Angiotensin II-Induced Relaxation of Anococcygeus Smooth Muscle via Desensitization of AT1 Receptor, and Activation of AT2 Receptor Associated with Nitric-Oxide Synthase Pathway

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Abstract
We evaluated the role of receptor desensitization, activation of AT2 receptors, and enzymatic degradation of angiotensin II (Ang II) by amino/neutral endopeptidases in rat anococcygeus smooth muscle (ASM) relaxation. Ang II (0.3 nM to 10 μM) produced contractions (Emax = 21.50 ± 5.73%) followed by passive relaxations (Emax reduced to 9.08 ± 2.55%). Contractions were inhibited (Emax = 13.67 ± 2.03%) by losartan (0.1 μM; AT1 antagonist) but not by PD123,319 [S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid] (0.1 μM; AT2 antagonist). Conversely, the passive relaxation was inhibited (Emax = 18.00 ± 3.45%) by PD123,319 but not by losartan. Ang II (0.3 μM to 100 μM) produced initial contractions (Emax = 11.49 ± 9.39%) followed by active relaxations [Imax (maximum inhibition elicited by the agonist) = 47.85 ± 4.23%] on strips precontracted by bethanechol (100 μM). A second administration of Ang II on the background of bethanechol (1 h later) resulted in stronger relaxations (Imax = 64.03 ± 5.47%) without the initial contractions. NG-Nitro-l-arginine methyl ester [nitric-oxide synthase (NOS) inhibitor], ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; guanylate cyclase inhibitor), PD123,319, and tetrodotoxin (neurotoxin) inhibited the relaxations. The presence of AT1 and AT2 receptors was confirmed by Western blot. Experiments with amastatin (1 μM) and thiorphan (1 μM), aminopeptidase, and neutral endopeptidase inhibitors, respectively, excluded the involvement of enzymatic degradation in Ang II-induced relaxation of ASM. In conclusion, the rat ASM relaxation by Ang II is the result of active and passive relaxations. The passive relaxation depends on desensitization of excitatory AT1 receptors, and the active relaxation is mediated by stimulation of AT2 receptors and activation of the neuronal NOS/soluble guanylate cyclase pathway.