Serotonin N-acetyltransferase Activity Research Articles

Effects of near-ultraviolet light on the nocturnal serotonin N-acetyltransferase activity of rat pineal gland

Effects of near-ultraviolet (UV-A; 325–390 nm, peak at 365 nm) light on the activity of the pineal serotonin N-acetyltransferase (NAT; a penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in rats. Acute exposure of dark-adapted animals to UV-A radiation produced a marked suppression of NAT activity of the pineal gland, the effect being dependent on exposure time. The decrease in the night-time NAT activity evoked by a 1-min pulse of UV-A light (as well as by a 15-s pulse of broad-band visible light) gradually deepened during the first 40 min of treatment of animals with constant darkness, then the enzyme activity began to rise reaching control values by 3 h. Treatment of rats with a protein synthesis inhibitor, cycloheximide, attenuated this night-driven reactivation of the pineal NAT activity. The presented results provide evidence that UV-A light is a powerful signal capable of controlling melatonin biosynthesis in rat pineal gland.

Melatonin and serotonin N-acetyltransferase activity in developing eggs of the cricket Gryllus bimaculatus

Melatonin ( N-acetyl-5-methoxytryptamine) and serotonin N-acetyltransferase (NAT), a key regulatory enzyme in melatonin synthesis, are present in the adults and larvae of several insect species, as well as in vertebrates. To determine when melatonin and NAT first appear in insects ontogenetically, melatonin levels and NAT-like activity were measured in developing eggs of the cricket Gryllus bimaculatus. When the eggs were incubated under a 12-h light/12-h dark (LD) cycle at 24–26°C, melatonin was detected in the egg extracts at all of the developmental stages examined. NAT-like activity was first found in the eggs 3 days after oviposition. From 5 to 11 days after oviposition, both NAT-like activity and melatonin levels showed significant day/night changes with the high levels occurring during the dark period of the LD cycle. By contrast, significant day/night changes were not detected in eggs just before hatching. To determine more detailed temporal changes, NAT-like activity was assayed in eggs 6 to 7 days after oviposition at 2- or 4-h intervals over a 48-h period. The activity in the eggs clearly exhibited a diurnal rhythm, peaking in the dark period of the LD cycle, and the rhythm persisted in constant darkness. These results suggest that the cricket egg (probably the embryo) synthesizes melatonin, and that its melatonin synthesis may fluctuate with a circadian rhythm. In addition, the results of the present study strongly suggest that a circadian clock controlling NAT activity functions in the cricket at the embryonic stage.

L-Aspartate-evoked inhibition of melatonin production in rat pineal glands

Our previous studies in rat indicated that pinealocytes secrete l-glutamate through microvesicle-mediated exocytosis to regulate negatively melatonin production. Recently, we further found that pinealocytes secrete l-aspartate through microvesicle-mediated exocytosis. In the present study, we investigated the role of l-aspartate in the melatonin production in isolated rat pineal glands. It was found that l-aspartate inhibits norepinephrine-stimulated melatonin production as well as serotonin N-acetyltransferase activity reversibly and dose-dependently, the concentrations required for 50% inhibition being 150 and 175 μM, respectively. l-Asparagine and oxaloacetate, metabolites of l-aspartate, had no effect on the melatonin production. These results suggest that pinealocytes use l-aspartate, as well as l-glutamate, as a negative regulator for melatonin production.

Effect of constant and fluctuating temperature on daily melatonin production by eyecups from Rana perezi.

We analysed the effect of daily temperature cycles in relation to constant temperature on day/night melatonin synthesis in frog eyecups in culture. Eyecups were cultured for 24 h under 12L:12D photoperiod and two thermal regimes, constant temperature (25, 15 and 5 degrees C) and thermoperiod (WL/CD, thermophase coinciding with photophase and cryophase coinciding with scotophase; and CL/WD, cryophase coinciding with photophase and thermophase coinciding with scotophase). A negative correlation between ocular serotonin N-acetyltransferase activity and culture temperature for both diurnal and nocturnal activities has been observed. This effect of increased ocular activity at low temperature is more pronounced than the well-known stimulatory effect of darkness, and it does not depend on the photoperiod phase. The lack of interactions between the phase of photoperiod and culture temperature indicates that the effects of both factors are independent. Night-time temperature is the key factor in determining the amplitude of the melatonin rhythm in the Rana perezi retina. However, daytime temperature can not counteract the inhibitory effect of light on ocular melatonin synthesis.

Chick pineal clock regulates serotonin N-acetyltransferase mRNA rhythm in culture.

Melatonin production in the chick 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. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.

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

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 approximately 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.

Diurnal fluctuation in the enzyme activity and the messenger RNA level of pineal serotonin N-acetyltransferase in normal and hereditary microphthalmic rats.

The enzyme activity and the messenger RNA level of pineal serotonin N-acetyltransferase were more than 20- and 50-fold higher, respectively, in the dark period than in the light period in normal rats. In hereditary microphthalmic rats, however, the serotonin N-acetyltransferase activity and its mRNA level did not undergo a great diurnal change through the light and dark periods. These results indicate that the diurnal rhythms of the activity and the mRNA level of serotonin N-acetyltransferase are not detected in the pineal gland of hereditary blind rats, suggesting free-running rhythms in individual animals due to desynchronization of their circadian rhythms by a lack of their optic nerve.

Cellular and Molecular Regulation of Serotonin N-Acetyltransferase Activity in Chicken Retinal Photoreceptors

Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and large changes in the activity of this enzyme appear to regulate the rhythm in melatonin synthesis. Recent advances have made it possible to study the mRNA encoding chicken AA-NAT, which has only been detected in the retina and pineal gland. Within the retina, AA-NAT mRNA is expressed primarily in photoreceptors. The levels of chicken retinal AA-NAT mRNA and activity exhibit 24-hour rhythms with peaks at night. These rhythms appear to reflect circadian clock control of AA-NAT mRNA abundance and independent effects of light and darkness on both mRNA levels and enzyme activity. The effects of darkness and light may occur through alterations in cAMP-dependent protein phosphorylation, which increases AA-NAT activity in photoreceptor cell cultures. The cAMP-dependent increase of AA-NAT enzyme activity reflects, at least in part, increased mRNA levels and inhibition of enzyme inactivation by a posttranslational mechanism. This review discusses a hypothetical model for the cellular and molecular regulation of AA-NAT activity by circadian oscillators and light in chicken retinal photoreceptor cells.

Pharmacological modifications in dopaminergic neurotransmission affect the quinpirole-evoked suppression of serotonin N-acetyltransferase activity in chick retina: an impact on dopamine D4-like receptors.

Dopamine (DA) plays an important role in the regulation of melatonin biosynthesis in retinas of several vertebrate species. In the retina of chick, the DA receptor controlling melatonin production represents a D4-like subtype. Stimulation of this receptor by quinpirole (QNP) results in a dose-dependent decline of the nighttime activity of serotonin N-acetyltransferase (NAT; a key regulatory enzyme in melatonin biosynthesis) and melatonin level of chick retina. The present study was undertaken to determine whether long-term treatment with antipsychotic drugs (clozapine-30 mg/kg, i.m.; sulpiride-100 mg/kg, i.m.; and raclopride-10 mg/kg, i.p., once daily for 21 days) and L-DOPA (80 mg/kg, i.p., once daily for 7 days) affects the response of the melatonin generating system of chick retina to the suppressive effect of QNP. Chronic administration to chicks of clozapine and sulpiride, but not raclopride, resulted in a markedly increased response of retinal NAT activity to the action of QNP. ED50 values for QNP were 3-times (clozapine) and 4-times (sulpiride) lower than those in the respective vehicle-treated control groups. On the other hand, QNP was significantly less potent in retinas of birds treated with L-DOPA than in control animals; the ED50 value for QNP was 3-times higher in birds injected with L-DOPA than in the vehicle-treated group. These results indicate that long-term treatment with clozapine, sulpiride and L-DOPA may modify the reactivity of D4-like DA receptors regulating NAT activity of chick retina. A possibility of modifications of circadian and electrophysiological processes within the eye following prolonged administration of DA-ergic drugs is discussed.

Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland.

In vertebrates, the circadian rhythm in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] drives the daily rhythm in circulating melatonin. We have discovered that expression of the AA-NAT gene in the rat pineal gland is essentially turned off during the day and turned on at night, resulting in a more than 150-fold rhythm. Expression is regulated by a photoneural system that acts through an adrenergic-cAMP mechanism in pinealocytes, probably involving cAMP response element-binding protein phosphorylation. Turning off AA-NAT expression appears to involve de novo synthesis of a protein that attenuates transcription. A approximately 10-fold night/day rhythm in AA-NAT messenger RNA occurs in the retina, and AA-NAT messenger RNA is also detected at low levels in the brain.

Are there rhythms in scleral precursor synthesis?

Australian and New Zealand Journal of OphthalmologyVolume 24, Issue S2 p. 45-47 Free Access Are there rhythms in scleral precursor synthesis? Malini Devadas BSc(Hons), Corresponding Author Malini Devadas BSc(Hons) Visual Sciences Group, Research School of Biological Sciences, Australian National University, ACT 2600.Malini Devadas.Search for more papers by this authorIan Morgan BSc, PhD, Ian Morgan BSc, PhD Visual Sciences Group, Research School of Biological Sciences, Australian National University, ACT 2600.Search for more papers by this author Malini Devadas BSc(Hons), Corresponding Author Malini Devadas BSc(Hons) Visual Sciences Group, Research School of Biological Sciences, Australian National University, ACT 2600.Malini Devadas.Search for more papers by this authorIan Morgan BSc, PhD, Ian Morgan BSc, PhD Visual Sciences Group, Research School of Biological Sciences, Australian National University, ACT 2600.Search for more papers by this author First published: May 1996 https://doi.org/10.1111/j.1442-9071.1996.tb00992.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 Bock G., Widdows K. Myopia and the control of eye growth. CIBA Foundation Symposium 155. Chichester , UK : Wiley, 1990; 1– 256. 2 Weiss S., Schaeffel F. Diurnal growth rhythms in the chicken eye: relation to myopia development and retinal dopamine levels. J Comp Physiol 1993; A172: 263– 70. 3 Rada J., Thoft A., Hassell J. Increased aggrecan (cartilage proteoglycan) production in the sclera of myopic chicks. DcvBiol 1991; 147: 303– 12. 4 Hamm H., Menaker M. Retinal rhythms in chicks: variations in melatonin and serotonin N-acetyltransferase activity. Proc Natl Acad Sci USA 1980; 77: 4998– 5002. 5 Sasaki M., Masuda A., Oishi T. Circadian rhythms of corneal mitotic rate, retinal melatonin and immunoreac-tive visual pigments, and the effects of melatonin on the rhythms in the Japanese quail. J Comp Physiol 1995; A176: 465– 71. 6 Nickla D., Wallman J. The diurnal rhythms of intraocular pressure and ocular elongation are altered in myopic chick eyes. Invest Ophthalmol Vis Sci 1995; 36: S413. 7 Chiu J., Mack A., Fernald R. Daily rhythms of cell proliferation in the teleost retina. Brain Res 1995; 673: 119– 25. 8 Morgan I., Boelen M. A retinal dark-light switch: a review of the evidence. Vis Neurosci 1996; (in press). 9 Stone R., Lin T., Iuvone P., Laties A. Retinal dopamine and deprivation myopia. Proc Natl Acad Sci USA 1989; 86: 704– 6. Citing Literature Volume24, IssueS2May 1996Pages 45-47 ReferencesRelatedInformation

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

Stimulation of a D 4-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 D 4-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 D 3/D 4 DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D 4-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retinas maintained in “eye-cup” preparations. It is suggested that D 4-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.

N-acetyl transferase activity during the photoperiodic-dependent Pieris brassicae development

A serotonin N-acetyl transferase (NAT) activity was detected in the brain of Pieris brassicae. The maximal head NAT activity appeared to occur during the wandering-pharate phase. The haemolymph level of serotonin, the selected NAT substrate, presented a maximum on the 3rd day of the 5th instar for both short- and long-photoperiod animals with a longer increase in short-photoperiod animals. The circadian rhythm of head NAT, determined on the same day, appeared bimodal and was affected by pteridines only when they were injected at “specific” times. At the last larval stage, the effect of either serotonin, N-acetylserotonin or melatonin was characterized by inactivity immediately after the injection, leading to death by starvation. In “underchilled” chrysalids, serotonin induced a diapause breakdown while melatonin led to mortality. These data suggest, besides the role of juvenile hormone, ecdysone, and polypeptidic hormones, a role for indolamines during the photoperiodic-dependent development of Pieris.

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

The aim of the present study was to examine the roles of membrane depolarization, calcium influx, and cyclic AMP synthesis in regulating the stability and inactivation of serotonin N-acetyltransferase activity (NAT) in cultured chick photoreceptor cells. NAT activity was induced by pretreating cells for 6 h with 1 microM forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivation) was determined. After induction, in the presence of cycloheximide, NAT activity declined with a half-life of approximately 30 min. The rate of inactivation was greatly reduced when depolarizing concentrations of K+, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine were added together with cycloheximide. The apparent increase in NAT stability caused by K+ was abolished by addition of EGTA or nifedipine and potentiated by Bay K 8644, indicating the involvement of Ca2+ influx through dihydropyridine-sensitive channels. MDL-12330A, and inhibitor of K(+)-stimulated cyclic AMP formation, blocked the effect of depolarizing concentrations of K+. This result suggests that the effect of Ca2+ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolarization-evoked Ca2+ influx and cyclic AMP formation have two roles in the regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein required for NAT activity. Second, they increase the half-life of the enzyme, presumably by regulating the turnover of existing enzyme molecules.

Gating of retinal inputs through the suprachiasmatic nucleus: Role of excitatory neurotransmission

The mammalian circadian clock, located in the hypothalamic suprachiasmatic nucleus (SCN) is important in the regulation of many circadian rhythms, including regulation of pineal gland metabolism and melatonin secretion. Transsection of the optic nerves, disrupting the retinohypothalamic pathway, lesion of the SCN, or lesion of the hypothalamic paraventricular nucleus (PVN) abolish the regulation of pineal serotonin N-acetyltransferase activity by light. Therefore, the pathways linking the retina and the pineal gland must be channelled from the retina through the SCN and the PVN. Many lines of evidence indicate that the major neurotransmitter in the retinal afferents is glutamate. The first aim was therefore to study the retinal target neurons by localising glutamate receptors in the rodent SCN. Using in situ hybridisation, we detected NMDA-RI and NMDA-R2C mRNA subunits in the SCN. Using immunocytochemistry, immunoreactivity for the AMPA type receptors GluR 1, GluR 2,3 and GluR 4 was also detected in the SCN. Presentation of a short light pulse during the subjective night [i.e. circadian time (CT) 14 or 19], when light induced phase-shifting of activity-rest cycles can be accomplished, also induces expression of the immediate early-genes c- fos and junB in the rodent SCN. The second aim was to use this cellular correlate of behavioural function to determine the location of potential retinal target neurons in the SCN, and to investigate the hypothesis that glutamatergic neurotransmission mediates the effects of light on the circadian system. Thus, the ability of the NMDA receptor antagonist MK-801 to block light-induced c- fos expression in the SCN was studied. In the rat, this antagonist blocked c - fos mRNA expression in a subpopulation of cells in the ventral SCN at doses of 6, but not 2 mg/kg. In contrast, in the hamster both doses blocked light-induced c- fos expression in the ventral SCN. These data provide support for the hypothesis that glutamate mediates effects of light in the SCN, although it appears that the complexes of NMDA receptor subunits, which are involved in light-induced expression of c fos after light, are relatively insensitive to MK-801. The diversity, heterogeneous distribution, and complexity of glutamate receptor subunits in the SCN suggest that processing of light pulses in the SCN is mediated by several cell types in the SCN. Via an integration process in the clock, the transmission of photic information takes place to other brain structures.

Melatonin biosynthesis in photoreceptor-enriched chick retinal cell cultures: role of cyclic amp in the K +-evoked, ca 2+ -dependent induction of serotonin n-acetyltransferase activity

The roles of cyclic AMP and calcium in the regulation of serotonin N-acetyltransferase (NAT) activity were studied in low density monolayer cultures of chick retinal photoreceptors and neurons. Photoreceptor-enriched recinal cell cultures were prepared from embryonic day 6 retinas and cultured for 6 days. NAT activity in these cultures could be induced by treatment with cyclic AMP protagonists, 8Br-cyclic AMP, forskolin, and 3-isobutyl-l-methylxanthine (IBMX), or by treatment with depolarizing concentrations of extracellular K +. The stimulatory effect of K +, which involves Ca 2+ influx through dihydropyridine-sensitive channels, was mediated at least in part by cyclic AMP, as indicated by the following observations. Depolarizing concentrations of K + stimulated the formation of cyclic AMP, and the stimulatory effects of K + on both cyclic AMP formation and on NAT activity were synergistically potentiated by the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX). MDL 12,330A, a putative adenylate cyclase inhibitor, inhibited K +-evoked cyclic AMP accumulation and induction of NAT activity over the identical concentration range. In contrast, MDL 12,300A failed to inhibit the induction of NAT elicited by 8Br-cyclic AMP. H-89, an inhibitor of cyclic AMP-dependent protein kinase, antagonized the induction of NAT activity by either forskolin or K + with equal potency for both stimuli. These results suggest that cyclic AMP plays an essential role in the induction of NAT activity that occurs as a consequence of membrane depolarization. Cyclic AMP and Ca 2+ may also interact at a step distal to adenylate cyclase. In cells treated with a low concentration of 8Br-cyclic AMP, increasing intracellular Ca 2+ with K + (20 or 45 mM) or the calcium ionophore A23187 elicited a stimulation of NAT activity that was slightly, but consistently greater than that expected from the additive effects of each treatment alone. The Ca 2+ channel agonist Bay K 8644, however, did not potentiate the effect of 8Br-cyclic AMP. In contrast to the interaction of calcium elevating agents and 8Br-cyclic AMP, Bay K 8644 or depolarizing concentrations of K + elicited a large, synergistic potentiation of NAT activity induced by treatments that raise the levels of endogenous cyclic AMP, forskolin and IBMX. These results indicate that the primary role of Ca 2+ influx in the induction of NAT activity in photoreceptor cells is to stimulate cyclic AMP formation, but a secondary role may be to potentiate the effect of cyclic AMP at a step distal to adenylate cyclase.

Preliminary Evidence of a Synergistic α1- and β1-Adrenoceptor Regulation of Rat Pineal Hydroxyindole-O-methyltransferase

The activity of serotonin N-acetyltransferase in the rat pineal is regulated through a synergistic action of norepinephrine on α1- and β1-adrenoceptors. Rat pineal hydroxyindole-O-methyltransferase (HIOMT) activity is also responsive to the β1-adrenoceptor, while melatonin synthesis and indoleamine release in certain species is apparently under α1-adrenoceptor control. The present study reports that the simultaneous administration of doxazosin (a selective α1-adrenergic antagonist) and metoprolol (a selective β1-adrenergic antagonist) reduces peak HIOMT activity significantly more than either agent alone, indicating that rat pineal HIOMT is also subject to synergistic α1- and β1-adrenergic regulation. The relationship between α1-adrenoceptor stimulation, serotonin release, and HIOMT activity is considered, following the observation that nocturnal HIOMT activity is elevated in serotonin-depleted pineal glands.

Properties of clock-controlled and constitutive N-acetyltransferases from chick pineal cells

The pineal gland synthesizes its hormone melatonin ( O-methyl- N-acetylserotonin) from serotonin. Acetyl-CoA; serotonin N-acetyltransferase (SNAT), the enzyme that catalyzes the committed step in this biosynthesis, is largely restricted to the pineal gland and is regulated by adrenergic and circadian mechanisms. Another enzyme, however, is not rhythmically regulated. (ANAT), having an apparently similar acitivity, is also present in the pineal. This enzyme, however, is not rhythmically regulated. SNAT activity of cultured chick pineal cells was obtained without ANAT after ammonium sulfate precipitation. ANAT activity was retained without SNAT activity after pre-incubation at 37°C. Thus, each enzyme could be examined independently. Overlap in substrate specificity between the two enzymes was minimal. Kinetic analysis of the separated enzyme activities revealed that while SNAT operates via a random or ordered bi bi mechanism, ANAT catalysis occurs through a ping pong bi bi mechanism with substrate inhibition by acetyl-COa. By size-exclusion chromatography, ANAT was confirmed to be 30–35 kDa, and SNAT was estimated at 15–20 kDa. Taken together, these results indicate that the two enzymes differ in their structure, reactivity. stability and mechanism of catalysis.

Serotonin N-acetyltransferase activity as a target for temperature in the regulation of melatonin production by frog retina

The adaptive mechanisms of serotonin N-acetyltransferase (NAT) activity in the regulation of melatonin synthesis in frog retina in the face of chronic and acute temperature changes have been investigated. We performed thermal acclimation experiments to test different environmental temperatures at two seasons of the year (summer and winter), followed by the set-up of an eyecup culture system to investigate the acute effects of temperature on NAT activity and melatonin production daily rhythms. Low temperature induced a significant increase in NAT activity, independent of both the time of the photocycle (midday or midnight) and the season of the year (winter or summer). Acute cold-induced stimulation of NAT activity may be associated with lower decreases in the enzyme synthesis rate, rather than decreases in the degradation rate. In contrast, acclimation to warm temperature (25 degrees C) stimulated ocular melatonin production. Nocturnal melatonin production in eyecups cultured at 25 degrees C was significantly higher than in eyecups cultured at 5 degrees C. We suggest that this discrepancy in thermal regulation of melatonin synthesis can be justified by a seasonal variation in serotonin content within the photoreceptor cells, which determines the thermal response of melatonin production through changes in NAT kinetics.

Stimulation of D 4-like dopamine receptor suppresses serotonin N-acetyltransferase activity but does not phase-shift the circadian oscillator in chick retina

We have recently reported that dopamine (DA) receptor-regulating serotonin N-acetyltransferase (NAT) activity in chick retina represents a D 4-like subtype. In this work, effects of stimulation of DA-D 4 receptors upon the circadian rhythm of retinal NAT activity were investigated in chicks. Systemic administration of quinpirole (QNP), a DA-D 4 receptor agonist, resulted in a potent suppression of the nighttime NAT activity of chick retina but did not significantly affect the phase of subsequent cycles of the rhythmic oscillation of the enzyme activity in constant darkness. 6-h pulses of white light caused an acute suppression of NAT activity in chick retina and, in contrast to QNP, resetted the phase of the free-running rhythm. It is suggested that, although D 4-like DA receptors in chick retina are involved in the regulation of NAT activity by the circadian clock, they do not appear to be implicated in the regulation of the clock by light.