Regulation of Melatonin Production by Pineal Photoreceptor Cells: Role of Cyclic Nucleotides in the Trout (Oncorhynchus mykiss)

Abstract: The light/dark cycle influences the rhythmic production of melatonin by the trout pineal organ through a modulation of the serotonin N -acetyltransferase (NAT) activity. In static organ culture, cyclic AMP (cAMP) levels (in darkness) and NAT activity (in darkness or light) were stimulated in the presence of forskolin, isobutylmethylxanthine, or theophylline. Analogues of cAMP, but not of cyclic GMP, induced an increase in NAT activity. Light, applied after dark adaptation, inhibited NAT activity. This inhibitory effect was partially prevented in the presence of drugs stimulating cAMP accumulation. In addition, cAMP accumulation and NAT activity increase, induced by forskolin, were temperature dependent. Finally, melatonin release, determined in superfused organs under normal conditions of illumination, was stimulated during the light period of a light/dark cycle by adding an analogue of cAMP or a phosphodiesterase inhibitor. However, no further increase in melatonin release was observed during the dark phase of this cycle in the presence of the drugs. This report shows for the first time that cAMP is a candidate as intracellular second messenger participating in the control of NAT activity and melatonin production by light and temperature.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Light Pulses (II)

Abstract: N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought responsible for melatonin circadian rhythms. The enzyme has properties of a circadian biological clock—its rhythm persists in constant conditions and it is precisely controlled by light and dark. Experiments are reported in which light pulses of 1 to 10 h duration were imposed on chicks during their dark-time. The effect of these pulses upon the NAT was measured and the effect of the pulses on subsequent NAT was also determined. The experiments support the conclusion that the amount and/or duration of dark-time NAT is limited. This finding is interpreted as supporting the idea that a fixed amount of some substance, an initiator, is synthesized during the subjective day.     

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.     

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.     

N-Acetylation of Serotonin Is Correlated with α2- but Not with β-Adrenergic Regulation of Cyclic AMP Levels in Cultured Chick Pineal Cells

We investigated the role of cyclic AMP (cAMP) in alpha 2- and possible beta-adrenergic regulation of arylalkylamine-N-acetyltransferase (NAT), the penultimate enzyme in the biosynthesis of melatonin. The study was performed on primary cultures of dispersed chick pineal cells. Electron microscopy indicated that approximately 70% of the dispersed cells were modified photoreceptors. A similar proportion of melatoninergic cells was detected by immunocytochemical labeling of hydroxyindole-O-methyltransferase, the final enzyme in the biosynthesis of melatonin. Adrenergic agonists caused a sustained 50% inhibition of forskolin-augmented cAMP levels and NAT activity, with an alpha 2-adrenergic potency order of UK 14,304 greater than or equal to clonidine greater than norepinephrine greater than phenylephrine. Noradrenergic inhibition of 3-isobutyl-1-methylxanthine-augmented cAMP levels and NAT activity was reversed by yohimbine (an alpha 2-adrenergic antagonist) but not by prazosin (an alpha 1-adrenergic antagonist). The alpha-adrenergic inhibition of cAMP accumulation and NAT activity was prevented by pertussis toxin. Addition of propranolol (a beta-adrenergic antagonist) was necessary to observe an inhibitory effect of norepinephrine on cAMP levels but not on NAT activity. Similarly, the beta-adrenergic agonist isoproterenol transiently increased cAMP levels but did not affect NAT activity. The data indicate that the alpha 2-adrenergic inhibition of NAT activity in chick pineal cells is strongly correlated with an inhibition of cAMP accumulation. The lack of beta-adrenergic effect on NAT suggests that beta-adrenoceptors might be on a subset of cells that do not produce melatonin or that the beta-adrenergic-induced increase in cAMP levels is too transient to affect NAT.     

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.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Rapid Phase Reversal and Response to Shorter than 24-Hour Cycles (IV)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is responsible for circadian rhythms in melatonin. The NAT activity rhythm has circadian properties such as persistence in constant conditions and precise control by light and dark. Experiments are reported in which chicks (Gallus domesticus), raised for 3 weeks in 12 h of light alternating with 12 h of dark (LD12:12), were exposed to 1-3 days of light-dark treatments during which NAT activity was measured in their pineal glands. (a) In LD12:12, NAT activity rose from less than 4.5 nmol/pineal gland/h during the light-time to 25-50 nmol/pineal gland/h in the dark-time. Constant light (LL) attenuated the amplitude of the NAT activity rhythm to 26-45% of the NAT activity cycle in LD12:12 during the first 24 h. (b) The timing of the increase in NAT activity was reset by the first full LD12:12 cycle following a 12-h phase shift of the LD12:12 cycle (a procedure that reversed the times of light and dark by imposition of either 24 h of light or dark). This result satisfies one of the criteria for NAT to be considered part of a circadian driving oscillator. (c) In less than 24-h cycles [2 h of light in alternation with 2 h of dark (LD2:2), 4 h of light in alternation with 4 h of dark (LD4:4), and 6 h of light in alternation with 6 h of dark (LD6:6)], NAT activity rose in the dark during the chicks' previously scheduled dark-time but not the previously scheduled light-time of LD12:12. In a cycle where 8 h of light alternated with 8 h of dark (LD8:8), NAT activity rose in both 8-h dark periods, even though the second one fell in the light-time of the prior LD12:12 schedule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of Melatonin Synthesis in Chicken Retina: Regulation of Serotonin N-Acetyltransferase Activity by Light, Circadian Oscillators, and Cyclic AMP

In chicken retinas, melatonin levels and the activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme of melatonin biosynthesis, are expressed as circadian rhythms with peaks of levels and activity occurring at night. In the present study, NAT activity was examined in retinas of embryonic and posthatch chicks to assess the ontogenic development of regulation of the enzyme by light, circadian oscillators, and the second messenger cyclic AMP. During embryonic development, NAT activity was consistently detectable by embryonic day 6 (E6). Significant light-dark differences were first observed on E20, and increased to a maximum amplitude of sixfold by posthatch day 3 (PH3). Circadian rhythmicity of NAT activity appears to develop at or prior to hatching, as evidenced by day-night differences of activity in constant darkness observed in PH1 chicks that had been exposed to a light-dark cycle in ovo only. NAT activity is regulated by a cyclic AMP-dependent mechanism. Activity was significantly increased by incubating retinas with forskolin or dibutyryl cyclic AMP as early as E7, and seven- to ninefold increases were observed following treatment with these agents on E14. Thus, development of the cyclic AMP-dependent mechanism for increasing NAT activity significantly precedes that of rhythmicity, suggesting that the onset of rhythmicity may be related to the onset of photoreception or development of the circadian oscillator in chick retina.     

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.     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.     

Different mechanisms of phase delays and phase advances of the circadian rhythm in rat pineal N-acetyltransferase activity

The circadian rhythm in rat pineal N-acetyltransferase (NAT) activity, which drives the rhythm in melatonin production, is controlled by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus. As the NAT rhythm has two well-defined phase markers--namely, the time of the evening activity rise and of the morning decline--it is suitable for studies of the entrainment of the pacemaker by environmental light. Phase delays of the NAT rhythm proceed more rapidly than phase advances. One day after a brief light pulse applied before midnight, or after a delay in evening lights-off, or a delay of a light-dark (LD) cycle, phase delays of the evening NAT rise result in almost corresponding delays of the morning NAT decline. Consequently, the NAT rhythm is phase-shifted, but its pattern does not change. One day after a brief light pulse applied past midnight, or after bringing forward morning lights-on, or after an advance of an LD cycle, the morning NAT decline is phase-advanced, but the evening rise is not phase-advanced at all or may even by phase-delayed. Consequently, the phase relationship between the evening NAT activity onset and the morning offset may be compressed considerably, and it may take several transient cycles before phase advances of the morning NAT decline are followed by corresponding advances of the evening NAT rise. Due to the phase-delaying effect of evening light on the NAT rise and to the phase-advancing effect of morning light on the NAT decline, the phase relationship between the NAT rise and the decline is compressed on long days and decompressed on short days. Different phase shifts of the evening NAT rise and of the morning decline, even in opposite directions, are consistent with the hypothesis of a complex, two-component (evening-morning, or E-M) pacemaker controlling the NAT rhythm. As the E-M phase relationship determines duration of the high night melatonin production, and the duration of the nocturnal melatonin pulse may convey information on daylength, the data are consistent with the internal coincidence model for photoperiodic time measurement.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Phase Shifting by Dark Pulses (III)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is thought to be responsible for melatonin circadian rhythms. The enzyme has circadian properties--its rhythm persists in constant conditions, and it is precisely controlled by light and dark. Experiments are reported in which 4-h light or dark pulses were imposed on chicks (Gallus domesticus) over a 24-h period. Pineal NAT profiles were measured during and subsequent to the pulses. The phase of the NAT cycle following pulses was plotted to obtain phase-response curves. Light pulses produced a maximum phase shift (advance of 5 h) 8 h after the expected time of lights-out; dark pulses produced a maximum phase shift (advance of 4 h) 3 h after the expected time of lights-out. Maximum phase delays (-2 h) occurred 1-2 h after the expected lights-out for light pulses and 8 h after expected lights-on for dark pulses.     

Does a biological clock reside in the eye of quail?

The site (intra- vs. extraocular) of the circadian clock driving an ocular melatonin rhythm in Japanese quail was investigated by alternately covering the left and right eyes of individual quail, otherwise held in constant light (LL), for 12-hr periods. This procedure exposed each eye to a light-dark (LD) 12:12 light cycle 180 degrees (12 hr) out of phase with the LD 12:12 light cycle experienced by the other eye. This protocol entrained the melatonin rhythm in the left eye of quail 180 degrees out of phase with the rhythm expressed in the right eye. These results are compatible with the hypothesis that an independent light-entrainable circadian pacemaker resides in each eye; they are incompatible with the hypothesis that a single (or functionally single) extraocular pacemaker drives the ocular melatonin rhythm in both eyes. However, the results are also compatible with a model in which two independent extraocular circadian pacemakers, each with an exclusive photic input from one eye, drive the ocular melatonin rhythm.     

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.     

Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle

Summary The rhythm in melatonin production in the rat is driven by a circadian rhythm in the pineal N-acetyltransferase (NAT) activity. Rats adapted to an artificial lighting regime of 12 h of light and 12 h of darkness per day were exposed to an 8-h advance of the light-dark regime accomplished by the shortening of one dark period; the effect of melatonin, triazolam and fluoxetine, together with 5-hydroxytryptophan, on the reentrainment of the NAT rhythm was studied.In control rats, the NAT rhythm was abolished during the first 3 cycles following the advance shift. It reappeared during the 4th cycle; however, the phase relationship between the evening rise in activity and the morning decline was still compressed.Melatonin accelerated the NAT rhythm reentrainment. In rats treated chronically with melatonin at the new dark onset, the rhythm had already reappeared during the 3rd cycle, in the middle of the advanced night, and during the 4th cycle, the phase relationship between the evening onset and the morning decline of the NAT activity was the same as before the advance shift. In rats treated chronically with melatonin at the old dark onset or in those treated with melatonin 8 h, 5 h and 2 h after the new dark onset during the 1st, 2nd and 3rd cycle, respectively, following the advance shift, the NAT rhythm reappeared during the 3rd cycle as well but in the last third of the advanced night only.Neither triazolam nor fluoxetine together with 5-hydroxytryptophan administered around the new dark onset facilitated NAT rhythm reentrainment after the 8-h advance of the light-dark cycle.Abbreviations NAT N-acetyltransferase - LD cycle light-dark cycle - CT circadian time - LD xy light dark cycle comprising x h of light and y h of darkness     

Effect of brefeldin A on melatonin secretion of chick pineal cells

Melatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.     

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.     

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.     

Twenty-four hour oscillation of cAMP in chick pineal cells: role of cAMP in the acute and circadian regulation of melatonin production

Chick pineal cells contain circadian oscillators that regulate a rhythm of melatonin biosynthesis. We explored the role of cAMP in regulating this melatonin rhythm. Chick pineal cells expressed a 24 hr oscillation of cAMP efflux with a waveform similar to that of melatonin. Elevation of cAMP in chick pineal cells stimulated melatonin. These results suggest that an oscillation of cAMP regulates the rhythm of melatonin. We investigated whether cAMP was a component of the circadian oscillator by determining the effects of 8-Br cAMP pulses on the phase of the circadian melatonin rhythm. Six hour pulses of 8-Br cAMP did not cause steady-state phase shifts of the rhythm. The acute regulation of melatonin by cAMP, the 24 hr oscillation of cAMP, and the inability of cAMP to phase-shift the melatonin rhythm strongly suggest that cAMP acts as an output signal of the circadian oscillator.     

Prostaglandins Stimulate Serotonin Acetylation in Chick Pineal Cells: Involvement of Cyclic AMP-Dependent and Calcium/Calmodulin-Dependent Mechanisms

Abstract: The effects of prostaglandins (PGs) on the activity of the rate-limiting enzyme of melatonin biosynthesis, aryl-alkylamine- N -acetyltransferase (NAT) were investigated on primary cultures of dispersed chick pineal cells. In indomethacin-treated cells, PGs caused a four-fold increase in NAT activity. This response was associated with an eightfold increase in cyclic AMP (cAMP) levels. The potency order of PGs was the same for NAT and for cAMP responses (PGE¹ > PGE² > PGF^2α≫ cloprostenol). However, each PG tested was 30- to 200-fold more potent to increase NAT activity than to stimulate cAMP accumulation. As a result, half-maximal stimulation of NAT by PGs was not associated with an increase in cAMP levels. Half-maximal stimulation of NAT by PGE¹ was highly sensitive to inhibition by a calcium/calmodulin antagonist (W-7). In contrast, maximal stimulation of NAT by PGE¹ as well as stimulations evoked by either forskolin or 8-bromo-cAMP were poorly sensitive to inhibition by W-7. These results indicate that an increase in cAMP levels may be responsible for the maximal stimulation of NAT evoked by PGs, whereas half-maximal stimulation of NAT by PGs would rely principally on a calcium/calmodulin-dependent mechanism.