Bushfield M, Savage A, Morris N J, Houslay M D
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Biochem J. 1993 Jul 1;293 ( Pt 1)(Pt 1):229-36. doi: 10.1042/bj2930229.
Both amylin and calcitonin-gene-related neuropeptide (CGRP) activated adenylate cyclase activity in hepatocyte membranes around 5-fold in a dose-dependent fashion, with EC50 values of 120 +/- 14 and 0.3 +/- 0.14 nM respectively. Whereas amylin exhibited normal activation kinetics (Hill coefficient, h approximately 1), CGRP showed kinetics indicative of either multiple sites/receptor species having different affinities for this ligand or a single receptor species exhibiting apparent negative co-operativity (h approximately 0.21). The CGRP antagonist CGRP-(8-37)-peptide inhibited adenylate cyclase stimulated by EC50 concentrations of either amylin or CGRP. Inhibition by CGRP-(8-37) was selective in that markedly lower concentrations were required to block the action of amylin (IC50 = 3 +/- 1 nM) compared with that of CGRP itself (IC50 = 120 +/- 11 nM). Dose-effect data for inhibition of CGRP action by CGRP-(8-37) showed normal saturation kinetics (h approximately 1), whereas CGRP-(8-37) inhibited amylin-stimulated adenylate cyclase activity in a fashion which was indicative of either multiple sites or apparent negative co-operativity (h approximately 0.24). Observed changes in the kinetics of inhibition by CGRP-(8-37) of CGRP, but not amylin-stimulated adenylate cyclase, at concentrations of agonists below their EC50 values militated against a model of two distinct populations of non-interacting receptors each able to bind both amylin and CGRP. A kinetic model is proposed whereby a single receptor, capable of being activated by both CGRP and amylin, obeys either a mnemonical kinetic mechanism or one of negative co-operativity with respect to CGRP but not to amylin. The relative merits of these two models are discussed together with a proposal suggesting that the activation of adenylate cyclase by various G-protein-linked receptors may be described by a mnemonical model mechanism.
胰淀素和降钙素基因相关神经肽（CGRP）均可使肝细胞膜中的腺苷酸环化酶活性以剂量依赖方式激活约5倍，其半数有效浓度（EC50）值分别为120±14和0.3±0.14 nM。胰淀素表现出正常的激活动力学（希尔系数，h约为1），而CGRP的动力学表明存在对该配体具有不同亲和力的多个位点/受体类型，或者是表现出明显负协同性的单一受体类型（h约为0.21）。CGRP拮抗剂CGRP - (8 - 37) - 肽可抑制由EC50浓度的胰淀素或CGRP刺激的腺苷酸环化酶。CGRP - (8 - 37)的抑制作用具有选择性，因为与CGRP本身（IC50 = 120±11 nM）相比，阻断胰淀素的作用所需浓度明显更低（IC50 = 3±1 nM）。CGRP - (8 - 37)抑制CGRP作用的剂量 - 效应数据显示出正常的饱和动力学（h约为1），而CGRP - (8 - 37)以表明存在多个位点或明显负协同性的方式（h约为0.24）抑制胰淀素刺激的腺苷酸环化酶活性。在激动剂浓度低于其EC50值时，观察到CGRP - (8 - 37)对CGRP而非胰淀素刺激的腺苷酸环化酶抑制动力学的变化，这不利于两个不相互作用的受体群体模型，每个群体都能够结合胰淀素和CGRP。提出了一个动力学模型，其中一个能够被CGRP和胰淀素激活的单一受体，对于CGRP遵循记忆动力学机制或负协同性机制之一，但对胰淀素不遵循。讨论了这两种模型的相对优点，并提出了一个建议，即各种G蛋白偶联受体对腺苷酸环化酶的激活可能由记忆模型机制来描述。
