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. 1999 Jul;127(5):1288-94.
doi: 10.1038/sj.bjp.0702658.

Ligand efficacy and potency at recombinant human MT2 melatonin receptors: evidence for agonist activity of some mt1-antagonists

R Nonno  1 , M Pannacci, V Lucini, D Angeloni, F Fraschini, B M Stankov
Affiliations

Ligand efficacy and potency at recombinant human MT2 melatonin receptors: evidence for agonist activity of some mt1-antagonists

R Nonno et al. Br J Pharmacol. 1999 Jul.

Abstract

NIH3T3 fibroblast cells transfected with the full-length coding region of the MT2 human melatonin receptor stably expressed the receptor that is coupled to a pertussis toxin-sensitive G protein and exhibits high affinity for melatonin (K(I) = 261 pM). The order of apparent affinity for selected compounds was: 4-phenyl-2-propionamidotetralin (4P-PDOT) > 2-phenylmelatonin > 2-iodomelatonin > 2-bromomelatonin > 6-chloromelatonin > or = melatonin > luzindole > N-acetyl-tryptamine > or = N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide (compound 6) > N-acetylserotonin. 4P-PDOT exhibited a very high selectivity (approximately 22,000 times) for the MT2 receptor with respect to the mt1 receptor subtype, as tested in comparative experiments with membrane preparations from NIH3T3 cells stably transfected with the human mt1 receptor. MT2 melatonin receptors mediated incorporation of [35S]-GTPgammaS into isolated membranes via receptor catalyzed exchange of [35S]-GTPgammaS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]-GTPgammaS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]-GTPgammaS binding by 250% over basal (taken as 100%). Luzindole did not increase basal [35S]-GTPgammaS binding but competitively inhibited melatonin-stimulated [35S]-GTPgammaS binding, thus exhibiting antagonist action. The other two mt1 antagonists used here, 4P-PDOT and N-[(2-phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT2 subtype, with relative intrinsic activities of 0.37 and 0.39, respectively. These findings show, for the first time, important differences in the intrinsic activity of analogues between the human mt1 and MT2 melatonin receptor subtypes.

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Figures

Figure 1
Figure 1
Competition curves of melatonin and its analogues for 2-[125I]-iodomelatonin (2-125Imel) binding to NIH3T3MT2 membranes. The data are representative of a single experiment with each point determined in duplicate. The rank order of apparent affinity was 2-phenylmelatonin> 2-bromomelatonin> 6-chloromelatonin⩾melatonin>N-acetyl-tryptamine⩾compound 6>N-acetylserotonin. The experiment was carried out as described in Methods, for 90 min at 37°C. Each point is the mean of duplicate determinations; the error bars indicate the standard deviation.
Figure 2
Figure 2
Comparison of the stimulation of [35S]-GTPγS binding to NIH3T3MT2 membranes by melatonin and its analogues. The data are representative of a single experiment with each point determined in triplicate. The order of agonist potency (with relative intrinsic activities obtained in this experiment reported in brackets) was: 4P-PDOT (0.37)>2-phenylmelatonin (0.98)>2-bromomelatonin (1.03)>6-chloromelatonin (0.97)>melatonin (1)>N-acetyl-tryptamine (0.82)>compound 6 (0.41)>N-acetylserotonin (0.78). Luzindole was without any effect on basal [35S]-GTPγS binding. The experiment was carried out as described in Methods, for 30 min at 37°C. Each point is the mean of triplicate determinations; the error bars indicate the standard error. Values represent percentage of basal binding, defined as 100%.
Figure 3
Figure 3
Comparison of competition curves for 4P-PDOT and luzindole to 2-[125I]-iodomelatonin (2-125Imel) binding to NIH3T3MT2 (closed symbols) and NIH3T3mt1 (open symbols) membranes. The data are representative of a single experiment in which each time point is determined in duplicate. Both 4P-PDOT and luzindole show higher affinity for the MT2 receptor subtype. Note the slope of the competition curve of 4P-PDOT (−0.58) in NIH3T3MT2 membranes with respect to the slope of luzindole (−0.91) in NIH3T3MT2 membranes and to the slope of 4P-PDOT (−0.96) and luzindole (−0.94) in NIH3T3mt1 membranes. The experiment was carried out as described in Methods, for 90 min at 37°C. Each point is the mean of duplicate determinations; the error bars indicate the standard deviation.
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