García-Pergañeda A, Pozo D, Guerrero J M, Calvo J R
Department of Medical Biochemistry and Molecular Biology, University of Seville School of Medicine, Spain.
J Immunol. 1997 Oct 15;159(8):3774-81.
We analyzed the melatonin signal transduction in human blood lymphocytes. High affinity melatonin receptors were identified by specific binding of 2-[125I]melatonin ([125I]MEL) to human lymphocyte membranes. Scatchard analysis of [125I]MEL binding revealed high affinity receptors, with a dissociation constant (Kd) of 0.45 nM and a binding capacity (Bmax) of 7.8 fmol/mg protein. Specific [125I]MEL binding was reduced markedly by GTP and its nonhydrolyzable analogues guanosine 5'-beta, gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-triphosphate) (GTP-gamma-S). Gpp(NH)p inhibited in a dose-dependent manner the [125I]MEL specifically bound to human lymphocyte membranes with a half-maximal inhibition (IC50) of 3.5 +/- 0.6 microM Gpp(NH)p. Treatment of human lymphocyte membranes with Gpp(NH)p increased the Kd value (Kd = 1.20 nM). Melatonin inhibited significantly and in a dose-dependent manner forskolin-stimulated cAMP production in intact human lymphocytes. Melatonin was able to stimulate diacylglycerol production in a dose-dependent manner in human lymphocyte membranes. Pertussis toxin treatment inhibited the specific [125I]MEL binding and blocked the ability of melatonin to both inhibit forskolin-stimulated cAMP production and stimulate diacylglycerol production. Pertussis toxin ADP-ribosylation and Western blot experiments demonstrated the protein expression of alpha i1/2, alpha i3/0, and beta gamma complexes of G proteins in human lymphocyte membranes. The results strongly suggest a pertussis toxin-sensitive melatonin signal transduction pathway in human lymphocytes that involves the inhibition of adenylyl cyclase and the stimulation of phospholipase C.
我们分析了褪黑素在人血淋巴细胞中的信号转导。通过2-[¹²⁵I]褪黑素（[¹²⁵I]MEL）与人淋巴细胞膜的特异性结合鉴定出高亲和力褪黑素受体。对[¹²⁵I]MEL结合进行Scatchard分析显示存在高亲和力受体，解离常数（Kd）为0.45 nM，结合容量（Bmax）为7.8 fmol/mg蛋白。GTP及其不可水解类似物鸟苷5'-β,γ-亚氨基三磷酸（Gpp(NH)p）和鸟苷5'-O-(3-三磷酸)（GTP-γ-S）可显著降低特异性[¹²⁵I]MEL结合。Gpp(NH)p以剂量依赖性方式抑制特异性结合到人淋巴细胞膜上的[¹²⁵I]MEL，半数最大抑制浓度（IC50）为3.5±0.6 μM Gpp(NH)p。用Gpp(NH)p处理人淋巴细胞膜会增加Kd值（Kd = 1.20 nM）。褪黑素可显著且以剂量依赖性方式抑制完整人淋巴细胞中福斯高林刺激的cAMP生成。褪黑素能够以剂量依赖性方式刺激人淋巴细胞膜中二酰基甘油的生成。百日咳毒素处理可抑制特异性[¹²⁵I]MEL结合，并阻断褪黑素抑制福斯高林刺激的cAMP生成以及刺激二酰基甘油生成的能力。百日咳毒素ADP-核糖基化和蛋白质印迹实验证明了人淋巴细胞膜中G蛋白的αi1/2、αi3/0和βγ复合物的蛋白表达。结果强烈提示人淋巴细胞中存在一条对百日咳毒素敏感的褪黑素信号转导途径，该途径涉及腺苷酸环化酶的抑制和磷脂酶C的刺激。
