Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

Differential regulation of arylalkylamine N-acetyltransferase activity in chicken retinal ganglion cells by light and circadian clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48?h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30-60?min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar ).     

Inhibitory modulation of photoreceptor melatonin synthesis via a nitric oxide-mediated mechanism

Nitric oxide (NO) has been suggested to have many physiological functions in the vertebrate retina, including a role in light-adaptive processes. The aim of this study was to examine the influence of the NO-donor sodium nitroprusside (SNP) on the activity of arylalkylamine-N-acetyltransferase (AA-NAT; EC. 2.3.1.87), the activity of which responds to light and reflects the changes in retinal melatonin synthesis—a key feature of light-adaptive responses in photoreceptors. Incubation of dark-adapted and dark-maintained retinas with SNP lead to the NO-specific suppression of AA-NAT activity, with NO suppressing AA-NAT activity to a level similar to that seen in the presence of dopaminergic agonists or light. Increased levels of cGMP appeared to be causally involved in the suppression of AA-NAT activity by SNP, as non-hydrolysable analogues of cGMP and the cGMP-specific phosphodiesterase (PDE) inhibitor zaprinast also significantly suppressed AA-NAT activity, while an inhibitor of soluble guanylate cyclase blocked the effect of SNP. While this chain of events may not be part of the normal physiology of the retina, it could be important in pathological circumstances that are associated with marked increase in levels of cGMP, as is found to be the case in certain forms photoreceptor degeneration, which are produced by defects in cGMP phosphodiesterase activity.     

Dopamine-dependent cyclic AMP generating system in chick retina and its relation to melatonin biosynthesis

Stimulation of a D₄-like dopamine (DA) receptors inhibits a cAMP-dependent increase in serotonin N-acetyltransferase activity and melatonin biosynthesis in the chick retina. In order to gain more insight into the molecular mechanisms underlying this suppressive action of DA, the effects of selective stimulation of the D2-family of DA receptors (including the D₄-subtype) on cAMP formation were examined in chick retina using two experimental approaches: measurements of adenylyl cyclase activity in retinal homogenates, and cAMP accumulation in eye cup preparations prelabeled with [³H]adenine. The DA-sensitive adenylyl cyclase system is well expressed in chick retina. DA increased both basal and forskolin-stimulated adenylyl cyclase activity. This effect of DA was antagonized by SCH 23390 (a blocker of D1-family of DA receptors) and not affected by sulpiride (a D2-family blocker). Incubation of retinal homogenates with quinpirole (a predominant agonist of D₃/D₄ DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D₄-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retinas maintained in “eye-cup” preparations. It is suggested that D₄-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.     

Turkey retina and pineal gland differentially respond to constant environment

Dynamics of rhythmic oscillations in the activity of arylalkylamine N-acetyltransferase (AA-NAT, the penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in the retina and pineal gland of turkeys maintained for 7 days in the environment without daily light-dark (LD) changes, namely constant darkness (DD) or continuous light (LL). The two tissues differentially responded to constant environment. In the retina, a circadian AA-NAT activity rhythm disappeared after 5 days of DD, while in the pineal gland it persisted for the whole experiment. No circadian rhythm was observed in the retinas of turkeys exposed to LL, although rhythmic oscillations in both AA-NAT and melatonin content were found in the pineal glands. Both tissues required one or two cycles of the re-installed LD for the full recovery of the high-amplitude AA-NAT rhythm suppressed under constant conditions. It is suggested that the retina of turkey is less able to maintain rhythmicity in constant environment and is more sensitive to changes in the environmental lighting conditions than the pineal gland. Our results indicate that, in contrast to mammals, pineal glands of light-exposed galliformes maintain the limited capacity to rhythmically produce melatonin.     

Regulation of retinal dopamine biosynthesis and tyrosine hydroxylase activity by light

Dopamine (DA)-containing neurons of the rat retina are apparently activated transsynaptically by photic stimulation. Exposure of dark-adapted rats to light increases retinal DA biosynthesis and metabolism. Associated with the light-evoked increase of DA biosynthesis is a rapid activation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. The activation of TH is characterized by an increased affinity of the enzyme for the pteridine cofactor. Because TH in dark-adapted retinas is apparently not saturated with cofactor, the light-evoked increase of affinity is probably responsible for the observed stimulation of DA biosynthesis. Cyclic AMP (cAMP)-dependent protein phosphorylation in vitro activates TH extracted from dark-adapted retinas, and phosphorylation-induced TH activation is very similar and not additive with light-evoked activation of the enzyme. Incubation of viable cell suspensions of dissociated retinas with 8-bromo cAMP also activates TH, which indicates the availability of sufficient cAMP-dependent protein kinase in the proper subcellular compartment to regulate the enzyme in situ. The DA-containing neurons of the rat retina are tonically inhibited in darkness, and evidence is presented that this tonic inhibition involves direct synaptic input to the DA neurons from gamma-aminobutyric acid-containing amacrine cells. The DA-containing neurons are also subject to feedback inhibition through DA receptors, and to modulation by alpha 2-adrenergic receptors.     

Differential Regulation of Arylalkylamine N-Acetyltransferase Activity in Chicken Retinal Ganglion Cells by Light and Circadian Clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48?h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30–60?min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar)     

Circadian rhythm of tryptophan hydroxylase activity in chicken retina

1. Retinal tryptophan hydroxylase activity in chickens (1-4 weeks old and embryos) was estimated by determination of levels of 5-hydroxytryptophan (5HTP) in retinas at defined intervals after inhibition of aromatic L-amino acid decarboxylase with m-hydroxybenzylhydrazine (NSD1015). 2. The relationship of tryptophan hydroxylase activity to photoperiod was explored. In chickens maintained on a 12-hr light: 12-hr dark cycle, a diurnal cycle in tryptophan hydroxylase activity was observed. Activity during middark phase was 4.4 times that seen in midlight phase. Cyclic changes in tryptophan hydroxylase activity persisted in constant darkness with a period of approximately 1 day, indicating regulation of the enzyme by a circadian oscillator. The phase of the tryptophan hydroxylase rhythm was found to be determined by the phase of the light/dark cycle. The relationship of the tryptophan hydroxylase rhythm to the light/dark cycle mirrors previously described rhythms of melatonin synthesis and serotonin N-acetyltransferase (NAT) activity in the retina. 3. Light exposure for 1 hr during dark phase suppressed NAT activity by 82%, while tryptophan hydroxylase activity was suppressed by only 30%. 4. Based on the differential responses of retinal NAT activity and tryptophan hydroxylase activity to acute light exposure during dark phase, it was predicted that exposure to light during dark phase would divert serotonin in the retina from melatonin biosynthesis to oxidation by MAO. In support of this, levels of 5-hydroxyindole acetic acid (5HIAA) in retina were found to be elevated approximately two-fold in chickens exposed to 30 min of light during dark phase. In pargyline-treated chickens, 2 hr of light exposure during dark phase was found to increase retinal serotonin levels by 64% over pargyline-treated controls. 5. Cyclic changes in tryptophan hydroxylase activity and NAT activity persisted for 2-3 days in constant light. Tryptophan hydroxylase activity at mid-night gradually decreased on successive days in constant light; on the first day of constant light, tryptophan hydroxylase activity at mid-night was 70% of activity seen during middark phase of the normal light/dark cycle and decreased further on subsequent days. In contrast, on each of 3 days of constant light, NAT activity at mid-night was approximately 15% of normal middark phase activity. 6. Cycloheximide completely inhibited the nocturnal increase in tryptophan hydroxylase activity when given immediately before light offset. The nocturnal increase in NAT activity was inhibited in a similar fashion. 7. Like the development of the NAT rhythm, cyclic changes of tryptophan hydroxylase activity in the retinas of chickens began on or immediately before the day of hatching. hatching.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.     

Diurnal patterns of dopamine release in chicken retina

The retinal dopaminergic system appears to play a major role in the regulation of global retinal processes related to light adaptation. Although most reports agree that dopamine release is stimulated by light, some retinal functions that are mediated by dopamine exhibit circadian patterns of activity, suggesting that dopamine release may be controlled by a circadian oscillator as well as by light. Using the accumulation of the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) in the vitreous as a measure of dopamine release rates, we have investigated the balance between circadian- and light control over dopamine release. In chickens held under diurnal light:dark conditions, vitreal levels of DOPAC showed daily oscillations with the steady-state levels increasing nine-fold during the light phase. Kinetic analysis of this data indicates that apparent dopamine release rates increased almost four-fold at the onset of light and then remained continuously elevated throughout the 12h light phase. In constant darkness, vitreal levels of DOPAC displayed circadian oscillations, with an almost two-fold increase in dopamine release rates coinciding with subjective dawn/early morning. This circadian rise in vitreal DOPAC could be blocked by intravitreal administration of melatonin (10 nmol), as predicted by the model of the dark-light switch where a circadian fall in melatonin would relieve dopamine release of inhibition and thus be responsible for the slight circadian increase in dopamine release. The increase in vitreal DOPAC in response to light, however, was only partially suppressed by melatonin. The activity of the dopaminergic amacrine cell in the chicken retina thus appears to be dominated by light-activated input.     

Effect of In Vivo Modulation of Membrane Docosahexaenoic Acid Levels on the Dopamine-Dependent Adenylate Cyclase Activity in the Rat Retina

Abstract: We have studied the effect of a dietary deprivation of n-3 fatty acids on the activity of the dopamine (DA)-de-pendent adenylate cyclase in the rat retina. Experiments were conducted in 6-month-old rats raised on semipurified diets containing either safflower oil (n-3 deficient diet) or soybean oil (control diet). The levels of docosahexaenoic acid [22:6 (n-3)] in retinal phospholipids were significantly decreased in n-3 deficient rats (35–42% of control levels). This was compensated by a rise in 22:5 (n-6), the total content of poly-unsaturated fatty acids (PUFA) remaining approximately constant. Adenylate cyclase activity was measured in retinal membrane preparations from dark-adapted or light-exposed rats. The enzyme activity was stimulated by DA and SKF 38393 in a light-dependent fashion. The activation was lower in rats exposed to light than in dark-adapted animals, suggesting a down-regulation of the DI DA receptors by light. The activation by guanine nucleotides and forskolin was also decreased in light-exposed rats. There was no significant effect of the dietary regimen on the various adenylate cyclase activities and their response to light. Furthermore, the guanine nucleotide- and DA-dependent adenylate cyclase activities of retinal membranes were found to be relatively resistant to changes in membrane fluidity induced in vitro by benzyl alcohol. The results indicate that in the absence of changes in total PUFA content, a decreased ratio of n-3 to n-6 fatty acids in membrane phospholipids does not significantly affect the properties of adenylate cyclase in the rat retina.     

Melatonin synthesis in the greenfrog retina in culture: II. Dopaminergic and adrenergic control

Serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels at night. Although the rhythmic properties of NAT and melatonin are similar in pineal gland and retina, great differences in the light perception and transmission mechanisms exist. We have analyzed the effects of adrenergic and dopaminergic agents on greenfrog (Rana perezi) eyecup culture, in order to identify the receptors involved in the regulation of retinal melatonin synthesis. A D2-like receptor is directly involved in the regulation of NAT activity and melatonin release in R. perezi retina. Quinpirole mimics the effect of light, reducing the darkness-stimulated NAT activity and melatonin release, while sulpiride antagonized these actions. Neither D1-agonist (SKF 38393) nor D1-antagonist (SCH 23390) had effect on NAT activity. However, a significant inhibition of darkness-evoked melatonin release was produced by SKF 38393 after 6 hours of culture. The beta- and antagonist1-agonists showed a clear inhibition. However, a direct effect of beta, alpha1 and D1-agonists on photoreceptors is unproven, being more probable that the adrenergic actions imply a non-photoreceptor retinal cell. In conclusion, eyecup culture of Rana perezi revealed a dopaminergic control of melatonin synthesis and a possible modulation of dopaminergic tone by adrenergic receptors. Melatonin release is a more sensitive parameter than NAT activity to the action of neuroactive agents, suggesting that melatonin synthesis can be regulated by more than one enzymatic step in Rana perezi.     

Differential Effects of Bromocriptine on Dopamine and Acetylcholine Release Modulatory Receptors

Rabbit neostriatal slices were prelabeled with [3H]dopamine (DA) and [14C]choline and then superfused. The electrical stimulation-evoked release of DA and of acetylcholine (ACh) was abolished by 0.33 microM tetrodotoxin and by low calcium concentrations (0.13 mM). Bromocriptine, a selective D2-DA receptor agonist, inhibited in a concentration-dependent manner the evoked overflow of DA and ACh, without affecting the basal efflux of both transmitters. The effects of bromocriptine were antagonized by sulpiride, a specific antagonist of D2-DA receptors. With stimulation at 0.3 Hz and 120 pulses, bromocriptine was eight times more potent in inhibiting the evoked overflow of DA (IC50: 11 nM) than that of ACh (IC50: 83 nM). Stimulations at 3 Hz and 360 pulses markedly reduced the potency of bromocriptine in inhibiting DA and ACh release, and diminished its selectivity for presynaptic receptors. These results indicate that DA receptors that modulate the release of DA and ACh are of the D2 subtype. The greater potency of bromocriptine at pre- than at postsynaptic sites suggests that these receptors may be different in quantity and/or quality [D2-alpha (presynaptic) versus D2-beta (postsynaptic)]. Finally, marked differences in the potency and efficacy of DA agonist actions on DA and ACh release modulatory receptors are obtained, depending on the parameters of stimulation used.     

Changes in light and dark periods affect the arylalkylamine N-acetyl transferase,melatonin activities and redox status in the head and hemolymph of nocturnal insect Spodoptera litura

The present study was conducted to describe the impact of circadian rhythm on melatonin levels and redox statusunder three photoperiods (12L:12D, 0L:24D, and 24L:0D) in head and hemolymph of Spodoptera litura. Melatonin is an powerful antioxidant and controls the reproduction of organisms. In this study, melatonin levels, Arylalkylamine N-acetyltransferase(AA-NAT), and antioxidant enzyme activities were analyzed. Results showed melatonin, AA-NAT levels in hemolymph were significantly (p < 0.05) higher during the dark period than during LL regime. HPLC chromatogram of the insect head and hemolymph showed 5 peaks while hemolymph showed 6 peaks in LD, and LLregimes. The day–night changes of melatonin increased the antioxidant enzymes (GST, CAT, POX) persisted in the insect hemolymph, but were suppressed by constant light. The present study leads us to speculate that synthesis and release of melatonin in the S.litura head occur as circadian rhythm and light has an inhibitory effect on melatonin synthesis.