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TN Ivanova, PM Iuvone; Development of Circadian Rhythms of Serotonin N-acetyltransferase Activity in Photoreceptor-enriched Cell Culture of Chick Embryo Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1357.
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Purpose: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is a key regulatory enzyme in melatonin biosynthesis. In chicken retina in vivo, AANAT mRNA and activity exhibit circadian rhythms with peaks at night. During in vivo development, daily fluctuations of AANAT activity are not observed until embryonic day 20, just prior to hatching. The purpose of the present study was to investigate the temporal development of light/dark oscillations and circadian clock regulation of AANAT activity in cultured retinal cells prepared from 6 and 8 day old embryos (E6, E8, respectively). Methods: The monolayer cultures were prepared from neural retinas of E6 and E8 chick embryos by a modification of the method of Adler et al. (1984). Cells were cultured for 5-14 days with 14 h light/ 10 h dark cycle. Culture medium was exchanged for first time on the 4th day in vitro, and every 2 days thereafter. AANAT activity was determined as described by Thomas et al (1990). The reaction product, N-acetyltryptamine, was determined by HPLC with fluorescence detection. Results: Photoreceptor-enriched cell cultures, prepared from E6 retinas and cultured under an LD cycle for 5-6 days, displayed prominent daily fluctuations on days 5 and 6 in vitro. Cultured under LD, activity was high at night and low during daytime, as it is in posthatch retina. E6 cells, cultured similarly for 5 days under LD followed by 2 days in DD failed to exhibit circadian fluctuations of AANAT activity in DD. However, a circadian rhythm of AANAT activity was observed in E6 cells cultured for 8 days in LD followed by 2 days in DD. A 2h light pulse in the middle of the night of the second day of DD suppressed AANAT activity, indicating that the enzyme activity in the cultured cells is acutely suppressed by light, as it is in posthatch chick retinas. A circadian fluctuation of AANAT was also observed in cells prepared from E8 retinas and cultured for 5 days in LD followed by 2 days in DD. Conclusion: Light-dark and circadian regulation of AANAT is an intrinsic property of retinal cells and develops more rapidly in vitro than in ovo. Light-dark regulation of AANAT activity appears to precede circadian clock control of enzyme activity.
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