Molecular and cellular pharmacologyComparative pharmacological studies of melatonin receptors: mt1, mt2 and mt3/qr2. tissue distribution of mt3/qr2 1
Introduction
Melatonin is an indole-derived neurohormone of long standing interest which is produced in the pineal gland and is derived from serotonin. The main feature of the pharmacodynamics of melatonin is its nocturnal synthesis and secretion. As a consequence, melatonin is suspected to relay the circadian rhythm and the information on the photoperiod to the peripheral organs for daily and seasonal physiological regulations. Furthermore, melatonin has a proven role in the sleep/wake cycle [1], and is involved in numerous physiological functions depending on the circadian rhythm, such as the immune [2] and the cardiovascular systems [3]. Many cellular targets of melatonin have been detected since 1986, after the synthesis of 2-iodomelatonin [4], a very potent melatonin agonist which was rapidly used as 2-[125I]-iodomelatonin for labeling tissue sections and performing pharmacological studies [5], [6]. Melatonin binding sites were initially detected in the central nervous system of rodents and chicken, and then, in numerous peripheral tissues of birds and mammals [6], [7], [8], suggesting their widespread distribution among tissues. Three melatonin receptor isoforms have been cloned to date. The Mel1a gene encodes the MT1 receptor [9], the Mel1b gene encodes the MT2 receptor [10] and the Mel1c was cloned from Xenopus laevis[11] but is not expressed in mammals [12]. The MT1 and MT2 receptors share a common seven-transmembrane predicted structure and the ability to transduce membrane signals via G-protein coupling [13], [14]. These two receptors also share a close pharmacological profile, with the following order of affinities 2-iodomelatonin > melatonin > 6-hydroxymelatonin ⪢ N-acetylserotonin ⪢ prazosin [15]. The MT1 and MT2 receptors are also characterized by subnanomolar affinities for melatonin and 2-iodomelatonin.
In addition to these high affinity melatonin receptors, there is evidence for a nanomolar melatonin binding site in Hamster brain [15], [16], [17] and kidney [18], [19], MT3. The ligands known to date of MT3 specificity over MT1 and MT2 include prazosin [17] and 5-methoxycarbonylamino-N-acetyltryptamine (MCA-NAT; [18]). Besides its original pharmacology, MT3 has always displayed very fast kinetics of ligand association/dissociation [16], [18], [19], raising difficulties for affinity measurements. Because of this property, MT3 has always been studied at low temperature (0° or 4°) in order to impair the fast ligand dissociation kinetics. Despite major studies conducted on MT3 binding sites, it remains that widely different experimental conditions may have occulted differences in MT1, MT2 and MT3 ligand specificities, especially with the large utilization of 2-[125I]-iodomelatonin as a radioligand, although 2-[125I]-iodomethoxy-carbonylamino-N-acetyltryptamine (2-[125I]-I-MCA-NAT) was proven to be a more specific tool [18]. Further, our recent description of the MT3 pharmacology [20] needs to be completed by a better assessment of the experimental conditions under which MT3 is best described, i.e. with the specific radioligand 2-[125I]-I-MCA-NAT and at 20°. The recent purification of MT3 and its identification as the quinone reductase 2 (QR2, EC 1.6.99.2), an enzyme related to the detoxifying enzyme quinone reductase 1, shed new lights on the pharmacological characterization of all the melatonin binding sites [20]. Indeed, melatonin binding sites can now be regarded both as a population of proteins encompassing genuine membrane receptor activity leading to intracellular signaling and, alternatively, as fugacious melatonin binding sites with yet unresolved function.
We describe in the present paper a new standard for the molecular pharmacology of MT3/QR2 receptor at higher temperature (as opposed to 4°), including comparisons with the MT1 and MT2 receptor pharmacology. Furthermore, we describe new compounds as potent as prasozin but more specific for the MT3 binding site. We also discuss the relevance of the comparison of MT3 binding and QR2 enzymatic activity after determining the tissue distribution of the two signals in different species.
Section snippets
Materials
2-[125I]-melatonin (2200 Ci/mmmol) was purchased from NEN, 2-IbMT (2-iodo-N-butanoyl-5-methoxy-tryptamine), 2-iodomelatonin, 2-phenylmelatonin, 4-P-PDOT (4-phenyl-2-propionamidotetraline), 6-chloromelatonin, DH-97 (N-pentanoyl-2-benzyltryptamine), luzindole (N-acetyl-2-benzyltryptamine), MCA-NAT (5-methoxycarbonylamino-N-acetyltryptamine) and S20760 (5-methoxy-N-cyclopropanoyl-tryptamine) were purchased from Tocris and all other reagents were obtained from Sigma-Aldrich. S24635, S25726 and
Determination of binding conditions on mt3 membranes
Using an original technique, based on the distribution of samples directly onto 96 well filter plates under constant vacuum, the filtration of samples and the subsequent rinsing of the filters lasted clearly less than one second, partly overcoming the rapidness of ligand dissociation from the MT3 binding sites. It was then possible to perform comparative binding assays on MT1, MT2, and MT3 membranes using 2-[125I]-iodomelatonin and 2-[125I]-I-MCA-NAT at different temperatures. Fig. 1 shows the
Discussion
In addition to the now well documented seven transmembrane domains G protein-coupled receptors, MT1 and MT2, several melatonin binding sites are presented as putative melatonin receptors. Among these are the MT3 binding site, the nucleus-associated site identified by 2-[125I]-melatonin binding [27], [28], [29], a site on adipocytes [30] and several other binding sites with original 2-[125I]-iodomelatonin binding properties (discussed in [19]). The MT3 melatonin binding site was originally
Acknowledgements
The authors are indebted to Sophie Lallier for technical assistance.
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Abbreviations: MCA-NAT, methoxy-carbonylamino-N-acetyltrypta- mine, 2-[125I]-I-MCA-NAT, 2-[125I]-iodomethoxy-carbonylamino-N-acetyltryptamine; 2-IbMT, 2-iodo-N-butanoyl-5-methoxytryptamine; 4-P-PDOT, 4-phenyl-2-propionamido-tetraline; DH97, N-pentanoyl-2-benzyltryptamine; S20760, 5-methoxy-N-cyclopropanoyl-tryptamine; S24635, N-[2-(5-carbamoylbenzofuran-3-yl)ethyl]-acetamide; S25726, N-methyl-(3-{2-[(cyclopropylcarbonyl)-amino]ethyl}benzo[b]furan-5-yl)carbamate; S26553, N-methyl-{1-[2-(acetylamino)ethyl]-naphthalen-7-yl}carbamate.