The ability of asymmetric dimethylarginine (ADMA) to block endothelium-dependent, nitric oxide-mediated relaxation in rat aorta is determined by the efficacy of the relaxant agonist

Mohammed Al-Zobaidy, William Martin. University of Glasgow, Glasgow, UK

The inhibitor of nitric oxide synthase, asymmetric dimethylarginine (NG, NG-dimethyl-L-arginine; ADMA), has recently evolved as one of the endogenous factors that control nitric oxide bioactivity (Anderssohn et al., 2010). We have shown that when the level of tone is matched in control and treated rat aortic rings, that ADMA has little effect on acetylcholine (ACh)-induced relaxation, whereas NG-nitro-L-arginine methyl ester (L-NAME) produces powerful blockade (Al-Zobaidy et al., 2010). Previous work has suggested that relaxant agonist efficacy is critical in determining the ability of nitric oxide synthase inhibitors to block endothelium-dependent relaxation (Martin et al., 1992). Consequently, the aim of this study was to determine if the poor blocking action of ADMA was related to the relaxant efficacy of ACh. This was tested by using a range of agonists (ACh, calcium ionophore A23187 and CGRP-1), by use of the M3 receptor partial agonist, butyrylcholine (BCh), and by lowering the apparent efficacy of ACh by use of the irreversible alkylating agent, phenoxybenzamine (PBZ; Martin et al., 1992).

Aortic rings from female Wistar rats weighing 150-200 g (Al-Zobaidy et al., 2010) were mounted under 10 mN resting tension in tissue baths containing Krebs solution at 37°C and gassed with 95% O2 and 5% CO2. Submaximal tension was induced using phenylephrine (PE) before obtaining relaxation to ACh (1 nM-10 µM), A23187 (1 nM-1 µM), CGRP-1 (0.1 nM-1 µM) or BCh (0.1-300 µM). When the effects of ADMA were examined on relaxation to these agents, care was taken to induce comparable levels of tone as in control experiments. In experiments to lower the apparent efficacy of ACh, tissues were treated with PBZ (3 µM for 30 minutes) then extensively washed. In these experiments, submaximal tone was induced using endothelin-1 (ET-1) before eliciting relaxation to ACh. Relaxations are expressed as % of PE- or ET-1-induced tone (mean ± SEM of n≥6 observations); with statistical differences determined by ANOVA followed by the Bonferroni test.

ACh and A23187 each produced powerful relaxation (81.7 ± 3.8 % and 96.2 ± 1.0 %,respectively) and these were unaffected by ADMA at 0.1 mM (Emax 84.3 ± 1.9 % and 91.0 ± 1.9 % for ACh and A23187, respectively). CGRP-1 had a lower Emax (57.6 ± 3.2 %) than ACh or A23187, and this was blocked by ADMA at 0.1 mM (Emax 15.2 ± 4.2 %, P< 0.001). BCh also had a lower Emax (72.6 ± 5.4 %,) than ACh or A23187, which was blocked in a concentration-dependent manner by ADMA (Emax 49.5 ± 4.9 %, P< 0.01 and 10.5 ± 3.8 %, P< 0.001 for 0.1 and 1 mM, respectively). Treatment with PBZ reduced the Emax for ACh from 87.9 ± 2.6 % to 63.3 ± 3.5 %, P<0.001, and this was further blocked in a concentration-dependent manner by ADMA (Emax 41.3 ± 3.9 % and 15.6 ± 2.3 % for 0.1 and 1 mM, respectively, both P< 0.001). Thus, the ability of ADMA to block endothelium-dependent, nitric oxide-mediated relaxation appears to be inversely proportional to the efficacy of the relaxant agonist. These findings may therefore explain why relaxation to the high efficacy agonists, ACh and A23187, are relatively resistant to blockade by ADMA.

Al-Zobaidy, M.J., et al., 2010. Br. J. Pharmacol., 160: 1476-1483.

Anderssohn, M., et al., 2010. Diabetes & Vascular Disease Research., 7 105–118.

Martin, G.R., et al., 1992. Br. J. Pharmacol., 105: 643-652.