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P.M. Iuvone, S.S. Chaurasia, T.N. Ivanova, J.H. Wessel III, R. Haque; Circadian rhythms of Ca2+/calmodulin–stimulated adenylyl cyclase mRNA expression and CREB phosphorylation in chicken retina . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4642.
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Purpose: Photoreceptor–enriched chick retinal cell cultures, entrained to a daily light–dark (LD) cycle, exhibit a circadian rhythm of cAMP level with highest levels at night (Ivanova and Iuvone, Brain Res. 991: 96, 2003). Depolarization–evoked cAMP synthesis in these cell cultures is Ca2+ dependent (Iuvone et al., J. Neurochem. 57: 615,1991), apparently involving Ca2+/calmodulin–stimulated adenylyl cyclase. The current study was conducted to determine if Ca2+/calmodulin–stimulated adenylyl cyclase transcript expression is circadian and if the rhythm of cAMP correlates with a rhythm of phosphorylation of the cAMP response element binding protein (CREB). Methods: Photoreceptor–enriched retinal cell cultures were prepared from embryonic day 6 chick neural retina and incubated under LD for 8 days. For in vivo experiments, one day–old chicks were entrained to LD for two weeks before use. The photoreceptor layer was isolated from frozen sections of retina by laser capture microdissection (LCM). Transcript levels for two Ca2+/calmodulin–stimulated adenylyl cyclases, AC1 and AC8, were measured by quantitative real–time RT–PCR. CREB phosphorylation was assessed by western blot analysis with pan and phospho–specific CREB antibodies. Results: In cell cultures exposed to LD, AC1 and AC8 transcripts were rhythmically expressed with peak levels during the daytime. These rhythms persisted for at least 1 day in constant darkness (DD), but the rhythm of AC8 was damped relative to that in LD. In vivo, retinal AC1 mRNA levels exhibited a circadian rhythm, with peak levels late in the daytime. The rhythmic expression of AC1 mRNA was also observed in photoreceptor cells isolated by LCM. CREB phosphorylation was high at night, compared to daytime, in LD and DD. Conclusions: AC1 mRNA is expressed as a daily rhythm that peaks late in the day. Given the temporal delay associated with translation and processing of the membrane bound enzyme, the AC1 rhythm could contribute to the circadian rhythm of cAMP, which peaks at night. CREB phosphorylation is also circadian and correlates temporally with the rhythm of cAMP. These rhythms could contribute to daily rhythms of melatonin synthesis and gene expression in photoreceptor cells.
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