Binding assays
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Cells stably expressing MT1 or MT2 receptors (CHO-hMelR7) were selected and cultured in Eagle’s Minimum Essential Medium-α (MEM-α) supplemented with 10% dialyzed fetal bovine serum (dFBS) under a 5% CO2/95% air atmosphere. Cells were harvested at confluence in Ca2+-Mg2+ free Hanks’ balanced salt solution containing 5 mM EDTA and collected by centrifugation. Cells were homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.7 at 25℃), washed twice, pelleted, and stored at -30℃ until the binding assays were conducted. Ramelteon and 40 pM 2-[125I]melatonin were mixed with the thawed homogenate in a total volume of 1 mL and incubated at 25℃ for 150 min. The reaction was terminated by addition of 3 mL of icecold buffer followed by vacuum filtration on a Whatman GF/B. The filter was washed twice and radioactivity was counted by a γ-counter. Nonspecific binding was defined as the binding in the presence of 10 mM melatonin.
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Applications
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In cats, ramelteon has a sleep-promoting action and does not appear to cause learning, memory, or motor function impairment, or to have rewarding properties [4]. In a clinical study, ramelteon decreases latency to persistent sleep and increases total sleep time and sleep efficiency in subjects with primary chronic insomnia [1].
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References:
[1]. Kato K, Hirai K, Nishiyama K, et al. Neurochemical properties of ramelteon (TAK-375), a selective MT1/MT2 receptor agonist. Neuropharmacology, 2005, 48(2): 301-310.
[2]. Nishiyama K, Hirai K. The melatonin agonist ramelteon induces duration-dependent clock gene expression through cAMP signaling in pancreatic INS-1 β-cells. PLoS One, 2014, 9(7): e102073.
[3]. Imbesi M, Uz T, Dzitoyeva S, et al. Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells. Neurosci Lett, 2008, 439(1): 34-36.
[4]. Miyamoto M, Nishikawa H, Doken Y, et al. The sleep-promoting action of ramelteon (TAK-375) in freely moving cats. Sleep, 2004, 27(7): 1319-1325.
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