Pelet C, Mironneau C, Rakotoarisoa L, Neuilly G
Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, URA CNRS 1489, Université de Bordeaux II, France.
Eur J Pharmacol. 1995 Jun 6;279(1):15-24. doi: 10.1016/0014-2999(95)00125-5.
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.
以氯沙坦（DuP 753）和PD 123319为代表的选择性联苯咪唑和四氢咪唑并吡啶拮抗剂已分别被证明可选择性地与血管紧张素AT1和AT2受体亚型结合。为了确定血管紧张素II受体的哪些亚型在哺乳动物门静脉平滑肌中表达，我们使用膜和条带制剂进行了[3H]血管紧张素II结合实验，然后进行收缩实验以研究这些结合位点的功能相关性。[3H]血管紧张素II的特异性结合具有高亲和力、可饱和性和可逆性。[3H]血管紧张素II的特异性结合被血管紧张素II和肽拮抗剂[Sar1，Ile8]血管紧张素II完全取代。氯沙坦（2-正丁基-4-氯-5-羟甲基-1-[(2'-(1H-四氮唑-5-基)联苯-4-基)-甲基]咪唑，钾盐）和DuP 532（2-正丙基-4-五氟乙基-1-[(2'-(1H-四氮唑-5-基)联苯-4-基)-甲基]咪唑-5-羧酸）对[3H]血管紧张素II结合的抑制是双相的，LIGAND曲线拟合分析显示有两个特异性结合位点群体。一个亚群占总结合的75%，对血管紧张素II、氯沙坦和DuP 532具有高亲和力，但对肽血管紧张素AT2受体拮抗剂CGP 42112A（N-α-烟酰基-Tyr-Lys-[N-α-CBZ-Arg]-His-Pro-Ile-OH）具有低亲和力，因此似乎与克隆的血管紧张素AT1受体亚型相同。其余25%的位点对氯沙坦的亲和力低近1000倍，对DuP 532的亲和力低6500倍，对PD 123319（S-1-[[4-(二甲基氨基)-3-甲基苯基]甲基]-5-二苯乙酰基-4,5,6,7-四氢-1H-咪唑并[4,5-c]吡啶-6-羧酸，二氟乙酸盐水合物）和CGP 42112A具有高亲和力，其Ki值与在血管紧张素AT1结合位点发现的氯沙坦和DuP 532的值在相同范围内（nM）。这些位点似乎是血管紧张素AT2受体。如在鸟苷5'-O-(3-硫代磷酸)或氟铝酸盐存在下[3H]血管紧张素II结合被80%抑制所示，只有血管紧张素AT1受体亚型与G蛋白相互作用。尽管血管紧张素II诱导的收缩被氯沙坦完全抑制，pA2值为8.8，但PD 123319使血管紧张素II诱导的收缩减少了20-25%，表明血管紧张素AT1和AT2受体亚型在门静脉平滑肌中均有功能。
