Mediation by M3-muscarinic receptors of both endothelium-dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat

Abstract

Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s} endothelium-dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR). Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium-dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of N(G)-nitro-L-arginine (to prevent the formation of nitric oxide). Endothelium-dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo-oxygenase activity). Responses to acetylcholine were assessed against the non-preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M1), methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3). The potency of acetylcholine in inducing endothelium-dependent contraction was 6.54 ± 0.07 (EC50). Atropine, pirenzepine, methoctramine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana-Schild plots to be (log M) 9.48 ± 0.07, 6.74 ± 0.22, 6.30 ± 0.20 and 9.3 ± 0.22 respectively. The potency of acetylcholine in inducing endothelium-dependent relaxation was 7.82 ± 0.09 (IC50). Atropine, pirenzepine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4-DAMP for the relaxation to acetylcholine were estimated from Arunlakshana-Schild plots to be (log M) 9.15 ± 0.23 and 9.63 ± 0.28, respectively. These results suggest that the muscarinic M3 receptor subtype mediates both endothelium-dependent relaxation and contraction to acetylcholine in SHR aorta.