Molecular cloning and pharmacological characterization of rat melatonin MT1 and MT2 receptors

Audinot, Valérie; Bonnaud, Anne; Grandcolas, Line; Rodriguez, Marianne; Nagel, Nadine; Galizzi, Jean‐Pierre; Balík, Aleš; Messager, Sophie; Hazlerigg, David G.; Barrett, Perry; Delagrange, Philippe; Boutin, Jean A.

doi:10.1016/j.bcp.2008.02.022

Cited by 43 publications

(46 citation statements)

References 35 publications

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“…The kinetics curves are presented in Fig. 1 We assessed a set of 24 compounds that were previously described in the literature [including 4-phenyl-2-propionamidotetraline (Dubocovich et al, 1997), luzindole (Dubocovich, 1988a), and ramelteon (Uchikawa et al, 2002)] or that were issued from our own medicinal chemistry programs (Depreux et al, 1994; Audinot et al, 2003Audinot et al, , 2008Mailliet et al, 2004;Devavry et al, 2012a,b;Ettaoussi et al, 2013;Legros et al, 2013Legros et al, , 2014 (Fig. 3) with a pK d 1 (site 1) of 10.35 6 0.03, a pK d 2 (site 2) of 9.25 6 0.13, a B max 1 (site 1) of 1.46 6 0.13 fmol·mg protein 21 , and a B max 2 (site 2) of 2.32 6 0.98 fmol·mg protein 21 (n 5 4).…”

Section: Cell Membrane Preparationmentioning

confidence: 99%

Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors

Legros¹,

Brasseur²,

Delagrange³

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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Melatonin exerts a variety of physiologic activities that are mainly relayed through the melatonin receptors MT 1 and MT 2 Low expressions of these receptors in tissues have led to widespread experimental use of the agonist 2-[125 I]-iodomelatonin as a substitute for melatonin. We describe three iodinated ligands:, which are specific ligands at MT 2 receptors, and125 I]-iodomelatonin with slightly different characteristics. Here, we further characterized these new ligands with regards to their molecular pharmacology. We performed binding experiments, saturation assays, association/dissociation rate measurements, and autoradiography using sheep and rat tissues and recombinant cell lines. Our results showed that [125 I]-S70254 is receptor, and can be used with both cells and tissue. This radioligand can be used in autoradiography. Similarly, DIV880, a partial agonist [43% of melatonin on guanosine 59-3-O-(thio)triphosphate binding assay], selective for MT 2 , can be used as a tool to selectively describe the pharmacology of this receptor in tissue samples. The molecular pharmacology of both human melatonin receptors MT 1 and MT 2 , using a series of 24 ligands at these receptors and the new radioligands, did not lead to noticeable variations in the profiles. For the first time, we described radiolabeled tools that are specific for one of the melatonin receptors (MT 2 ). These tools are amenable to binding experiments and to autoradiography using sheep or rat tissues. These specific tools will permit better understanding of the role and implication in physiopathologic processes of the melatonin receptors.

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Section: Cell Membrane Preparationmentioning

confidence: 99%

Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors

Legros¹,

Brasseur²,

Delagrange³

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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“…To provide a better understanding of the physiological functions of MT 1 and MT 2 receptor subtypes in animal models, a comparison of pharmacological properties of ligands for melatonin receptor subtypes from different species was recently reported. [13,14] The results of these studies demonstrated that rat MT 1 and MT 2 receptors have high homology to human MT 1 (88 %) and MT 2 (78 %) receptors, including amino acids that have been shown to be involved in ligand binding. Furthermore, comparison of binding affinities at rat MT 1 and MT 2 receptors of several melatonin ligands with the data obtained at hMT 1 and hMT 2 receptors revealed a significant statistical correlation, particularly for MT 2 sites.…”

Section: Introductionmentioning

confidence: 95%

“…Furthermore, comparison of binding affinities at rat MT 1 and MT 2 receptors of several melatonin ligands with the data obtained at hMT 1 and hMT 2 receptors revealed a significant statistical correlation, particularly for MT 2 sites. [14] In the SCN, the MT 1 receptor is associated with suppression of neuronal electrical activity, while the MT 2 receptor induces a phase shift of neuronal firing. Moreover, the MT 1 receptor inhibits prolactine secretion and induces vasoconstriction, whereas the MT 2 receptor activation promotes vasodilation and modulation of the immune system.…”

Section: Introductionmentioning

confidence: 99%

N‐(Anilinoethyl)amides: Design and Synthesis of Metabolically Stable, Selective Melatonin Receptor Ligands

et al. 2009

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The class of N-(anilinoethyl)amides includes melatonin receptor ligands with varied subtype selectivity and intrinsic activity. One of these ligands, the MT(2)-selective partial agonist UCM765 (N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide), had evidenced hypnotic effects in rodents at doses > or =40 mg kg(-1) (s.c.), in spite of its sub-nanomolar affinity for human melatonin receptors. Supposing that its low in vivo potency could be due, at least in part, to metabolic liability in rat liver, UCM765 was incubated with rat liver S9 fraction and rat, mouse, or human microsomes, and the major metabolites were identified by LC-MS, synthesized, and in vitro tested for their affinity toward MT(1) and MT(2) receptors. The obtained information was exploited to design novel analogues of UCM765 that are more resistant to in vitro oxidative degradation, while maintaining a similar binding profile. The analogue UCM924 (N-{2-[(3-bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide) displayed a binding profile similar to that of UCM765 on cloned human receptors (MT(2)-selective partial agonist) and a significantly longer half-life in the presence of rat liver S9 fraction.

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“…In the post-genomic era, the ORF regions of rat membrane melatonin receptors can be deduced from the rat genome. Recently, pharmacological properties of the receptors were characterized using genomically identified ORF sequences [10]. However, genomically identified sequences lack information about UTRs that contain the signal motifs involved in posttranscriptional regulation and mRNA decay [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Gene structures, biochemical characterization and distribution of rat melatonin receptors

et al. 2008

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G-protein coupled receptors for the pineal hormone melatonin have been partially cloned from rats. However, insufficient information about their cDNA sequences has hindered studies of their distribution and physiological responses to melatonin using rats as an animal model. We have cloned cDNAs of two rat membrane melatonin receptor subtypes, melatonin receptor 1a (MT1) and melatonin receptor 1b (MT2), using a rapid amplification of cDNA end (RACE) method. The rat MT1 and MT2 cDNAs encode proteins of 353 and 364 amino acids, respectively, and show 78-93% identities with the human and mouse counterparts. Stable expression of either rat MT1 or MT2 in NIH3T3 cells resulted in high affinity 2-[ 125 I]-iodomelatonin ( 125 I-Mel) binding (K d = 73.2 ± 9.0 and 73.7 ± 2.9 pM, respectively), and exhibited a similar rank order of inhibition of specific 125 I-Mel binding by five ligands (2-iodomelatonin [ melatonin [ 6-hydroxymelatonin [ luzindole [ N-acetyl-5-hydroxytryptamine). RT-PCR analysis showed that MT1 is highly expressed in the hypothalamus, lung, kidney, adrenal gland, stomach, and ovary, while MT2 is highly expressed in the hippocampus, kidney, and ovary. We also performed multi-cell RT-PCR to examine the expression of mRNAs encoding MT1 and MT2 in adult GnRH neurons. MT1 was weakly expressed in male GnRH neurons, and was less expressed in the female neurons. MT2 expression was undetectable in GnRH neurons from either sex. This study delineates the gene structures, fundamental properties, and distribution of both rat melatonin receptor subtypes, and may offer opportunities to assess the physiological significance of melatonin in rats.

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Molecular cloning and pharmacological characterization of rat melatonin MT1 and MT2 receptors

Cited by 43 publications

References 35 publications

Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors

Alternative Radioligands for Investigating the Molecular Pharmacology of Melatonin Receptors

N‐(Anilinoethyl)amides: Design and Synthesis of Metabolically Stable, Selective Melatonin Receptor Ligands

Gene structures, biochemical characterization and distribution of rat melatonin receptors

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