Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine

Retinas of rats, rabbits, chicks and carp possess enzymes, i.e. serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), which convert serotonin (5-HT) to melatonin, NAT activity and melatonin levels, but not HIOMT activity, show distinct circadian rhythms, with peak values occurring during the dark (night) phase of the 12 h light-dark cycle. Exposure of the animals to light at night inhibited the night-stimulated NAT activity. Treatment of rats and rabbits with the dopaminergic agonist, apomorphine, inhibited the retinal NAT activity. Dopamine levels in the rabbit retina showed diurnal variations, with higher contents seen during the light phase of both the 12 h light-dark cycle with lights on between 06:00–18:00, and that with reversed periods of illumination (lights on between 18:00–06:00). Melatonin potently inhibited the electrically-evoked calcium-dependent release of [³H]dopamine from pieces of retina from both albino and pigmented rabbits. Our results indicate that the light-regulated melatonin-generating system does operate in the vertebrate retina. The present data, together with other findings, suggest that in the retina there is an antagonistic interplay between melatonin and dopamine. Thus, melatonin inhibits dopamine synthesis in, and release from, the retinal dopaminergic cells, whilst dopamine inhibits the night (dark)-stimulated melatonin formation by decreasing NAT activity. Since light increases metabolic activity of the retinal dopaminergic cells (it enhances the amine synthesis, levels and release), it seems likely that the retinal dopamine plays a role of a “light” messenger in the inhibition of melatonin synthesis. It is suggested that an interplay between melatonin and dopamine in the retina is responsible for regulation of those retinal events which follow circadian rhythmicity, and/or are dependent on light-dark conditions.     

Melatonin Increases Serotonin N-Acetyltransferase Activity and Decreases Dopamine Synthesis in Light-Exposed Chick Retina: In Vivo Evidence Supporting Melatonin-Dopamine Interaction in Retina

The administration of melatonin, either peripherally (0.01-10 mg/kg) or intraocularly (0.001-10 mumol/eye), to light-exposed chicks dose-dependently increased serotonin N-acetyltransferase (NAT) activity in retina but not in pineal gland. The effect of melatonin was slightly but significantly reduced by luzindole (2-benzyl-N-acetyltryptamine), and not affected by two other purported melatonin antagonists, N-acetyltryptamine and N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23). The elevation of the enzyme activity induced by melatonin was substantially stronger than that evoked by 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, or 5-methoxytryptamine. The melatonin-evoked rise in the retinal NAT activity was counteracted by two dopamine D2 receptor agonists, quinpirole and apomorphine, and prevented by the dopamine D2 receptor blocker spiroperidol, and by an inhibitor of dopamine synthesis, alpha-methyl-p-tyrosine. Melatonin (0.1-10 mg/kg i.p.) dose-dependently decreased the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), as well as the DOPAC/dopamine ratio, in chick retina but not in forebrain. The results obtained (1) indicate that melatonin in vivo potently inhibits dopamine synthesis selectively in retina, and (2) suggest that the increase in retinal NAT activity evoked by melatonin in light-exposed chicks is an indirect action of the compound, and results from the disinhibition of the NAT induction process from the dopaminergic (inhibitory) signal. The results provide in vivo evidence supporting the idea (derived on the basis of in vitro findings) that a mutually antagonistic interaction between melatonin and dopamine operates in retinas of living animals.     

GABA and the regulation of serotonin N-acetyltransferase activity in amphibian retina—II. The role of dopamine

Retinal melatonin biosynthesis is regulated in part by the activity of serotonin N-acetyltransferase (NAT), which increases in dark-adapted, but not light-exposed, retinas at night. Using an in vitro eye cup preparation from the African clawed frog (Xenopus laevis), we have obtained evidence indicating that dopamine and gamma-aminobutyric acid (GABA) interact in the regulation of the nocturnal rise in NAT activity. Increases of NAT activity induced by the GABA agonist muscimol were suppressed by dopamine. Spiperone, a D2 dopamine receptor antagonist, and muscimol separately increased NAT activity, but were not additive in their effects. Inhibition of NAT activity by the GABA antagonist picrotoxin was blocked by spiperone. Additionally, muscimol decreased concentrations of dopamine and its principle metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in light exposed retinas, while picrotoxin increased retinal DOPAC levels in darkness. These data suggest that in darkness, activation of GABA receptors inhibits dopamine secretion, consequently releasing NAT-synthesizing cells from a tonic inhibitory influence.     

N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin levels in the Harderian glands of the female Syrian hamster: changes during the light:dark cycle and the effect of 6-parachlorophenylalanine administration

The activities of NAT and HIOMT and the melatonin content of the Harderian glands of female Syrian hamsters were studied. When hamsters were kept under a light:dark cycle of 14:10 (lights on at 06.00 h), NAT activity exhibited a sharp, short term rise at one hour after lights on. Simultaneously, the activity of HIOMT, which forms melatonin, exhibited a rapid decline. Melatonin levels, like HIOMT activity, also showed a precipitous drop at one hour after light onset. After the respective changes, both NAT and HIOMT activity reverted back to night time levels. Melatonin levels remained depressed for several hours but by 1400 h (8 hours after lights on), nighttime melatonin values were re-established. Treatment of female hamsters with PCPA, a trytophan hydroxylase inhibitor, led to depressed levels of Harderian melatonin without affecting the activities of either NAT or HIOMT.     

Rat serum stimulates serotonin N-acetyltransferase activity in pineal monolayer cultures

The effects of rat serum on serotonin N-acetyltransferase (NAT) activity and indole synthesis in monolayer cultures of neonatal rat pineal glands was examined. The addition of 5% rat serum to these cultures resulted in stimulation of NAT activity equal to that obtained with optimal concentrations of the adrenergic agonist norepinephrine (NE). Rat serum also increased the synthesis of both N-acetylserotonin and melatonin from tryptophan. Stimulation of NAT activity by rat serum was partially blocked by metoprolol and propranolol, but not by phentolamine or butoxamine. The serum factor responsible for the stimulation was stable to both freezing and boiling. No significant amounts of epinephrine, norepinephrine, or dopamine were detected in the serum.     

Changes in pineal indoleamine metabolism in vitamin A deficient rats

Weanling, male rats were fed a vitamin A deficient (VAD) diet from 20 to 77 days of age. The circadian rhythms of the precursors and metabolites of pineal melatonin were measured along with the activity of N-acetyltransferase (NAT). Significant decreases in peak melatonin levels (0100 hours) and in nightime NAT activity (0100 and 0300 hours) were found in the pineals of the VAD rats. In contrast, the contents of serotonin, 5-hydroxytryptophan and 5-hydroxyindole acetic acid were only moderately affected by the deficiency. Daily administration of 25 micrograms melatonin from 20 to 74 days of age markedly reduced NAT activity in control and VAD rats. These data suggest that NAT activity is more sensitive to chronic VAD than any other parameters of melatonin metabolism.     

Serotonin N-acetyltransferase (NAT) induction in mammalian retina: role of cyclic AMP and calcium ions.

In retinas and pineal glands of rat, rabbit and hen, activities of the penultimate (and key regulatory) enzyme in melatonin biosynthesis, serotonin N-acetyltransferase (NAT), display distinct diurnal variations, with high and low values during dark and light phase of a 12-h dark: 12-h light illumination cycle. Two-hour incubation (during daytime hours in light) of isolated pineal glands of the studied vertebrates, or the retinas, with 50 microM forskolin (plus 100 microM 3-isobutyl-1-methylxanthine, IBMX-a phosphodiesterase inhibitor), and 1 mM dibutyryl-cAMP, markedly increased the tissue NAT activity. The same procedures significantly enhanced the enzyme activity of rat retina in light, however, only during nighttime hours. The forskolin (+ IBMX)-induced increase of NAT activity in rat retina was significantly lower in a calcium-free medium, and substantially enhanced when calcium concentration was raised from 1.3 mM to 3.9 mM. Treatment of rats with IBMX or aminophylline, and rabbits with aminophylline, increased NAT activity in their pineal glands irrespective of the time of the day, whereas both phosphodiesterase inhibitors significantly increased the enzyme activity of rat retina only when injected during the subjective dark hours. It is concluded that, by analogy to vertebrate pineal gland, in vertebrate retina an increase of NAT activity (and consequently melatonin formation), stimulated both physiologically (i. e. at night), or pharmacologically, involves a cAMP- and calcium dependent process of the enzyme induction.     

Evidence for a D2 dopamine receptor in frog retina that decreases cyclic AMP accumulation and serotonin N-acetyltransferase activity

The regulation of serotonin N-acetyltransferase (NAT) activity and cyclic AMP accumulation in the retina of the African clawed frog (Xenopus laevis) was studied using an in vitro eye cup preparation. Retinal NAT, a key enzyme in the synthesis of melatonin, is expressed as a circadian rhythm with peak activity at night. The increase of NAT activity at night appears to be mediated by cyclic AMP and is suppressed by light. Dopamine inhibits the nocturnal increase of retinal NAT activity; approximately 80% inhibition was observed with 1 microM dopamine. Dopamine at 1 microM did not stimulate retinal cyclic AMP accumulation. The effect of dopamine on NAT activity was antagonized by the D2-selective receptor antagonists spiperone and metoclopramide, but not by the putative D1 selective antagonist SCH 23390. The nocturnal rise in NAT activity was inhibited by LY 171555, a putative D2 selective agonist, but not by SKF 38393, a putative D1 selective agonist. LY 171555 also decreased cyclic AMP accumulation in eye cups incubated under similar conditions. Dopamine inhibited the stimulation of NAT activity in light by 3-isobutylmethylxanthine, but not that by dibutyryl cyclic AMP, suggesting that dopamine acts by decreasing cyclic AMP formation in the NAT-containing cells. Thus, the effects of dopamine on NAT activity may be mediated by a receptor with the pharmacological and biochemical characteristics of a D2 receptor.     

Day-to-day variation in pineal serotonin N-acetyltransferase activity in stressed and non-stressed male Sprague-Dawley rats

Previous studies involving physical-immobilization stress in laboratory rats have yielded inconsistent results with respect to melatonin synthesis in the pineal gland. As melatonin formation undergoes circadian and infradian rhythms, the aim of the present study was to examine whether stress experiments exhibit day-to-day variation. Toward this end, groups of male Sprague-Dawley rats were stressed by physical immobilization on eight consecutive days, respectively, or left relatively undisturbed, and killed. The pineal gland was rapidly dissected out and serotonin N-acetyltransferase (NAT) activity and melatonin levels were measured. NAT activity was significantly depressed on experimental days 1, 3 and 5, and slightly depressed on day 7. In addition, both in control and experimental animals NAT activity exhibited statistically significant differences between experimental days. Pineal melatonin levels were less variable. On experimental days 3 and 6 immobilization led to a significant increase of pineal melatonin levels. These results show that day-to-day variation is an important factor that influences the outcome of stress experiments and represent another example that NAT activity and pineal melatonin levels do not always show corresponding changes.     

Regulation and possible role of serotonin N-acetyltransferase in the retina

The activity of retinal serotonin N-acetyltransferase (NAT) (arylamine acetyltransferase, EC 2.3.1.5), the penultimate enzyme in melatonin biosynthesis, exhibits properties of a circadian rhythm comparable to that seen in the pineal gland. Using an eye cup preparation we have found that circadian properties persist in vitro, which indicates that an endogenous circadian oscillator controlling NAT is present in the eye. Nighttime increases in NAT activity are suppressed by light, protein synthesis inhibitors, and catecholamines. In light, NAT activity is induced by conditions expected to increase intracellular levels of cyclic AMP (cAMP). This suggests that catecholamines and cAMP are normally involved in the regulation of NAT. Circadian indoleamine metabolism may play a role in the control of rhythmic photoreceptor metabolism as evidenced by the observation that melatonin and related compounds are potent activators of disk shedding.     

UV radiation elevates arylalkylamine N-acetyltransferase activity and melatonin content in the two-spotted spider mite, Tetranychus urticae

Ultraviolet (UV) radiation produces reactive oxygen species (ROS) in mammals, where melatonin plays the role of a ROS scavenger. The melatonin synthetic enzyme arylalkylamine N-acetyltransferase (NAT) is a significant element in a possible ROS removal system. Changes in NAT activity and melatonin content were determined in the two-spotted spider mite Tetranychus urticae by irradiating it with monochromatic light using the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki, Japan. The NAT activity and melatonin content were suppressed by blue light (450nm). No effects of red light (650nm) on the NAT activity and melatonin content were observed. UV radiation had intensity-dependent dual effects on the NAT activity and melatonin content. In the UV-B (300nm) treatment, the NAT activity and melatonin content were suppressed at the intensity below 1mumolm(-2)s(-1) but elevated when the intensity was as high as 10mumolm(-2)s(-1). In the UV-A (350nm) treatment, the melatonin content was elevated when the intensity was as high as 10mumolm(-2)s(-1), though the NAT activity and melatonin content were suppressed at the intensity below 10 and 1mumolm(-2)s(-1), respectively. Elevation of the NAT activity and melatonin content by high intensity UV irradiation may indicate that the UV signals initiate melatonin synthesis for ROS removal in mites.     

Injections of alpha-melanocyte stimulating hormone affect pineal serotonin, melatonin and N-acetyltransferase activity

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.     

Bromocriptine prevents the castration-induced rise in porphyrin concentration in the harderian glands of the male Syrian hamster, Mesocricetus auratus

The Harderian glands in Syrian hamsters exhibit a striking sexual dimorphism. Male Harderian glands show two cell types and low levels of porphyrins and melatonin. Of the enzymes involved in the synthesis of melatonin, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) show high and low activity levels, respectively. Female Harderian glands show but one cell type and have high porphyrin and melatonin levels, low NAT activity, and high HIOMT activity. In castrated males, the Harderian glands exhibit a female pattern of morphology, porphyrin levels, and indoleamine metabolism. In an attempt to determine whether prolactin in involved in this sexually dimorphic response of the Harderian glands, intact and castrated male and intact female hamsters were injected daily with 500 micrograms of bromocriptine, a dopamine agonist. Bromocriptine led to reduced serum prolactin levels in all groups. It had no apparent effect on the Harderian glands of intact males. In contrast, in castrated males bromocriptine prevented the postcastrational rise in porphyrin levels but had no effect on NAT or HIOMT activities. In females, bromocriptine treatment had no effect on porphyrin concentrations or HIOMT activity; it led to a statistically significant increase in NAT activity. We propose that testosterone inhibits Harderian porphyrin synthesis while dopamine or prolactin stimulates it.     

Photoperiodic control of rat pineal serotonin N-acetyl-transferase activity

Adult male albino rats were acclimated to constant light (light:dark-LD-24:0) or to darkness interrupted with brief periods of light at 6 h intervals (LD 1/4:5 3/4 X 4) concurrently with rats maintained in a LD 14:10 photoperiodic cycle. The activity and rhythmicity of pineal serotonin N-acetyltransferase (NAT) was examined at regular intervals for 24 hours in rats maintained in the experimental photoperiods and compared to pineal NAT activity and rhythmicity in rats maintained in the LD 14:10 photoperiod. The results indicate that constant light is capable of depressing nocturnal levels of rat pineal NAT and obliterating the pineal NAT rhythm. Likewise, rats subjected to darkness interrupted with brief periods of light at 6 h intervals experienced a similar response in pineal NAT activity to animals subjected to constant light, i.e., pineal NAT activity was persistently low and the rhythmicity was obliterated. The results are discussed relative to the hypothesis that the pineal NAT activity responds to an endogenous rhythm in photoperiodic time measurement. The evidence herein suggests that the time of occurrence of environmental light in the photoperiod is more important in determining pineal NAT activity and/or rhythmicity than is the total amount of darkness or the dark to light ratio to which animals may be subjected.     

Simultaneous determination of N-acetyltransferase activity, hydroxyindole-O-methyl-transferase activity, and melatonin content in the pineal gland of the Syrian hamster

The activities of serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin content were measured in Syrian hamster pineal glands at 2-hr intervals over a period of 24 hr. NAT and HIOMT are the two enzymes which catalyze the formation of melatonin from serotonin. The use of micromethods for determination of the enzyme activities allowed concurrent measurement of NAT and melatonin or HIOMT and melatonin in the same gland. HIOMT activity showed no significant diurnal rhythm whereas NAT activity and melatonin content exhibited distinct peak values late in the dark phase as described previously. Despite an apparent parallelism between the NAT activity rhythm and melatonin content, no correlation exists between these parameters in single pineal glands.     

The organochlorine insecticide 1,2,3,4,5,6-hexachlorocyclohexane (lindane) but not 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) augments the nocturnal increase in pineal N-acetyltransferase activity and pineal and serum melatonin levels

The effect of organochlorine insecticides lindane (1,2,3,4,5,6-hexachlorocyclohexane) and DDT (1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane) were studied in terms of their effects on the rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during the day (2000h) and at night (2300 and 0100h). Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Nocturnal NAT activity was increased after lindane administration; likewise, lindane augmented pineal and serum melatonin levels at 2300h. Conversely, DDT was without a statistically significant effect on either NAT activity or on pineal or serum melatonin levels. Neither lindane nor DDT significantly influenced pineal HIOMT values either during the day or at night. Likewise, neither insecticide consistently influenced pineal levels of either 5-HTP, 5-HT or 5-HIAA. The results indicate that the organochlorine insecticide, lindane, modifies pineal melatonin synthesis in vivo.     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.     

Two-phase response of rat pineal melatonin to lethal whole-body irradiation with gamma rays.

Male Wistar rats adapted to artificial light:dark (LD) regimen 12:12 h were whole-body irradiated with a single dose of 9.6 Gy of gamma rays and sham/irradiated in the night in darkness. The rats were examined 60 min, 1, 3 and 5 days after exposure between 22:00 and 01:30 h in the darkness. The results obtained indicate a two-phase reaction of pineal melatonin after the lethal irradiation of rats: the decline of melatonin concentration early after the exposure (at 60 min) with unchanged serotonin N-acetyltransferase (NAT) activity followed by an increase of melatonin synthesis, accompanied by an increase of pineal and serum melatonin on day 5 after the exposure. NAT activity was increased on day 3 after the exposure. Serum corticosterone concentrations in irradiated rats were increased 60 min and 3 days after exposure. With respect to the antioxidant, immunomodulating and stress-diminishing properties of melatonin, we consider the increase in melatonin synthesis during later periods after irradiation as part of adaptation of the organism to overcome radiation stress.     

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.