title: Physiological exposure to melatonin supersensitizes the cyclic adenosine 3',5'-monophosphate-dependent signal transduction cascade in Chinese hamster ovary cells expressing the human mt1 melatonin receptor.
abstract: Here, we report the effects of short exposure to melatonin on the human mt1 (h mt1) melatonin receptor-mediated signaling in Chinese hamster ovary (CHO) cells, and the consequences of an exposure that resembles the physiological pattern of melatonin release on cAMP-mediated signal transduction. Short exposure (10 min) of h mt1 melatonin receptors to melatonin (400 pM) inhibited forskolin-stimulated cAMP formation, cAMP-dependent protein kinase activity, and phosphorylation of the cAMP response element-binding protein. However, treatment of mt1-CHO cells with melatonin in a manner that closely mimics the in vivo activation of melatonin receptors (i.e. 400 pM melatonin for 8 h to mimic darkness) resulted in a supersensitization of the cAMP-dependent signal transduction cascade during the period of withdrawal (i.e. 16 h without melatonin to mimic the light cycle of a diurnal photoperiod). During the period of withdrawal, forskolin induced a time-dependent (1-16 h) increase in cAMP formation (approximately 200% of control cells). This effect of melatonin was dependent on the presence of the h mt1 melatonin receptor, as no potentiation of forskolin-induced cAMP formation was observed in CHO cells transfected only with the neomycin resistance plasmid. The time-dependent increase in forskolin-stimulated cAMP levels resulted in a potentiation of cAMP-dependent protein kinase activity 1 h after withdrawal (approximately 130% of control cells; P < 0.05) and in the number of cells containing the phosphorylated form of cAMP response element-binding protein (approximately 75% of cells at 1 and 16 h compared with 30% in control cells; P < 0.05). An increase in the undissociated state (G alphabetagamma) of Gi proteins may underlie this phenomenon as demonstrated by the increase in pertussis toxin-catalyzed ADP-ribosylation of G proteins (217 +/- 48% of control; P < 0.05) after melatonin withdrawal. This increase in the ribosylation was not due to an up-regulation of Galpha(i) protein, as no significant change in Galpha(i) protein levels occurred at this time. We demonstrated that activation of the h mt1 melatonin receptor in a manner that resembles the physiological pattern of melatonin exposure alters signaling, as potentiation of cAMP-mediated signal transduction events is observed after hormone withdrawal. The CHO cells expressing the human melatonin receptor may provide an in vitro cellular model in which to investigate the putative signaling mechanisms leading to gene regulation by melatonin.
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Witt-Enderby
authors.forename: M I
authors.initials: MI
authors.lastname: Masana
authors.forename: M L
authors.initials: ML
authors.lastname: Dubocovich
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008550:Melatonin / Q000008:administration & dosage / Q000494:pharmacology*; D037342:Pertussis Toxin; D010766:Phosphorylation / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D044094:Receptors, Melatonin; D012266:Ribose / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology*; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D012266:Ribose; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D008550:Melatonin