Stankov B, Biella G, Panara C, Lucini V, Capsoni S, Fauteck J, Cozzi B, Fraschini F
Institute of Physiopathology and Therapy of Pain, University of Milan, Italy.
Endocrinology. 1992 Apr;130(4):2152-9. doi: 10.1210/endo.130.4.1312448.
The cortex of the rabbit (Oryctolagus cuniculus) is rich in melatonin binding sites, and particularly abundant is the parietal cortex. Consequently, we characterized the putative melatonin receptor in the parietal cortex by a series of in vitro ligand-receptor binding experiments and biochemical and electrophysiological studies. The in vitro saturation and competition experiments demonstrated that the binding in the crude cortical membrane preparations was of high affinity and specificity. Guanine nucleotides (GDP, GTP, and GTP gamma S) inhibited the specific 2-[125I]iodomelatonin binding in a dose-dependent manner. Coincubation with a nonhydrolyzable GTP analog provoked a shift in the binding affinity; the numerical values of the Kd increased from 20-30 to 200-600 pM. Melatonin, in nanomolar concentrations, was able to inhibit the forskolin-stimulated accumulation of cAMP in parietal cortex explants, and preincubation with pertussis toxin counteracted this effect of melatonin. Apparently, the melatonin binding site in the rabbit parietal cortex is linked to its second messenger via a pertussis toxin-sensitive G-protein, probably of the inhibitory Gi class, similar to what has been described for different parts of the brain of other vertebrates. The experiments on the spontaneous firing activity of single neurons in the third to fourth layer of the parietal cortex in anesthetized animals showed that melatonin and its potent agonist 2-iodomelatonin exhibited gamma-aminobutyric acid (GABA)-like effects and were able alone, in nanomolar concentrations, to significantly slow the neuronal firing activity. Moreover, both melatonin and 2-iodomelatonin potentiated the effect of GABA on the neuronal activity, leading to powerful inhibition of the tested neurons. Undoubtedly, the binding site in the rabbit parietal cortex possesses all of the characteristics of a functional receptor. We suggest that melatonin is involved in the control of fundamental cortical functions and that it acts in concert with GABA, one of the two major inhibitory neurotransmitters in the central nervous system.
家兔（穴兔）的皮质富含褪黑素结合位点，其中顶叶皮质尤为丰富。因此，我们通过一系列体外配体 - 受体结合实验以及生化和电生理研究，对顶叶皮质中的假定褪黑素受体进行了表征。体外饱和和竞争实验表明，在粗制皮质膜制剂中的结合具有高亲和力和特异性。鸟嘌呤核苷酸（GDP、GTP和GTPγS）以剂量依赖性方式抑制特异性2 - [125I]碘褪黑素结合。与不可水解的GTP类似物共同孵育会引起结合亲和力的变化；Kd的数值从20 - 30 pM增加到200 - 600 pM。纳摩尔浓度的褪黑素能够抑制福斯高林刺激的顶叶皮质外植体中cAMP的积累，并且用百日咳毒素预孵育可抵消褪黑素的这种作用。显然，家兔顶叶皮质中的褪黑素结合位点通过一种对百日咳毒素敏感的G蛋白与其第二信使相连，该G蛋白可能属于抑制性Gi类，这与在其他脊椎动物大脑的不同部位所描述的情况类似。对麻醉动物顶叶皮质第三至第四层单个神经元的自发放电活动进行的实验表明，褪黑素及其强效激动剂2 - 碘褪黑素表现出γ - 氨基丁酸（GABA）样效应，并且仅在纳摩尔浓度下就能显著减慢神经元的放电活动。此外，褪黑素和2 - 碘褪黑素均增强了GABA对神经元活动的作用，导致对受试神经元的强力抑制。毫无疑问，家兔顶叶皮质中的结合位点具有功能性受体的所有特征。我们认为褪黑素参与了皮质基本功能的调控，并且它与GABA协同作用，GABA是中枢神经系统中两种主要的抑制性神经递质之一。
