Gating of the cAMP Signaling Cascade by the Circadian Clock in Mammalian Retina

Abstract

Abstract: : Purpose: Melatonin is synthesized in retinal photoreceptor cells and acts as a neuromodulator imparting photoperiodic information to the retina. The synthesis of melatonin is controlled by an ocular circadian clock and by light and darkness in a finely tuned mechanism that insures that melatonin is synthesized and acts only at night in darkness. The aim of this study was to identify the circadian gating mechanism(s) responsible for the time-of-day-dependent stimulation of melatonin biosynthesis by darkness. Methods: Rat retinas were cultured in a flow-through apparatus for 24-hours and then exposed to a period of darkness (6 hours) beginning at different times of the day (ZT0-12). Melatonin levels in the medium were measured by radioimmunoassay (RIA). mRNA levels of type 1 adenylyl cyclase (AC1) were measured by real-time quantitative RT-PCR and cAMP levels were measured by RIA. Results: Light suppresses melatonin biosynthesis at all times of day. When retinas were exposed to darkness for 6 h at different times of the day, induction of melatonin synthesis was observed with incubations ending at ZT 12, 15, and 18, but not at ZT 6 or 9, suggesting that the induction of melatonin synthesis in darkness is gated by the circadian clock. However, when the retinas were exposed to 6-h of darkness with 0.5 µM of 8p-CPT-cAMP, melatonin release was significantly increased at all times tested. This result demonstrates that the mechanisms gating the synthesis of melatonin during the day are located upstream of PKA, possibly at the level of adenylyl cyclase (AC). The expression of AC1 mRNA and the levels of cAMP in rat retina fluctuate as circadian rhythms, indicating that the circadian clock modulates the cAMP signaling cascade. This effect was localized to photoreceptor cells by demonstrating a robust AC1 transcript rhythm in laser capture microdissected inner segments and outer nuclear layer. The clock gene products BMAL1 and CLOCK activate an AC1 promoter-luciferase reporter in transfected retinal cells via interaction with an E-box enhancer in the proximal promoter. Conclusions: A circadian clock in the retina gates melatonin synthesis in part by regulating the expression of AC1 and the synthesis of cAMP in photoreceptor cells. This gating is effected through E box-mediated transcriptional activation of the AC1 gene. The circadian control of the cAMP signaling cascade indicates that the clock has a more general and profound impact on photoreceptor functions than previously thought.

Keywords: circadian rhythms • melatonin • photoreceptors