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P.M. Iuvone, S.S. Chaurasia, J. Bogenpohl, T. Ivanova, N. Pozdeyev, A. Visser, R. Haque; Rhythmic Melatonin Synthesis and Circadian Clock Gene Expression in Cultured Chicken Retinal Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3991.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Circadian clocks in chicken retina regulate melatonin biosynthesis, iodopsin expression, cFos expression, cAMP synthesis, and a variety of other biochemical and physiological processes in photoreceptor cells and inner retinal neurons. Circadian clocks in many systems consist of self–sustaining transcriptional–translational feedback loops, but little is known about the organization of the core molecular clockwork driving circadian rhythms in chicken retinal photoreceptor cells. The present study was conducted to investigate the temporal characteristics of melatonin synthesis and circadian clock gene expression in photoreceptor–enriched chick retinal cell cultures entrained to a daily light–dark cycle. Methods: Retinal cells were prepared from 6–day–old chick embryos and cultured under a 14 h light : 10 h dark (LD) daily cycle of illumination for at least 8 days prior to sampling. Melatonin release in the culture medium was measured by HPLC with fluorescence detection. Transcripts for arylalkylamine N–acetyltransferase (aanat), bmal1, mop4, cry1, per2, and clock were measured by quantitative real–time RT–PCR. Levels of these transcripts were normalized to those of the house–keeping gene hypoxanthine–guanine phosphoribosyltransferase. Results: In cultures exposed to the LD cycle of illumination, melatonin release and the abundance of aanat mRNA, a key regulatory enzyme in melatonin biosynthesis, were low during the daytime and increased in the darkness, peaking late in the night. Transcripts of clock genes were also rhythmically expressed. Bmal1 and mop4 were low in the morning and the first half of the day, peaking near the transition time from light to darkness, and then decreased late in the night. Clock mRNA was low during the daytime and high at night. Cry1 and per2 transcripts increased rapidly after light onset in the morning and were low at night. All of these transcripts, except clock, continued to cycle through at least 2 days in constant darkness, although the rhythm of per2 was damped. Conclusions: Previous studies have shown that the aanat gene promoter contains an E–box that is activated by BMAL/CLOCK and BMAL/MOP4 heterodimers. CRY1 and PER2 block the effects of these heterodimers. The temporal pattern of expression of the clock genes is consistent with a role for BMAL/CLOCK or BMAL/MOP4 in the activation of aanat transcription at night and for CRY1 and PER2 in the inhibition of aanat transcription during the daytime.
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