Abstract
Abstract: :
Purpose: Cyclic GMP-gated cationic channels (CNGCs) are essential for phototransduction in the vertebrate retina. In chick retinal cones, the affinity of CNGCs for cGMP is under circadian control, and is higher during the subjective night than during the subjective day. This effect persists on the second day of constant darkness (DD) after entrainment to 12:12 hr LD cycles in cells maintained either in vivo or in vitro. Circadian modulation of cGMP affinity is driven by rhythms in the activity of the MAP kinase Erk, which is maximally active during the subjective night. Here we examine possible steps that intervene between the photoreceptor circadian oscillator and Erk. Methods: Properties of CNGCs were determined by inside-out patch clamp recordings from cultured chick retinal photoreceptors on the second day of DD after LD 12:12 entrainment in vivo or in vitro. In vitro entrainment occurred in a culture chamber equipped with lights and timers. Embryonic chick cones were dissociated at E6, which results in a highly enriched population of cones, which contain large oil droplets and rudimentary outer segments. Other endpoints were based on immunochemical assays of Erk and Ras activation. Results: Application of the cAMP protagonist forskolin, or the cAMP analog 8CPT-cAMP caused a significant (p < 0.05) increase in cGMP affinity of CNGCs when they are applied to cones during the subjective day (CT4-7) but not during the subjective night (CT16-19). Forskolin also stimulates cone Erk activity. This effect is greater during the subjective day than the subjective night. Conversely, the adenylate cyclase inhibitor MDL12330A caused a decrease in CNGC affinity (p < 0.05) when applied at CT16-19, but had no effect at CT4-7. Dopamine and the D2 agonist quinpirole, which also inhibit adenylate cyclase in cones, caused a similar effect. A second potential mechanism for Erk activation entails small GTPases such as Ras, Rac, and Rap. Ras, in particular, causes activation of the protein kinase Raf, leading to activation of MEK1 and stimulation of Erk. We have observed that Ras activity in cone photoreceptors is much greater during the subjective night (CT16-22) than the subjective day (CT4-10) in chick cones free-running on the second day of DD. Conclusion: Cyclic AMP and Ras may contribute to circadian control of the MAP kinase Erk, and modulation of cGMP-gated channels of cones.
Keywords: 445 ion channels • 517 photoreceptors • 349 circadian rhythms