title: Pharmacological characterization of human recombinant melatonin mt(1) and MT(2) receptors.
abstract: We have pharmacologically characterized recombinant human mt(1) and MT(2) receptors, stably expressed in Chinese hamster ovary cells (CHO-mt(1) and CHO-MT(2)), by measurement of [(3)H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt(1) and MT(2) receptors with pK(D) values of 9.89 and 9.56 and B(max) values of 1.20 and 0.82 pmol mg(-1) protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt(1) and MT(2) receptors, a number of putative antagonists had substantially higher affinities for MT(2) receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt(1) and CHO-MT(2) cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC(50) 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT(2) receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt(1) receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt(1) and CHO-MT(2) cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA(2) values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of G(i)/G(o) G-proteins. This is the first report of the use of [(3)H]-melatonin for the characterization of recombinant mt(1) and MT(2) receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.
authors.affiliation: Receptor Pharmacology Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts, SG1 2NY.
authors.forename: C
authors.initials: C
authors.lastname: Browning
authors.forename: I
authors.initials: I
authors.lastname: Beresford
authors.forename: N
authors.initials: N
authors.lastname: Fraser
authors.forename: H
authors.initials: H
authors.lastname: Giles
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D007553:Isotope Labeling; D007700:Kinetics; D008550:Melatonin / Q000031:analogs & derivatives / Q000378:metabolism; D037342:Pertussis Toxin; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044094:Receptors, Melatonin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D005576:Colforsin; C044556:2-iodomelatonin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D008550:Melatonin