Nocturnal N-acetyltransferase Activity Research Articles

Melatonin synthesis in the greenfrog retina in culture: I. Modulation by the light/dark cycle, forskolin and inhibitors of protein synthesis

Melatonin is synthesized in the pineal gland and the retina of vertebrates. Retinal serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels during the dark phase of the photocycle. In some vertebrates, these retinal NAT and melatonin rhythms are maintained in vitro. The aim of present work is to develop an eyecup culture system for the greenfrog ( Rana perezi), suitable to analyze the mechanisms of regulation of melatonin synthesis by simultaneous determination of NAT activity and melatonin release. The R. Perezi eyecups released melatonin to the culture medium in a rhythmic manner at least over a 27-h period under photocycle conditions. Nat activity and melatonin rhythms were similar to that observed in vivo under natural environmental conditions. Rana perezi retina exhibits a pronounced photosensitivity in vitro. Forskolin increased up to 2-fold the NAT activity and 4-fold the melatonin production at any lighting conditions. The addition of the translation inhibitor, cycloheximide, to the medium reduced significantly both nocturnal NAT activity and melatonin release, suggesting that de novo protein synthesis is produced daily during darkness. Actinomycin D, a transcription inhibitor, needs a longer time of action, because pre-existing mRNA must be depleted before the inhibition of melatonin release can be observed. The eyecup culture system is highly sensitive to light and chemical factors, which makes it particularly suitable as a model for the neurochemical analysis of melatonin biosynthesis in the retina of Rana perezi.

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.

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.

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.

Clonidine in vivo mimics the acute suppressive but not the phase‐shifting effects of light on circadian rhythm of serotonin N‐acetyltransferase activity in chick pineal gland

Comparative in vivo studies on effects of pulses of light and clonidine, a selective agonist of alpha 2-adrenergic receptors, on the circadian rhythm of serotonin N-acetyltransferase (NAT) activity in chick pineal gland were performed. Six-hour pulses of white light caused an acute suppression of the nocturnal NAT activity and phase-dependent phase shifts of the circadian rhythm of the enzyme activity relative to controls. Systemic administration of clonidine acutely suppressed NAT activity of chick pineal gland, but did not affect the phase of subsequent cycles in constant darkness. These results give further support to the concept based on in vitro studies that alpha 2-adrenergic receptors are involved in regulation of melatonin biosynthesis in chick pineal gland by a mechanism distal to the pacemaker that generates the circadian melatonin rhythm.

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

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

Effects of light and an alpha-2-adrenergic agonist on serotonin N-acetyltransferase activity in chick pineal gland

Pineal serotonin N-acetyltransferase (NAT) is the enzyme that catalyzes the production of N-acetylserotonin from serotonin and is the rate limiting step in the biosynthesis of melatonin in the chick pineal gland. Chick pineal NAT activity is decreased by light and by noradrenergic agents that act at the alpha-2-adrenergic receptor. Light-induced inhibition of nocturnal NAT activity can be demonstrated by exposing 4-day-old chicks to light, or by exposing pineal gland explants cultured in vitro either to light or to UK 14,304 (an alpha-2-adrenergic agonist). The inhibition by either light or UK 14,304 is preceded by a transient, but consistent rise in NAT activity. The paradoxical, transient light-induced rise in enzyme activity is mimicked by UK 14,304 which suggests a similarity in mechanisms of light and adrenergic signal transduction associated with the regulation of NAT activity.

Ontogeny of Light-Induced Decrease of N-Acetyltransferase Activity in Explanted Chick Pineal Glands

The ontogeny of chick pineal serotonin N-acetyltransferase (NAT) activity was investigated in explanted chick pineal glands at 4, 10 and 21 d of age. Nocturnal levels of the enzyme and the response of the enzyme to light exposure were determined in pineal glands maintained in short-term culture at each age. The results indicate that nocturnal NAT activity was increased in the glands from older birds. Nocturnal levels of NAT activity at the time of the initiation of the experiment were threefold greater in glands from 21-d-old birds as compared to that in glands from 4-day-old chicks. The response to light was similar in all three ages examined; light induced a significant decrease in NAT activity within 60 min in explanted glands from 4-d-old chicks and within 180 min in the glands from the 10- and 21-d-old chicks. A paradoxical transient increase in enzyme activity occurred immediately (within 5 min) following light exposure which was significant in the glands from the youngest chicks, and present, but more variable, in the older chicks. These data indicate that the nocturnal enzyme activity is greater in glands from older birds, but that light exposure of explanted glands initiates a transient rise followed by a decrease in NAT activity at all three ages.

Rapid recovery of pineal function after partial denervation: a possible role for heteroneuronal uptake of transmitter in modulating synaptic efficacy

The pineal gland is innervated by sympathetic neurons whose cell bodies are located in the two superior cervical ganglia and whose axons reach the gland via the two internal carotid nerves (ICNs). Bilateral decentralization of the superior cervical ganglia, produced by lesioning both cervical sympathetic trunks (CSTs), abolishes the circadian rhythm in the activity of the pineal enzyme serotonin N-acetyltransferase (NAT). We have examined the effects on NAT activity of unilaterally cutting the ICN or the CST. During the first night after either operation, nocturnal NAT activity was reduced by 75% compared to controls. However, during the second night after unilaterally cutting the ICN, NAT activity was restored to control values, and normal enzyme activity was seen in these lesioned animals for up to 1 month after this operation. On the other hand, following unilateral decentralization of one superior cervical ganglion, enzyme activity was reduced for at least 5 months. The high enzyme activity in animals with one ICN cut was abolished by cutting the contralateral CST, indicating that the recovery of NAT activity depended on the remaining intact sympathetic neurons. Electrical stimulation of the intact ICN during the daytime in animals in which the contralateral ICN was cut produced an increase in pineal NAT activity which was greater than the increase seen when similar stimulation was performed in sham-operated animals or in animals in which the contralateral superior cervical ganglion had been decentralized. The time course of the recovery of nocturnal NAT activity after unilateral denervation of the pineal gland was similar to the time course of the decrease in norepinephrine uptake sites in the gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Sympathetic reinnervation of the pineal gland after postganglionic nerve lesion does not restore normal pineal function

The activity of the enzyme serotonin N-acetyltransferase (NAT) in the rat pineal gland exhibits a large circadian rhythm, with peak activity occurring at night. This rhythm is dependent on stimulation of the pineal gland by neurons whose cell bodies are in the superior cervical ganglia and whose axons reach the gland via the internal carotid nerves (ICNs). Two days after both ICN were cut, crushed, or frozen, nighttime NAT activity was decreased by 90%. The remaining low level of enzyme activity was not affected by decentralization of the superior cervical ganglia. Thus, this enzyme activity did not depend on the activity of neurons in these ganglia. Bilaterally lesioning the ICN also abolished the neuronal uptake of norepinephrine in the pineal, further indicating that the sympathetic innervation of the gland had been destroyed. Three months after crushing both ICNs, nighttime NAT activity was only 20% of control values. However, in these animals, bilateral decentralization of the superior cervical ganglion reduced this low level of NAT activity by 90%. Thus, NAT activity, although low, was again dependent on sympathetic nerve stimulation. In contrast to this rather small recovery of nocturnal NAT activity, the norepinephrine uptake capacity of the gland recovered to 60% of control values. A similar discrepancy between the extent of recovery of NAT activity and of norepinephrine uptake was observed when the ICNs were frozen rather than crushed. To determine to what extent the sympathetic nerves that had reinnervated the pineal gland in these lesioned animals were capable of regulating NAT activity, their cervical sympathetic trunks were stimulated electrically at 5 Hz for 3 hr during the daytime.(ABSTRACT TRUNCATED AT 250 WORDS)