Angiotensin II receptor subtypes and contractile responses in portal vein smooth muscle

The selective biphenylimidazole and tetrahydroimidazopyridine antagonists exemplified by losartan (DuP 753) and PD 123319 have been shown to bind selectively to angiotensin AT1 and AT2 receptor subtypes, respectively. To characterize which subtypes of angiotensin II receptors are expressed in mammalian portal vein smooth muscle, we performed, using both membrane and strip preparations, [3H]angiotensin II binding experiments and then contraction experiments to investigate the functional relevance of these binding sites. Specific binding of [3H]angiotensin II was of high affinity, saturable and reversible. Specific binding of [3H]angiotensin II was completely displaced by angiotensin II and the peptide antagonist [Sar1,Ile8]angiotensin II. The inhibition of [3H]angiotensin II binding by losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)- methyl]imidazole, potassium salt) and DuP 532 (2-n-propyl-4-pentafluoroethyl-1-[(2'-(1H-tetrazol-5-yl)biph enyl-4-yl)- methyl]imidazole-5-carboxylic acid) was biphasic and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation represented 75% of the total binding and showed high affinity for angiotensin II, losartan and DuP 532, but low affinity for the peptide angiotensin AT2 receptor antagonist CGP 42112A (N-alpha-nicotinoyl-Tyr-Lys-[N-alpha-CBZ-Arg]-His-Pro-Ile-OH) and thus appeared identical to the cloned angiotensin AT1 receptor subtype. The remaining 25% of the sites showed nearly 1000-fold lower affinity for losartan, 6500-fold lower affinity for DuP 532 and high affinity for PD 123319 (S-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-diphenylacetyl- 4,5,6,7-tetrahydro-1H-imidazo-[4,5-c] pyridine-6-carboxylic acid, difluoroacetate monohydrate) and CGP 42112A, with values of Ki in the same range (nM) as those found for losartan and DuP 532 at angiotensin AT1 binding sites. These sites appear to be angiotensin AT2 receptors. Only the angiotensin AT1 receptor subtype interacted with G-proteins, as indicated by the 80% inhibition of [3H]angiotensin II binding in the presence of guanosine 5'-O-(3-thiophosphate) or fluoroaluminates. Although the angiotensin II-induced contraction was completely inhibited by losartan with a pA2 value of 8.8, PD 123319 reduced the angiotensin II-induced contraction by 20-25%, indicating that both angiotensin AT1 and AT2 receptor subtypes are functional in portal vein smooth muscle.