PHARMACOLOGICAL CHARACTERIZATION OF THE HUMAN MELATONIN MEL(1A) RECEPTOR FOLLOWING STABLE TRANSFECTION INTO NIH3T3 CELLS

1 Mouse fibroblasts (NIH3T3) transfected with the full-length coding r egion of the Mel,, melatonin receptor stably expressed the receptor, c oupled to a pertussis toxin-sensitive G-protein(s) and exhibiting high affinity and adequate pharmacological profile. 2 The receptor protein had the tendency of a strong coupling to the G-protein and therefore low-affinity state was induced by uncoupling the receptor from its G-p rotein in presence of high concentrations of NaCl (500-700 mM) and/or GTP gamma S (100 mu M). Thereafter, the affinity of a series of melato nin analogues was determined to both, high- and low-affinity receptor states, thus providing a basis for the prediction of their efficacy, a ccording to the ternary complex model. 3 The cells were subsequently u sed to study the agonist-induced G-protein activation, determined by c alculating the rate of GDP-GTP exchange measured in presence of S-35-l abelled GTP gamma S. The natural ligand melatonin induced a significan t increase in the GDP-GTP exchange rate, the presence of GDP and NaCl being necessary to observe this effect. 4 The full agonists 2-phenylme latonin, 2-bromomelatonin and 6-chloromelatonin equally induced an inc rease of the GDP-GTP exchange. 5-Hydroxy-N-acetyltryptamine activated the GTP-GDP exchange to a much lesser extent (53%) than melatonin, thu s behaving as a partial agonist. As predicted by the model, the melato nin antagonist phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide) was without effect on basal G protein activation. Coincubation of this com pound with melatonin induced a dose-dependent rightward shift in the m elatonin concentration-effect curve, thus exhibiting the behaviour of a competitive and surmountable antagonist. 5 Using the equation propos ed by Venter (1997) we were able to determine that there were no 'spar e' receptors in the system. Therefore, the approach proposed in the pr esent work can be successfully used for the determination of 'drug act ion' at the level of the human Mel(1a) melatonin receptor and evaluati on of the efficacy of new selective melatonin analogues.