO release, vasodilatation induced by ACh or UK 14,304 showed no apparent changes in time, pointing to independent regulatory mechanisms. The selective inhibition by wortmannin implies a role for the PI3K/Akt pathway in the regulation of basal eNOS activity in the mouse aorta.