Pineal N-acetyltransferase Activity Research Articles

Opposite effects of photoperiod and temperature on circadian rhythms of pineal arylalkylamine N-acetyltransferas (AANAT) activity and circulating levels of thyroid hormones in airbreathing catfish, Clarias gariepinus

ABSTRACT Effects of different photoperiods (9L-15D, 12L-12D and 15L-9D at a constant temperature 25 ± 1°C) and simulated temperatures (15°C, 25°C and 35°C under a common photoperiod 12L-12D) were investigated on the circadian rhythms of plasma levels of thyroid hormones [thyroxine (T4) and triiodothyronine (T3)] and pineal arylalkylamine N-acetyltransferase (AANAT) activity in the fish, Clarias gariepinus during winter and summer seasons. Irrespective of the seasons, plasma levels of thyroid hormones (THs) and pineal AANAT activity exhibited circadian rhythm under all the photoperiodic regimes and simulated temperatures. The levels of THs were found to be directly related to the length of photoperiod as well as temperature. In contrast, pineal AANAT activity was inversely related to the length of photoperiod and temperature. On the basis of these findings, we suggest that photoperiod and temperature jointly regulate the inverse temporal relationship between the circadian rhythms of plasma levels of THs and pineal AANAT activity in C. gariepinus.

Eight hours of nocturnal 915 MHz radiofrequency identification (RFID) exposure reduces urinary levels of melatonin and its metabolite via pineal arylalkylamine N-acetyltransferase activity in male rats

Purpose: We investigated the effects of whole-body exposure to the 915 MHz radiofrequency identification (RFID) on mela-tonin biosynthesis and the activity of rat pineal arylalkylamine N-acetyltransferase (AANAT).Materials and methods: Rats were exposed to RFID (whole-body specific absorption rate, 4 W/kg) for 8 h/day, 5 days/week, for weeks during the nighttime. Total volume of urine excreted during a 24-h period was collected after RFID exposure. Uri-nary melatonin and 6-hydroxymelatonin sulfate (6-OHMS) was measured by gas chromatography-mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. AANAT enzyme activity was measured using liquid biphasic dif-13 fusion assay. Protein levels and mRNA expression of AANAT was 14 measured by Western blot and reverse transcription polymerase 15 chain reaction (RT-PCR) analysis, respectively.Results: Eight hours of nocturnal RFID exposure caused a sig-nificant reduction in both urinary melatonin (p = 0. 003) and 6-OHMS (p = 0. 026). Activity, protein levels, and mRNA expres-sion of AANAT were suppressed by exposure to RFID (p < 0. 05).Conclusions: Our results suggest that nocturnal RFID exposure can cause reductions in the levels of both urinary melatonin and 6-OHMS, possibly due to decreased melatonin biosynthesis via suppression of Aanat gene transcription in the rat pineal gland.

Seasonality of pineal gland activity and immune functions in chickens

The immunomodulatory action of melatonin in different animal species is already well known, although the mechanism(s) by which the indoleamine influences the immune system have yet to be fully elucidated. Previously, we have shown both anti-inflammatory and opioid-mediated influence of exogenous melatonin on thioglycollate-induced peritonitis in young chickens. In the present study, the kinetics of peritonitis and splenocyte proliferation were compared in chickens reared in both seasons under the same L:D 12:12 conditions. These two aspects of the immune response were correlated with the diurnal rhythm of pineal gland function, measured by the activity of N-acetyltransferase (NAT), a key enzyme in melatonin biosynthesis. The results revealed seasonal changes in the circadian rhythm of pineal NAT activity occurring in parallel to the natural local geophysical seasons. These changes appeared to influence the development of peritonitis and splenocyte responsiveness to mitogenic stimulation in vitro. Moreover, the existence of bidirectional communication between the pineal gland and the activated immune system was supported by the decreased activity of pineal NAT in chickens with peritonitis compared with control birds.

Melatonin and jet lag syndrome: experimental model and clinical implications.

What is the effect of melatonin on jet lag syndrome? Jet lag desynchronizes the internal sleep-wakefulness cycle with the environmental light/dark cycle. Advance (but not delay) of light onset is known to abolish pineal N-acetyltransferase activity and urine excretion of 6-sulphatoxymelatonin. Measurements of pineal serotonin, the substrate of melatonin biosynthesis; N-acetylserotonin (NAS), the immediate melatonin precursor; and melatonin (high-performance liquid chromatography-fluorimetric method) in the animal (rat) model of jet lag revealed that prolonged delay of dark-phase onset disrupted the rhytms in comparable ways as the advance of light-phase onset. Advance of dark phase onset resulted in less severe disturbances of rhythms as compared with the advance of light phase onset. Melatonin, but not NAS, injections at the beginning of a new dark period accelerated recovery of NAS and melatonin, but not serotonin, rhythms. Spontaneously hypertensive rats were more sensitive to advance of light onset and less responsive to melatonin injections than normotensive rats. NAS and methylene blue, an inhibitor of monoamine oxidase A, attenuated light-induced disruption of NAS but not melatonin rhythms. We draw the following conclusions from our data: the beginning of the dark period may be preferable to the beginning of light period as the arrival time on eastward flights; the efficacy of melatonin in alleviating jet lag may be enhanced by administering it before, during and after rapid transition through time zones; and hypertension may exaggerate jet lag syndrome.

Effects of Diazepam and Its Metabolites on Nocturnal Melatonin Secretion in the Rat Pineal and Harderian Glands. A Comparative In Vivo and In Vitro Study

We investigated the effects of diazepam (DZP) and its three metabolites: nordiazepam (NZP), oxazepam (OZP), and temazepam (TZP) on pineal gland nocturnal melatonin secretion. We looked at the effects of benzodiazepines on pineal gland melatonin secretion both in vitro (using organ perifusion) and in vivo in male Wistar rats sacrificed in the middle of the dark phase. We also examined the effects of these benzodiazepines on in vivo melatonin secretion in the Harderian glands. Neither DZP (10−5–10−6 M) nor its metabolites (10−4–10−5 M) affected melatonin secretion by perifused rat pineal glands in vitro. In contrast, a 10−4 M suprapharmacological concentration of DZP increased melatonin secretion of perifused pineal glands by 70%. In vivo, a single acute subcutaneous administration of DZP (3 mg/kg body weight) significantly affected pineal melatonin synthesis and plasma melatonin levels, while administration of the metabolites under the same conditions did not. DZP reduced pineal melatonin content (−40%), N-acetyltransferase activity (−70%), and plasma melatonin levels (−40%), but had no affects on pineal hydroxyindole-O-methyltransferase activity. Neither DZP nor its metabolites affected Harderian gland melatonin content. Our results indicate that the in vivo inhibitory effect of DZP on melatonin synthesis is not due to the metabolism of DZP. The results also show that the control of melatonin production in the Harderian glands differs from that observed in the pineal gland.

The Induction of Low Nocturnal Secretion of Melatonin Caused by Reverse Feeding Rhythms Depends on Availability of Macronutrient Diets

The present study investigated the effect of single chow diet and two-way dietary choice between a protein-rich and a carbohydrate-rich diet ingested during free access or daytime access (08.00-16.00 h) on the rhythms of ingestion and on nocturnal levels of serum melatonin and pineal N-acetyltransferase activity in rats. Animals with free access to the single or the choice diets displayed a circadian rhythmicity of total food intake with peaks at the beginning, the middle and the end of the dark phase. Rats fed the dietary choice displayed a preference for carbohydrate at the beginning, and a preference for protein at the middle and the end of the dark phase. Under daytime feeding access, both dietary conditions resulted in only one peak of total intake at the beginning of the access period. Rats fed the choice diets on the daytime feeding schedule initially preferred carbohydrate to protein but this preference disappeared subsequently. Nocturnal peaks of serum melatonin and pineal NAT activity were lower with daytime access to dietary choice compared to ad libitum access. The results suggest that in a two-way selection of macronutrients, feeding cycle can directly act on the rhythms expressed within the pineal gland.

Food access schedule and diet composition alter rhythmicity of serum melatonin and pineal NAT activity

This study investigated the effect of dietary composition and food access schedule on the rhythmicity of serum melatonin and pineal N-acetyltransferase (NAT) activity. Wistar rats maintained on a 12:12 h light–dark cycle were assigned to two dietary groups: a group fed rat chow and a group fed a choice between a protein-rich and a carbohydrate-rich diet. Each dietary group was further divided based on feeding schedule, with food available between 0800 and 1600 h or ad lib access to food. Regardless of dietary condition, total food and carbohydrate intake of rats having free access to food was higher than under the restricted food access schedule. Protein intake of rats fed the dietary choice was lower with the restricted access than in the free access. In rats fed the dietary choice, melatonin levels and NAT activity were significantly decreased with restricted access compared to free access. Such results were not found in rats offered restricted chow. This study suggests that the rhythms of melatonin secretion and NAT activity can be altered by dietary composition.

Ontogenetic effects of MAO-A inhibition on rat pineal n-acetylserotonin and melatonin during the first month of neonatal life.

Inhibitors of monoamine oxidase A (MAO-A) but not MAO-B stimulate the activity of pineal serotonin N-acetyltransferase (AANAT) in the adult rat pineal leading to increased formation of N-acetyl serotonin (NAS) and melatonin (MEL). The pineal gland of the neonatal rat has AANAT activity, but the second enzyme in melatonin biosynthesis, HIOMT (hydroxyindole-O-methyltransferase) converting NAS to MEL, is absent during the first week of neonatal life. In this study we examined the effects of acute clorgyline treatment in vitro and in vivo, on pineal indoles over the first month of neonatal life. The results show that clorgyline stimulates NAS production by pineal both in vitro and in vivo from day five on with a marked increase between day 14 and day 21. In contrast, MEL is not increased until day 21, with a sharp rise thereafter. Copyright 2000 John Wiley & Sons, Ltd.

50-Hz SINUSOIDAL MAGNETIC FIELD EFFECT ONIN VITROPINEALN-ACETYLTRANSFERASE ACTIVITY

Some studies have shown a decrease in pineal N-acetyltransferase (NAT) activity and/or blood melatonin concentration in rodents exposed to extremely low-frequency (ELF) and low magnetic flux density electromagnetic fields. The mechanism/s involved in such effects are not known. It has been hypothesized that the magnetic fields (MF) could act on the pineal gland directly and/or indirectly through the retina. The aim of this work was to study whether MFs could modify NAT activity through a direct effect on the gland. Pineal glands obtained from rats sacrificed in the middle of the dark period were exposed during a 1-h incubation to 10-, 100-, or 1,000-μT, 50-Hz, sinusoidal MFs. The results showed that the glands exposed to the highest magnetic flux density responded with a significant decrease in NAT activity. The data obtained from these experiments support the idea that the pineal gland can be directly affected by ELF electromagnetic fields.

Light‐induced changes in rabbit pineal gland n‐acetyltransferase activity

N‐acetyltransferase (NAT) activity was determined in different ages of New Zealand White rabbit pineal gland using 2‐aminofluorene and p‐aminobenzoic acid as substrates and it was assayed by high pressure liquid chromatography. Rabbits of different ages were either sacrificed during the light phase, exposed to darkness or light for 1 min during the dark phase of the lighting cycle, returned to their cages in darkness for 30 min and then sacrificed. Pineal gland NAT activity in animal nocturnally exposed to 1 min of light was inhibited in animals 1 ‐day‐old of age. Nocturnal light exposure did not inhibit enzyme activity in 1‐day‐old rabbit, even though these animal displayed clear light : dark differences in pineal gland NAT activity. Nocturnal light exposure also did not inhibit night time levels of NAT activity in 1‐day‐old animals who had been bilaterally enucleated. The result suggested that this effect is retinally mediated. Pre‐treatment of 1‐day‐old and 60‐day‐old animals with the isoproterenol (beta‐noradrenoreceptor agonist drug), prevented the nocturnal light‐induced inhibition of NAT activity. The different sensitivity of 60‐day‐old and 1‐day‐old animals to different illuminances or durations of nocturnal light exposure, was that the duration or intensity of light exposure was enable to inhibit nocturnal NAT activity. The NAT activity was at least 3.2‐ to 4.6‐fold greater in 1‐day‐old rabbits compared to 60‐day‐old rabbits. Kinetic constants for arylamine NAT activity in pineal gland from rabbits were determined. Km and Fmax values for 2‐aminofluorene were 2.6‐fold higher for light exposure than for no light exposure rabbits. This is the first demonstration of the retina‐pineal gland pathway appears light‐induced changes in pineal glands of animals in 1‐day‐old of ages or older; but this pathway does not function in 60‐day‐old rabbits like manner in 1‐day‐old rabbits.

Age-related differences in serum melatonin and pineal NAT activity and in the response of rat pineal to a 50-HZ magnetic field

In a previous study we have shown that exposure to a 50-Hz sinusoidal magnetic field decreased serum melatonin concentration and pineal enzyme activities in young rats (9 weeks). In the present study we looked for the effect of a magnetic field of 100 μT on serum melatonin and pineal NAT activity in aged rats and compared them to young rats. We hypothesized that aging may change sensitivity of rats to a magnetic field. Two groups of Wistar male rats [aged rats (23 months) and young rats (9 weeks)] were exposed to 50-Hz magnetic fields of 100 μT for one week (18h/day). The animals were kept under a standard 12:12 light: dark cycle with a temperature of 25° C and a relative humidity of 45 to 50%. Control (sham-exposed) animals were kept in a similar environment but without exposure to a magnetic field. The animals were sacrificed under red dim light. Serum melatonin concentration and pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities were studied. Our results showed that sinusoidal magnetic fields altered the production of melatonin (28% decrease; P < 0.05) through an inhibition of pineal NAT activity (52% decrease; P < 0.05) in the young rats whereas no effect was observed in aged ones. On the other hand, when comparing data from control animals between young and aged rats, we observed that serum melatonin level and NAT activity, but not HIOMT activity, decreased in aged rats (decrease by about 38% and 36% respectively). Our data strongly suggest that old rats are insensitive to the magnetic field.

Arginine vasotocin activates phosphoinositide signal transduction system and potentiates N-acetyltransferase activity in the rat pineal gland

The pineal gland is innervated by pinealopetal peptidergic fibers originating in the hypothalamic nuclei which release arginine vasopressin (AVP) and arginine vasotocin (AVT) from their endings. Since the mechanism of AVT action on the pineal signal transduction and melatonin synthesis has not been determined so far, we examined the effect of AVT on the phosphoinositide signalling system and the N-acetyltransferase (NAT) activity in the rat pineal gland. The effect of AVP 4–9 fragment and AVP analogue desmopressin was also tested. The phosphoinositide signalling system was studied by measuring 32P labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2) which reflects PI cycle activation. AVT (10 −5 and 10 −4 M) induced a significant increase in 32P labelling of PI, PIP and PIP 2. The AVT mediated activation of the PI signal cascade was supressed by the vasopressin V 1 receptor antagonist. The desmopressin and AVP 4–9 fragment were without the effect on PI signalling. To assess the AVT role in the melatonin synthesis we studied the daily pattern of the pineal NAT activity in rats treated by AVT (10 μg/100 g b.w). AVT application in the dark period of the day significantly increased nocturnal NAT activity. It can be summarized that AVT activates PI signalling system and potentiates NAT activity in the rat pineal gland.

Possible involvement of beta-adrenergic receptors in the enhancement of nocturnal pineal N-acetyltransferase activity due to parathion administration

The purpose of the present study was to examine the effects of administration of sublethal doses of O, O-diethyl- O- p-nitrophenyl phosphorothioate (parathion) on serum epinephrine (EPI) and norepinephrine (NE), as well as on night-time rat pineal melatonin synthesis, both in the presence and absence of propranolol, a β-adrenergic receptor antagonist. In the first experiment, two groups of adult albino rats were administered parathion orally (1.08 and 2.17 mg/kg/day; the total received by each animal was 6.5 and 13.0 mg/kg body weight over 6 days); another two groups received corn oil only. Animals were killed at 23:00 and 01:00 h by decapitation. Serum EPI was augmented at 01:00 h, but NE was increased at 01:00 and 23:00 h due to administration of the high dose of parathion (13 mg/kg). In the second experiment, two groups of adult male albino rats were administered parathion orally (13 mg/kg); another two groups received an intraperitoneal injection of propranolol (20 mg/kg body weight, 1 h before the lights were turned off). In addition, two groups were given a saline injection. Four hours after darkness onset, pineal N-acetyltransferase (NAT) activity as well as pineal and serum melatonin levels were measured. Parathion by itself significantly augmented nocturnal pineal NAT activity and serum melatonin levels in otherwise untreated rats; the insecticide was ineffective in reference to this enzyme when it was given in conjunction with the β-adrenergic receptor antagonist propranolol. The augmentation of NAT activity by parathion also caused significant reduction in pineal serotonin (5-HT); again, this response was blocked by propranolol treatment. The results are consistent with the idea that parathion influences pineal 5-HT metabolism either at the level of the β-adrenergic receptor or via the sympathetic innervation to the pineal gland.

Inconsistent suppression of nocturnal pineal melatonin synthesis and serum melatonin levels in rats exposed to pulsed DC magnetic fields.

The purpose of these experiments was to determine whether the exposure of rats at night to pulsed DC magnetic fields (MF) would influence the nocturnal production and secretion of melatonin, as indicated by pineal N-acetyltransferase (NAT) activity (the rate limiting enzyme in melatonin production) and pineal and serum melatonin levels. By using a computer-driven exposure system, 15 experiments were conducted. MF exposure onset was always during the night, with the duration of exposure varying from 15 to 120 min. A variety of field strengths, ranging from 50 to 500 microT (0.5 to 5.0 G) were used with the bulk of the studies being conducted using a 100 microT (1.0 G) field. During the interval of DC MF exposure, the field was turned on and off at 1-s intervals with a rise/fall time constant of 5 ms. Because the studies were performed during the night, all procedures were carried out under weak red light (intensity of <5 microW/cm2). At the conclusion of each study, a blood sample and the pineal gland were collected for analysis of serum melatonin titers and pineal NAT and melatonin levels. The outcome of individual studies varied. Of the 23 cases in which pineal NAT activity, pineal melatonin, and serum melatonin levels were measured, the following results were obtained; in 5 cases (21.7%) pineal NAT activity was depressed, in 2 cases (8.7%) studies pineal melatonin levels were lowered, and in 10 cases (43.5%) serum melatonin concentrations were reduced. Never was there a measured rise in any of the end points that were considered in this study. The magnitudes of the reductions were not correlated with field strength (i.e., no dose-response relationships were apparent), and likewise the reductions could not be correlated with the season of the year (experiments conducted at 12-month intervals under identical exposure conditions yielded different results). Duration of exposure also seemed not to be a factor in the degree of melatonin suppression. The inconsistency of the results does not permit the conclusion that pineal melatonin production or release are routinely influenced by pulsed DC MF exposure. In the current series of studies, a suppression of serum melatonin sometimes occurred in the absence of any apparent change in the synthesis of this indoleamine within the pineal gland (no alteration in either pineal NAT activity or pineal melatonin levels). Because melatonin is a direct free radical scavenger, the drop in serum melatonin could theoretically be explained by an increased uptake of melatonin by tissues that were experiencing augmented levels of free radicals as a consequence of MF exposure. This hypothetical possibly requires additional experimental documentation.

Differential adrenergic regulation of rat pineal cyclic AMP production and N-acetyltransferase activity during postnatal development: involvement of G alpha s and G alpha i1-2 proteins.

We have studied why rat pineal N-acetyltransferase (NAT) activity is relatively insensitive to isoproterenol in young rats when compared with adult rats. We report that activation by isoproterenol of pineal cyclic AMP production and NAT activity is higher in adult than in 2-week-old rats. However, the effect of dibutyryl cyclic AMP, which enters the pinealocyte and duplicates the effect of cyclic AMP, on NAT activity was similar at both ages. Moreover, we found that both alpha- and beta-adrenergic receptors are highly specific at both ages, since the binding of the specific radioligands used to their receptors could be displaced only by their corresponding agonists and antagonists. However, we observed differences between pineals from young and adult rats when several families of the alpha subunit of G-proteins were studied in cell membranes. ADP-ribosylation and immunoblot studies have shown clear differences in both 42 and 45 kDa forms of the Gs alpha Both forms exhibit low values in pineals from 2-week-old animals when compared with 6-week-old. We also show that the later appearance of both Gs alpha forms is roughly similar to the potent activation of cyclic AMP production and NAT activity in adult rats when compared with young rats. In conclusion, the results presented suggest that the relative lack of sensitivity of rat pineal gland to beta-adrenergic receptor agonists early in the postnatal development may be explained by the low levels of membrane Gs alpha, rather than postreceptor-mediated mechanisms or changes in the characteristics of the beta-adrenergic receptors on the pinealocyte membrane.

Light-induced changes in frog pineal gland N-acetyltransferase activity

N-Acetyltransferase (NAT) activity was determined in the pineal gland of frogs ( Rana tigrina) of different ages using 2-aminofluorene and p-aminobenzoic acid as substrates, and assayed by high-pressure liquid chromatography. Frogs of different ages were either killed during the light phase or exposed to darkness or light for 1 min during the dark phase of the lighting cycle, then returned to their cages in darkness for 30 min before being killed. The pineal gland NAT activity of 1-month-old frogs was inhibited when the animal was nocturnally exposed to 1 min of light. Nocturnal light exposure did not inhibit NAT activity in 1-month-old frogs, even though these animal displayed clear light-dark differences in pineal gland NAT activity. Nocturnal light exposure did not inhibit night-time levels of NAT activity in 1-month-old animals which had been bilaterally enucleated, thus suggesting that this effect is retinally mediated. Pretreatment of 1-month-old and 6-month-old animals with isoproterenol (a beta-adrenoceptor agonist drug) prevented the nocturnal light-induced inhibition of NAT activity. From the different sensitivity of 1-month-old and 6-month-old animals to different intensities or durations of nocturnal light exposure it was found that the duration or intensity of light exposure was not able to inhibit nocturnal NAT activity. The NAT activity was at least 4–5-fold greater in 1-month-old frogs than in 6-month-old frogs. This is the first demonstration of the retino-pineal gland pathway that appears to produce light-induced changes in pineal glands of frogs 1-month-old or older, but this pathway only functions in 1-month-old frogs, and does not appear to function in 6-month-old frogs.

Photoperiod-dependent correlation between light-induced SCN c-fos expression and resetting of circadian phase.

In rodents, brief light pulses that shift the phase of the circadian locomotor rhythm also increase c-fos gene expression in the suprachiasmatic nucleus (SCN), site of an endogenous clock that regulates such rhythmicity. Because the magnitude of photic phase shifts varies when light pulses are applied at different time points over the course of the subjective night, we examined the degree of SCN c-fos gene expression after single 30-min light pulses were delivered at time points that spanned the early and late subjective night in rats maintained in either short (8:16-h light-dark cycle) or long (16:8-h light-dark cycle) photoperiods. The light-induced level of c-fos mRNA and the number of cells expressing immunoreactive c-Fos protein were measured in the SCN by in situ hybridization and immunohistochemistry, respectively, and compared with the magnitude of the corresponding phase shifts of the circadian rhythm of pineal N-acetyltransferase (NAT) activity. We found a robust correlation between c-fos photoinduction and NAT phase shifts, but this relationship was dependent on photoperiod. The degree of c-fos gene expression was strongly correlated with the magnitude of NAT phase advances and delays under the short photoperiod and with phase advances under the long photoperiod, but not with phase delays under the long photoperiod. The data suggest that c-fos gene expression in the SCN may be involved in the photic resetting of the pineal NAT rhythm. Under the long photoperiod, however, the magnitude of phase delays may be limited by the functional state of the circadian pacemaking system.

Effects of nerve growth factor on splenic norepinephrine and pineal N-acetyl-transferase in neonate rats exposed to alcohol in utero: Neuroimmune correlates

Prenatal alcohol exposure (FAE) has been associated with multiple anomalies, including a selective developmental delay of sympathetic innervation in lymphoid organs. Sympathetic neurons require nerve growth factor (NGF) for their development and maintenance, and recent evidence has suggested that alcohol impairs the synthesis and/or biological activity of NGF in selected central and peripheral neurons. Thus, the present study examined the hypothesis that NGF administration to FAE rats during early postnatal development would reverse some of the peripheral sympathetic deficits. Neonate rats, FAE and the corresponding control cohorts, received daily treatments of NGF or cytochrome C (0.3 mg/kg; s.c.) for various time intervals, and were killed 24hr or 10 days after the last treatment. The measured parameters included norepinephrine (NE) concentrations in the spleen and heart, which receive noradrenergic innervation from the coeliac ganglion and the superior cervical ganglion (SCG), respectively. In addition, we measured the activity of pineal N-acetyltransferase (NAT), the rate-limiting enzyme of melatonin biosynthesis, which depends on sympathetic innervation from the SCG. The data show that chronic, but not acute, NGF treatments reversed the FAE-related deficits in splenic NE concentrations as well as in pineal NAT activity in a time- and age-dependent manner. Sympathetic neurons play an important role in immune modulation. Thus, the altered splenic NE levels and pineal NAT activity may play a role in immune deficits associated with exposure to alcohol in utero.

Changes in Pineal Indoleamines in Rats after Single Melatonin Injections: Evidence for a Diurnal Sensitivity to Melatonin

We recently determined that melatonin stimulated serotonin (5-HT) secretion from rat pineal glands by increasing 5-HT release from the pinealocytes (microM melatonin concentrations) and by inhibiting 5-HT uptake in the pineal sympathetic nerve endings (mM melatonin concentrations). The present study investigated whether a single melatonin injection could alter the content of indoleamines in the rat pineal gland, as well as its possible dependence on the daytime of administration. Melatonin (150 micrograms/kg) was i.p. injected at 8 time points (11.00 h, 14.00 h, 17.00 h, 20.00 h, 23.00 h, 02.00 h, 05.00 h and 08.00 h) to rats kept in 12:12 h light:dark cycle (lights on at 07.00 h). Melatonin injections in the afternoon (17:00 h) and late in the nighttime (02.00 h and 05.00 h) decreased pineal 5-HT content 90 min later. The levels of 5-hydroxyindoleacetic acid (5-HIAA) were also decreased 90 min after the melatonin treatment at 14.00 h, 17.00 h and 02.00 h. The effect of melatonin on 5-HT content was a long-fasting effect (still evident after 180 min) only when injected at 02.00 h, whereas 5-HIAA levels were found to be decreased 180 min after melatonin treatment at 14.00 h and 23.00 h. No changes in these compounds were detected 240 min after melatonin treatment. Moreover, melatonin did not change 5-hydroxytryptophan levels at any of the daytime points studied. By contrast, 90 min after the injection of melatonin at 20.00 h, an increased content of pineal N-acetylserotonin was observed. This effect of melatonin could be mediated through a phase alteration of the pineal N-acetyltransferase activity rhythm by acting on the suprachiasmatic clock, although a direct melatonin effect on the pineal rhythmic function cannot be excluded. The effects of the hormone on 5-HT and 5-HIAA contents agree with previous findings on the inhibitory effect of pharmacological doses of melatonin on pineal 5-HT uptake, which presumably would result in a decreased intraneuronal content of 5-HT and its acid metabolite. These data point to an acute regulatory action of exogenous melatonin on the pineal melatonin synthesis pathway which seems to be limited to two daytime phases; the afternoon-early evening period and the second half of the night.

Partial characterization of serotonin N - acetyltransferases from northern pike (Esox lucius, L.) pineal organ and retina: effects of temperature.

In vertebrates, the nocturnal rise in pineal organ and retinal melatonin synthesis results from the increase in the activity of the serotonin N-acetyltransferase (NAT), a cAMP-dependent enzyme. In the fish pineal organ in culture, light and temperature act in a similar manner on cAMP content and NAT activity. It is not known whether the effects of temperature are mediated through cAMP or through modifications of NAT kinetics. The present study was designed: (1) to find out whether NAT activity from pineal organ homogenates is similar to NAT activity from pineal organs in culture, with regard to variations in temperature, and (2) to compare NAT activity from the pineal organ and the retina. Pineal organ and retinal NAT activity increased linearly with protein concentrations. Higher activities were obtained with 0.2 mol/l of phosphate buffer, pH 6. Higher molarity or a higher pH induced a decrease in retinal and pineal organ NAT activity: retinal NAT was more sensitive than pineal organ NAT to changes in molarity, whereas the opposite held true as far as pH was concerned. Pineal organ and retinal NAT obeyed the Michaelis-Menten equation with respect to increasing concentrations of acetyl-coenzyme A. With increasing concentrations of tryptamine: (1) pineal organ NAT activity increased in a manner suggesting positive co-operativity, (2) retinal NAT displayed, after an initial increase, inhibition by substrate. The kinetics of the reactions were temperature dependent. Maximal activities were reached at 18/20 degrees C in the pineal organ and at 37 degrees C in the retina. The present study is the first to describe the optimum conditions for the assay of NAT activity in homogenates from the retina of fish and from the pineal organ of poikilotherms, and also the first to compare some characteristics of NAT activity from these two analogous organs. Our results suggest that the effects of temperature on melatonin production are mediated, at least in part, through modifications of NAT kinetics. Future studies will aim to clarify whether the activities measured in the pineal organ and retinal homogenates reflect the presence of one or of several enzymes.