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T.N. Ivanova, P.M. Iuvone; Circadian and Photic Regulation of cAMP: A Mechanism for Coupling the Circadian Oscillator to Melatonin Synthesis in Cultured Chicken Photoreceptor Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2870.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Melatonin biosynthesis in chick retina is controlled by an endogenous circadian oscillator that regulates the expression and activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin biosynthetic pathway. AANAT activity is high at night in darkness and low during the daytime. Light inhibits the circadian rhythm of AANAT activity by transcriptional and post-transcriptional mechanisms. Circadian and photic regulation of AANAT activity is an intrinsic property of retinal cells that develops in vitro in cultured photoreceptor cells. The aim of the present study was to investigate the role of cAMP in the circadian and photic regulation of AANAT activity. Methods: Retinal cells were prepared from 6-day-old chicken embryos and cultured under a 14 h light and 10 h dark (LD) cycle for 8 days, followed by exposure constant darkness (DD). AANAT activity and cAMP levels were measured in cell homogenates, day and night in LD and DD. Results: cAMP levels in cultured cells were higher at night than during the day in LD and DD conditions. AANAT activity fluctuated with a similar daily pattern. The adenylyl cyclase inhibitor, MDL12330A, and PKA inhibitors, Rp-cAMPS and H89, reduced the nocturnal increase of AANAT activity, supporting a causal link between the rhythms of cAMP level and AANAT activity. Nitrendipine, a Ca2+ channel blocker, inhibited the nocturnal increase of AANAT activity in LD and DD. In contrast, Bay K 8644, a Ca2+ channel agonist, enhanced AANAT activity in LD and DD. The effect of Bay K 8644 was blocked the adenylyl cyclase inhibitor, MDL 12330A, suggesting that the effect of the channel activator is mediated by Ca2+-dependent stimulation of cAMP formation. Light exposure (2h) in the middle of the night under DD reduced both cAMP level and AANAT activity. The effect of light on AANAT activity was antagonized by 8Br-cAMP, a cell permeable analogue of cAMP, and by lactacystin, a selective inhibitor of proteasomal proteolysis. Conclusions: A retinal circadian oscillator and light control the rhythmic synthesis of cAMP, which in turn stimulates AANAT activity. The circadian control of cAMP appears to be one of the mechanisms for coupling the circadian oscillator to rhythmic melatonin production by photoreceptor cells. Light exposure suppresses AANAT activity at night by reducing cAMP, leading to the proteasomal degradation of the enzyme.
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