Pretreat ment with physiological concentrations of melatonin resulted in a supersensitization of human ml_1a melatonin receptor signalling in stably transfected cho cells

The hormone melatonin, released from the pineal gland in a arcadian fashion, regulates many biological processes including circadian rhythms through activation of melatonin receptors. In humans, circulating levels of melatonin are highest during the night (80 pg/ml) and lowest during the day (10 pg/ml). Here, we report the effects of night levels of melatonin on CHO cells stably transfected with the human ML_1A melatonin receptor (ML_1A - CHO). Cells maintained in a serum-free media were exposed to melatonin (93 pg/ml) for 8h followed by different wash periods up to 16h. This treatment resulted in increases in both 2-[¹²⁵/]iodomelatonin binding (160% of control-untreated cells) and in forskolin-induced cAMP formation (200% of control) in a timedependent manner following removal of melatonin. To determine one of the potential mechanisms underlying such a phenomenon, G-protein levels were assessed by ADP ribosylation studies. Under the conditions previously described, pertussis toxin labelling of G-proteins was increased whereas cholera labelling of G-proteins was unchanged. We conclude that melatonin pretreatment of (ML_1A - CHO) cells increased 2- [¹²⁵/]iodomelatonin binding by increasing the heterotrimeric state of Gi proteins. This decrease in the dissociated state of Gi would remove a tonic inhibition on adenylyl cyclase, thus enhancing forskolin-stimulated cAMP formation. Supported by MH 42922 to MLD and F32 HL08965 to PAW-E.