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 (E_max = 21.50 ± 5.73%) followed by passive relaxations (E_max reduced to 9.08 ± 2.55%). Contractions were inhibited (E_max = 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 (E_max = 18.00 ± 3.45%) by PD123,319 but not by losartan. Ang II (0.3 μM to 100 μM) produced initial contractions (E_max = 11.49 ± 9.39%) followed by active relaxations [I_max (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 (I_max = 64.03 ± 5.47%) without the initial contractions. N^G-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.