Tahara A, Saito M, Sugimoto T, Tomura Y, Wada K, Kusayama T, Tsukada J, Ishii N, Yatsu T, Uchida W, Tanaka A
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan.
Br J Pharmacol. 1998 Dec;125(7):1463-70. doi: 10.1038/sj.bjp.0702220.
Three subtypes of human (h) arginine vasopressin (AVP) receptors, hV1A, hV1B and hV2, were stably expressed in Chinese hamster ovary (CHO) cells and characterized by [3H]-AVP binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. Scatchard analysis of saturation isotherms for the specific binding of [3H]-AVP to membranes, prepared from CHO cells transfected with hV1A, hV1B and hV2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.39, 0.25 and 1.21 nM and a maximum receptor density (Bmax) of 1580 fmol mg(-1) protein, 5230 fmol mg(-1) protein and 7020 fmol mg(-1) protein, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Pharmacological characterization of the transfected human AVP receptors was undertaken by measuring the relative ability of nonpeptide AVP receptor antagonists, YM087, OPC-21268, OPC-31260, SR 49059 and SR 121463A, to inhibit binding of [3H]-AVP. At hV1A receptors, the relative order of potency was SR49059>YM087>OPC-31260>SR 121463A> >OPC-21268 and at hV2 receptors, YM087=SR 121463A>OPC-31260>SR 49059> >OPC-21268. In contrast, the relative order of potency, at hV1B receptors, was SR 49059> >SR 121463A=YM087=OPC-31260=OPC-21268. In CHO cells expressing either hV1A or hV1B receptors, AVP caused a concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 1.13 nM and 0.90 nM, respectively. In contrast, stimulation of CHO cells expressing hV2 receptors resulted in an accumulation of cyclic AMP with an EC50 value of 2.22 nM. The potency order of antagonists in inhibiting AVP-induced [Ca2+]i or cyclic AMP response was similar to that observed in radioligand binding assays. In conclusion, we have characterized the pharmacology of human cloned V1A, V1B and V2 receptors and used these to determine the affinity, selectivity and potency of nonpeptide AVP receptor antagonists. Thus they may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of AVP.
人精氨酸加压素（AVP）受体的三种亚型，即hV1A、hV1B和hV2，在中国仓鼠卵巢（CHO）细胞中稳定表达，并通过[3H]-AVP结合研究进行了表征。此外，还研究了表达的受体蛋白与多种信号转导途径的偶联。对用hV1A、hV1B和hV2受体转染的CHO细胞制备的膜上[3H]-AVP特异性结合的饱和等温线进行Scatchard分析，得出表观平衡解离常数（Kd）分别为0.39、0.25和1.21 nM，最大受体密度（Bmax）分别为1580 fmol mg(-1)蛋白、5230 fmol mg(-1)蛋白和7020 fmol mg(-1)蛋白。希尔系数与1无显著差异，表明与同质、非相互作用的受体群体结合。通过测量非肽AVP受体拮抗剂YM087、OPC-21268、OPC-31260、SR 49059和SR 121463A抑制[3H]-AVP结合的相对能力，对转染的人AVP受体进行了药理学表征。在hV1A受体处，效力的相对顺序为SR49059>YM087>OPC-31260>SR 121463A>>OPC-21268；在hV2受体处，YM087=SR 121463A>OPC-31260>SR 49059>>OPC-21268。相比之下，在hV1B受体处，效力的相对顺序为SR 49059>>SR 121463A=YM087=OPC-31260=OPC-21268。在表达hV1A或hV1B受体的CHO细胞中，AVP引起细胞内Ca2+浓度（[Ca2+]i）呈浓度依赖性增加，EC50值分别为1.13 nM和0.90 nM。相比之下，刺激表达hV2受体的CHO细胞导致环磷酸腺苷积累，EC50值为2.22 nM。拮抗剂抑制AVP诱导的[Ca2+]i或环磷酸腺苷反应的效力顺序与放射性配体结合试验中观察到的相似。总之，我们已经表征了人克隆的V1A、V1B和V2受体的药理学特性，并利用这些特性确定了非肽AVP受体拮抗剂的亲和力、选择性和效力。因此，它们可能被证明是进一步研究AVP的生理和病理生理作用的有价值工具。
