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C.P. Ribelayga, S.C. Mangel; Circadian Clock Control of Fish Cone Horizontal Cell Light Responses: Characterization of the Adenosine Clock Pathway . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2312.
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In the fish retina, a circadian clock regulates cone horizontal cell (HC) light responses so that cone input dominates during the day and rod input dominates at night (Wang and Mangel, 1996). To achieve this effect, the clock uses several outputs, including adenosine. Specifically, the effects of the retinal clock at night are mediated in part by an increase in the extracellular level of adenosine and the subsequent activation of A2, but not A1 adenosine receptors. We therefore analyzed the means by which the clock controls extracellular adenosine.
Pharmacological agents were applied to superfused goldfish neural retinas at different times of the circadian cycle. Adenosine in the superfusate or in the retinal homogenate was quantified using reversed–phase HPLC with fluorescence detection.
Adenosine is synthesized extracellularly from the conversion of ATP and AMP via 5’–nucleotidase and/or intracellularly from AMP. The relative concentration of adenosine between the intra– and extracellular compartments sets the direction and intensity of the transmembrane flux of adenosine and thus the level of extracellular adenosine. Application of NBTI, an adenosine transport blocker, during the subjective day increased the level of extracellular adenosine, a finding consistent with a flux of adenosine directed from the extracellular space toward the intracellular compartment. During the subjective night, NBTI had little effect on extracellular adenosine but did not decrease it, thus indicating that the flux is always directed inward regardless of the phase of the circadian cycle. Application of GMP, a 5’–nucleotidase blocker, in the presence of NBTI during subjective day and night, decreased adenosine levels to an undetectable value, thus confirming the extracellular origin of extracellular adenosine. In addition, adenosine content was higher during the subjective night compared to the subjective day, thus indicating that it is under clock control. Finally, 30 min application of melatonin or spiperone plus SCH23390 during the subjective day, or luzindole or dopamine during the night, had no effect on extracellular adenosine.
Our observations show that the clock increases intracellular adenosine at night. The resultant decrease in the inwardly–directed flux of adenosine enhances the extracellular accumulation of adenosine and the activation of adenosine A2 receptors. This latter event contributes to the rod dominance of HCs at night. Our results also suggest that the adenosine and the melatonin/dopamine systems constitute separate clock pathways in the retina.
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