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JS Dalal, B-A Battelle; Circadian Regulation of Visual Arrestin mRNA Levels: A Role for cAMP and PKA . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1368.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Circadian rhythms impact many retinal functions including gene expression in photoreceptors. Our purpose was to examine the mechanisms underlying the circadian regulation of the arrestin gene. Visual arrestin (Varr) mRNA levels in Limulus lateral eye (LE) fall during the subjective night in response to activation of an octopaminergic circadian neural input from the brain. Octopamine (OCT) increases cAMP levels in photoreceptors. We tested whether the fall in varr mRNA can be caused by the OCT- stimulated rise in cAMP and activation of cAMP-dependent protein kinase (PKA). Methods: Varr mRNA levels in control and experimental LEs from the same animal were compared by Northern Blot analyses using 18S ribosomal RNA as the loading control. LEs were dissected from animals during the late afternoon and incubated overnight in organ culture medium. The next morning control eyes were incubated in the phosphodiesterase inhibitor IBMX (1mM); experimental eyes were incubated in one of the following: 40mM octopamine (OCT), 40 mM OCT + IBMX, 10mM 8-bromo cAMP + IBMX, 8-bromo cAMP + IBMX + 30mM H-89 a PKA inhibitor. Results: In eyes incubated for 4 hrs with OCT alone there was a small (15%) reduction in varr mRNA; in eyes incubated with OCT or 8-bromo cAMP + IBMX there was a 50% reduction. The reduction in varr mRNA produced at 4 hrs by 8-bromo cAMP + IBMX was blocked by the addition of the PKA inhibitor H-89. In other experiments, incubation for 2hr with actinomycin D alone (25ug/ml), a potent inhibitor of transcription, produced a 50% decrease in the varr mRNA levels. Conclusion: Varr mRNA in Limulus LEs has a relatively short half-life. The fall in varr mRNA in these eyes during the subjective night,which is driven by circadian efferent input, can be mimicked by OCT and cAMP. The activation of PKA also appears to be involved. Experiments in progress will determine whether this reduction in varr mRNA levels is produced by a decrease in transcription or the degradation of transcripts.
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