Romolo Nonno

The melatonin receptor in the human brain: cloning experiments and distribution studies

The adult human cerebellum expresses melatonin receptors with high density in the external zone of the molecular layer. Cloning of the receptor cDNA isolated by RT-PCR from human cerebellar specimens and sequencing analysis of the full-length coding region revealed that the receptor protein is encoded by a transcript identical to that recently cloned from the human hypothalamus (Mel1a). In situ hybridization using an antisense cRNA-probe demonstrated that the melatonin receptor mRNA is localized in the cerebellar granule cells. Mapping of the messenger by RT-PCR with Mel1a specific primers in different areas of the human brain disclosed a quite widespread distribution of the transcript, although expressed at very low levels. Semi-quantitative comparison between the different brain regions allowed to establish the following relative mRNA abundance: cerebellum > or = occipital cortex > or = parietal cortex > temporal cortex > thalamus > frontal cortex > or = hippocampus. No mRNA was detected in white blood cells.

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

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 (KI=261 pM). The order of apparent affinity for selected compounds was: 4‐phenyl‐2‐propionamidotetralin (4P‐PDOT)>2‐phenylmelatonin>2‐iodomelatonin>2‐bromomelatonin>6‐chloromelatoninmelatonin>luzindole>N‐acetyl‐tryptamineN‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide (compound 6)>N‐acetylserotonin. 4P‐PDOT exhibited a very high selectivity (∼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]‐GTPγS into isolated membranes via receptor catalyzed exchange of [35S]‐GTPγS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [35S]‐GTPγS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [35S]‐GTPγS binding by 250% over basal (taken as 100%). Luzindole did not increase basal [35S]‐GTPγS binding but competitively inhibited melatonin‐stimulated [35S]‐GTPγS 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.

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.

Pharmacological characterization of the human melatonin Mel1a receptor following stable transfection into NIH3T3 cells

Mouse fibroblasts (NIH3T3) transfected with the full‐length coding region of the Mel1a melatonin receptor stably expressed the receptor, coupled to a pertussis toxin‐sensitive G‐protein(s) and exhibiting high affinity and adequate pharmacological profile. 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‐protein in presence of high concentrations of NaCl (500–700 mM) and/or GTPγS (100 μM). Thereafter, the affinity of a series of melatonin analogues was determined to both, high‐ and low‐affinity receptor states, thus providing a basis for the prediction of their efficacy, according to the ternary complex model. The cells were subsequently used to study the agonist‐induced G‐protein activation, determined by calculating the rate of GDP‐GTP exchange measured in presence of 35S‐labelled GTPγS. The natural ligand melatonin induced a significant increase in the GDP‐GTP exchange rate, the presence of GDP and NaCl being necessary to observe this effect. The full agonists 2‐phenylmelatonin, 2‐bromomelatonin and 6‐chloromelatonin equally induced an increase of the GDP‐GTP exchange. 5‐Hydroxy‐N‐acetyltryptamine activated the GTP‐GDP exchange to a much lesser extent (53%) than melatonin, thus behaving as a partial agonist. As predicted by the model, the melatonin antagonist (N‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide) was without effect on basal G protein activation. Coincubation of this compound with melatonin induced a dose‐dependent rightward shift in the melatonin concentration‐effect curve, thus exhibiting the behaviour of a competitive and surmountable antagonist. Using the equation proposed by Venter (1997) we were able to determine that there were no ‘spare’ receptors in the system. Therefore, the approach proposed in the present work can be successfully used for the determination of ‘drug action’ at the level of the human Mel1a melatonin receptor and evaluation of the efficacy of new selective melatonin analogues.

2-[125I]Iodomelatonin binding sites in the bovine hippocampus are not sensitive to guanine nucleotides

The discrete distribution and pharmacological characteristics of melatonin binding sites in the bovine hippocampus were determined. Autoradiography revealed the presence of melatonin binding sites in the stratum lacunosum-molecularis of the hippocampus (CA1), stratum molecularis of the subiculum and in the enthorhinal cortex. Analysis of the kinetic parameters demonstrated that the binding was stable and reversible, represented by a single class high affinity binding sites (Kd 40 pM, Bmax = 3.9 fmol/mg protein). However, 2-iodomelatonin and 2-bromomelatonin inhibited 2-[125I]iodomelatonin binding in a biphasic manner. The presence of 4 mM CaCl2 did not cause changes in the affinity constant values. Finally, experiments performed with GTP gamma S revealed that binding affinity was not decreased even with high concentrations of the nucleotide. These findings show that 2-[125I]iodomelatonin binding sites in the bovine parahippocampal-hippocampal region possess some binding features not common to melatonin receptors described so far; moreover they seem not to be linked to a regulatory G-protein.

A new melatonin receptor ligand with mt1‐agonist and MT2‐antagonist properties

It has been difficult, so far, to obtain melatonin analogs possessing high selectivity for the respective melatonin receptors, mt1 and MT2. In the present work, we report the synthesis and pharmacological characterization of a new compound N‐{2‐[5‐(2‐hydroxyethoxy)‐1H‐indol‐3‐yl)] ethyl} acetamide or 5‐hydroxyethoxy‐N‐acetyltryptamine (5‐HEAT). To assess the activity of the compound, the following tests were performed: affinity determination for the high‐ and low‐affinity receptor states (2‐[ I]iodomelatonin binding), potency and intrinsic activity in inducing G protein activation ([ S]GTPγS binding assay). 5‐HEAT showed little selectivity for the mt1 receptor, with pKi values of 7.77 for mt1 and 7.12 for the MT2 receptors, respectively. 5‐HEAT was able to differentiate between the high‐ and the low‐affinity receptor states in the mt1 but not in the MT2 receptor. 5‐HEAT induced a high level of G protein activation when acting through the mt1 receptor, with a relative intrinsic activity of 0.92. On the contrary, it elicited only minimal MT2 receptor‐mediated G protein activation, with a relative intrinsic activity of 0.16, and was also able to inhibit the melatonin‐induced MT2 receptor‐mediated G protein activation, with a pKB value of 7.4. In conclusion, it appears that 5‐HEAT possesses very different efficacies at the two melatonin receptors, behaving as a full melatonin receptor agonist at the mt1 and as an antagonist/weak partial agonist at the MT2 receptor. Therefore, it is a promising ligand for use in functional studies aimed at distinguishing between the effects mediated by the different melatonin receptors in the human.

Methods for the Evaluation of Drug Action at the Human Melatonin Receptor Subtypes

NIH3T3 fibroblast cells transfected with the full-length coding regions of the mt₁ and MT₂ human melatonin receptors stably expressed the receptor, coupled to a pertussis-toxin-sensitive G protein and exhibiting high affinity for melatonin. Both mt₁ and MT₂ melatonin receptors mediated the incorporation of [³⁵S]GTPγS into isolated membranes via receptor-catalyzed exchange of [³⁵S]GTPγS for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [³⁵S]GTPγS incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [³⁵S]GTPγS binding. Luzindole did not increase basal [³⁵S]GTPγS binding but competitively inhibited melatonin-stimulated [³⁵S]GTPγS binding, thus exhibiting antagonist action. Two other mt₁ antagonists, 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. For the first time, these findings show important differences in analogue intrinsic activity between the human mt₁ and MT₂ melatonin receptor subtypes.

Distribution and characterization of the melatonin receptors in the hypothalamus and pituitary gland of three domestic ungulates

Abstract: With some exceptions, in most of the mammals the pituitary pars tuber‐alis and the hypothalamic suprachiasmatic nuclei are reportedly the main targets for the pineal hormone melatonin. However, it is not known if the conspicuous diversity in the distribution pattern of melatonin binding sites in these areas depicts differences in reproductive behavior observed in the seasonally breeding species in the temperate zones. We explored the distribution and the characteristics of melatonin binding sites in the hypothalamus and pituitary of three species (bovine, horse, and donkey) different in terms of seasonal reproductive competence. The topographical localization, investigated by in vitro autoradiography, revealed 2‐[125I]iodomelatonin binding sites only in the pituitary gland in all three species, primarily in the pars tuberalis (PT), but also in the pars distalis (PD) and pars intermedia (PI). Kinetic, inhibition, and saturation studies, performed by means of in vitro binding, revealed presence of a single class high affinity binding sites. The Kd values, melatonin, and 2‐iodomelatonin Kj values were in the low picomo‐lar range. Coincubation with GTP7S inhibited 2‐[125 I]iodomelatonin binding, demonstrating that these putative receptors are linked to a G protein in their signal‐transduction pathway. The hypothalamus was devoid of specific binding. In conclusion, the results suggest that in these species, the hypophysis may be a principal target for the melatonin action on the reproductive system.