Are Dopamine D4 Receptors Essential for Circadian Regulation of Ca2+/CaM-Stimulated Adenylyl Cyclases in Mouse Retina?

Abstract

Purpose:: In retina and photoreceptor cell cultures, cAMP is regulated in a circadian fashion, with high levels at night. Dopamine D4 receptors and light have been shown previously to regulate the same pool of cAMP in photoreceptor cells. The present study was conducted to investigate the role of dopamine D4 receptors in regulating the expression of Ca²⁺/calmodulin (CaM)-stimulated adenylyl cyclases in mouse retina.

Methods:: C57Bl/6 mice and dopamine D4 receptor null (Drd4^-/-) mice on a C57Bl/6 background were entrained to a 12h light / 12h dark cycle (LD) and subsequently kept in constant (24 h / day) darkness (DD) for two days. Retinas were dissected at various times on the last day of LD and the second day of DD. Transcripts encoding ADCY1 and ADCY8 were quantified by real-time RT-PCR. Adenylyl cyclase activity in retinal membrane preparations was determined by measuring the conversion of [α-³²P]ATP to [³²P]cAMP. ADCY8 protein was measured in retinal membrane fractions by Western blotting.

Results:: The levels of Adcy1 and Adcy8 transcripts in retinas exposed to LD are relatively low during the night and increase during the early daytime, peaking ~ 0-4 hours after light onset in the morning. Adcy1 and Adcy8 transcript levels continue to vary in a rhythmic pattern on the second day of DD, indicative of circadian clock control. A circadian rhythm of Ca²⁺/CaM- stimulated adenylyl cyclase activity was observed in mouse retina, with high activity at night and low activity during the day. ADCY8 protein levels also showed a daily pattern of expression with high levels at night. The circadian rhythms of Adcy1 and Adcy8 mRNA levels and Ca²⁺/CaM- stimulated adenylyl cyclase activity observed in the wild type mouse retina was absent from retinas of Drd4^-/- mice. Transcript and activity levels were low and arrhythmic in Drd4^-/- retinas. Daily subcutaneous injections of L745870, a D4 receptor antagonist, for 1 week abolished the circadian expression of Adcy1 and Adcy8 mRNAs in retinas of wild type mice.

Conclusions:: Circadian clocks in mouse retina control expression of mRNAs encoding the Ca²⁺/CaM-stimulated adenylyl cyclases. Activation of dopamine D4 receptors appears to be essential for circadian expression of the Ca²⁺-stimulated cAMP regulatory system in retina.

Keywords: circadian rhythms • retina • dopamine