Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells

doi:10.1016/j.brainres.2003.08.003

Brain Research

Volume 991, Issues 1–2, 21 November 2003, Pages 96-103

https://doi.org/10.1016/j.brainres.2003.08.003 Get rights and content

Abstract

Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway. In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in photoreceptor cells. AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night. In the present study, we investigated the circadian and photic regulation of adenosine 3′,5′-monophosphate (cAMP) in cultured retinal cells entrained to a daily light–dark (LD) cycle, as well as the role of Ca²⁺ and cAMP in the regulation of AANAT activity. Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light. This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD). These changes in cAMP appear to be causally related to control of AANAT activity. Adenylyl cyclase and protein kinase A inhibitors suppress the noctural increase of AANAT in DD, while 8Br-cAMP augments it. The nocturnal increase of AANAT activity also involves Ca²⁺ influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca²⁺ channel agonist. The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca²⁺ influx, cAMP formation, and AANAT activity in retinal cells. Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels. The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin. These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in photoreceptor cells.

Introduction

Melatonin is an internal regulator in the timing of circadian physiology in vertebrates [8]. Melatonin is produced in the pineal gland [41] and in retinal photoreceptor cells [7], [23], [34]. A key regulatory enzyme in melatonin synthesis is arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase; AANAT; EC 2.3.1.87), which plays a unique “time-keeping role as the molecular interface between the environment and the hormonal signaling of time” [22]. In chicken retina, AANAT expression in photoreceptor cells exhibits daily fluctuations with high levels at night in the dark and low levels during the day [6], [35]. The daily fluctuations of AANAT mRNA and activity are driven by an endogenous circadian oscillator and by light [6], [27], [34], but the molecular mechanisms coupling circadian oscillators and light to changes of AANAT activity in photoreceptor cells are incompletely understood.

Previous studies on photoreceptor-enriched chick embryo retinal cell cultures demonstrated that Ca²⁺ influx and elevated intracellular adenosine 3′,5′-monophosphate (cAMP) levels stimulate AANAT activity [3], [20], [25], [31]. However, these studies were conducted on immature cell cultures under conditions that do not support light-driven and circadian changes of AANAT activity and the role of these second messengers in the physiological regulation of AANAT activity in chick photoreceptor cells has remained unexplored. A recent report described culture conditions that support the photic regulation of AANAT activity [29]. In a further refinement of these conditions, we found that photoreceptor-enriched cell cultures, exposed to a daily light–dark cycle for 8 days, exhibit circadian and photic regulation of AANAT activity, recapitulating regulation in vivo [36]. The purpose of the present study was to investigate the regulation of cAMP level in retinal cultures entrained to a light–dark cycle and to examine the potential roles of intracellular cAMP and Ca²⁺ in the regulation of AANAT activity under these conditions.

Section snippets

Cell preparation and culture

Monolayer cultures of retinal cells were prepared from neural retinas of 6-day-old chicken embryos (E6) and incubated by a modification [36] of the method of the Adler et al. [1]. Neural retinas were dispersed in 0.25% trypsin and cells (∼3.4×10⁶ cells) were plated on 35-mm Primaria™ culture plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) in medium 199 containing 20 mM HEPES, linoleic acid–BSA 110 μg/ml, 2 mM glutamine, penicillin G (100 U/ml), and 10% fetal bovine serum. Cells were

Circadian rhythm of cAMP level in cultured retinal cells

As illustrated in Fig. 1, photoreceptor-enriched cell cultures incubated under a daily light–dark (LD) cycle displayed prominent daily fluctuations of cAMP, peaking at night on DIV 8 (ZT 10 vs. ZT 20, p<0.001). Similar cultures, incubated for an additional 36 h under constant (24 h/day) darkness (DD), displayed circadian fluctuations of cAMP level (ZT 10 vs. ZT 20, p=0.033) with reduced amplitude compared to that in LD. The cAMP levels during the daytime under DD on DIV 9 and 10 were

Discussion

The principal discovery reported in this manuscript is that cAMP levels in photoreceptor-enriched retinal cell cultures are controlled in a circadian fashion that, in turn, influences the circadian and photic control of AANAT activity. cAMP level is high at night and low during the daytime in cells exposed to a daily LD cycle. This day–night difference in cAMP level persists in constant darkness, indicative of circadian control.

The biosynthesis of melatonin in photoreceptor cells is regulated

Acknowledgements

The authors thank Dr. Rashidul Haque for advice on cAMP measurements and James Wessel for laboratory assistance. This research was supported by a grant from the National Institutes of Health EY04864. A preliminary report of some of these data was presented at the 2003 Meeting of the Association for Research in Vision and Ophthalmology.

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¹: Current address: Department of Cell Biology, Emory University, Atlanta, GA 30322, USA.

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Research reportCircadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells

Abstract

Introduction

Section snippets

Cell preparation and culture

Circadian rhythm of cAMP level in cultured retinal cells

Discussion

Acknowledgements

Neurochem. Int.

J. Biol. Chem.

Prog. Retin. Eye Res.

TINS

J. Biol. Chem.

Exp. Eye Res.

Neurochem. Int.

Biophys. J.

Brain Res.

Brain Res.

Neuron

J. Biol. Chem.

Neuron

Neuron

Anal. Biochem.

Brain Res.

Brain Res.

Expression of cone-like properties by chick embryo retina cells in glial-free monolayer cultures

J. Cell Biol.

Melatonin biosynthesis in cultured chick retinal photoreceptor cells: calcium and cyclic AMP protect serotonin N-acetyltransferase from inactivation in cycloheximide-treated cells

J. Neurochem.

Ionic channels of the inner segment of tiger salamander cone photoreceptors

J. Gen. Physiol.

Avian melatonin synthesis: photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina

J. Neurochem.

DNA elements of the type 1 adenylyl cyclase gene locus enhance reporter gene expression in neurons and pinealocytes

Eur. J. Neurosci.

Increased levels of 3′,5′-cyclic adenosine monophosphate induced by cobaltous ion or 3-isobutylmethylxanthine in the incubated mouse retina: evidence concerning location and response to ions and light

J. Neurochem.

Tryptamine and some related molecules block the accumulation of a light-sensitive pool of cyclic AMP in the dark-adapted, dark-incubated mouse retina

J. Neurochem.

Inositol-1,4,5-trisphosphate receptors in the vertebrate retina

Curr. Eye Res.

Cyclic nucleotide levels in normal and biologically fractionated mouse retina: effects of light and dark adaptation

J. Neurochem.

Circadian regulation of vertebrate photoreceptors: rhythms in the gating of cationic channels

A cationic channel regulated by a vertebrate intrinsic circadian oscillator

Nature

Calcium spikes in toad rods

J. Physiol. (Lond.)

Research report
Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells