Browning C, Beresford I, Fraser N, Giles H
Receptor Pharmacology Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts, SG1 2NY.
Br J Pharmacol. 2000 Mar;129(5):877-86. doi: 10.1038/sj.bjp.0703130.
We have pharmacologically characterized recombinant human mt(1) and MT(2) receptors, stably expressed in Chinese hamster ovary cells (CHO-mt(1) and CHO-MT(2)), by measurement of [(3)H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt(1) and MT(2) receptors with pK(D) values of 9.89 and 9.56 and B(max) values of 1.20 and 0.82 pmol mg(-1) protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt(1) and MT(2) receptors, a number of putative antagonists had substantially higher affinities for MT(2) receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt(1) and CHO-MT(2) cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC(50) 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT(2) receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt(1) receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt(1) and CHO-MT(2) cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA(2) values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of G(i)/G(o) G-proteins. This is the first report of the use of [(3)H]-melatonin for the characterization of recombinant mt(1) and MT(2) receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.
我们通过测量[³H] - 褪黑素结合以及福司可林刺激的环磷酸腺苷（cAMP）生成，对稳定表达于中国仓鼠卵巢细胞（CHO - mt(1)和CHO - MT(2)）中的重组人mt(1)和MT(2)受体进行了药理学特性分析。[³H] - 褪黑素与mt(1)和MT(2)受体结合，其解离常数（pK(D)）值分别为9.89和9.56，最大结合量（B(max)）值分别为1.20和0.82 pmol mg⁻¹蛋白质。虽然大多数褪黑素受体激动剂对mt(1)和MT(2)受体具有相似的亲和力，但一些假定的拮抗剂对MT(2)受体具有显著更高的亲和力，包括鲁辛朵（11倍）、GR128107（23倍）和4 - P - PDOT（61倍）。在CHO - mt(1)和CHO - MT(2)细胞中，褪黑素均以浓度依赖性方式抑制福司可林刺激的环磷酸腺苷积累（pIC(50)分别为9.53和9.74），在100 nM时分别导致环磷酸腺苷生成抑制83%和64%。测定了一系列褪黑素受体激动剂的效力。在MT(2)受体上，褪黑素、2 - 碘褪黑素和6 - 氯褪黑素基本等效，而在mt(1)受体上，这些激动剂的效力顺序为2 - 碘褪黑素>褪黑素>6 - 氯褪黑素。在CHO - mt(1)和CHO - MT(2)细胞中，褪黑素受体拮抗剂鲁辛朵以浓度依赖性方式拮抗褪黑素诱导的对福司可林刺激的环磷酸腺苷生成的抑制作用，其拮抗常数（pA(2)）值分别为5.75和7.64。用百日咳毒素预处理细胞可消除褪黑素介导的反应，这与G(i)/G(o) G蛋白的激活一致。这是首次使用[³H] - 褪黑素对重组mt(1)和MT(2)受体进行特性分析的报告。我们的结果表明，这些受体亚型具有不同的药理学特征。
