Pineal N-acetyltransferase Research Articles

Carbaryl-induced changes in indoleamine synthesis in the pineal gland and its effects on nighttime serum melatonin concentrations

The effects of different doses of chronically administered carbaryl on rat pineal N-acetyltransferase (NAT) activity, hydroxyindole- O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during darkness (2300 h and 0100 h) when pineal melatonin synthesis is high were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Carbaryl was administered at total doses (over 6 days) of either 50, 125 or 250 mg/kg by gastric gavage. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light/dark cycles of 14:10 with lights off at 2100 h. Pineal NAT and HIOMT activities and pineal melatonin were increased at 0100 h following carbaryl administration at all three doses. Conversely, serum melatonin was increased at 2300 h after the 250 mg/kg dose of carbaryl while all three doses of the pesticide reduced serum melatonin levels at 0100 h. Pineal 5-HTP, 5-HT and 5-HIAA levels were usually increased at 2300 h but unaffected at 0100 h. The results indicate that carbaryl has significant effects on pineal melatonin synthesis and secretion.

Chronic administration of sublethal doses of carbaryl increases pineal N-acetyltransferase and hydroxyindole-O-methyltransferase activities and serum melatonin levels

The purpose of this study was to examine the effects of chronic administration of sublethal doses of carbaryl on pineal melatonin synthesis. N-methyl 1-naphthylcarbamate (carbaryl) (8.33 mg/kg B.W. daily) was administered orally to adult male albino rats for 6 successive days. Nocturnal (0100) N-acetyltransferase and hydroxyindole-O-methyltransferase activities were increased (roughly 75% and 60%, respectively) by carbaryl administration; likewise, carbaryl augmented serum melatonin levels at 2300. Pineal tryptophan. 5-hydroxytryptophan, serotonin, and 5-hydroxindole acetic acid levels were unaffected at all three time points. The results indicate that the carbamate pesticide, i.e., carbaryl, modifies pineal melatonin synthesis in vivo.

Effects of Diazepam Administration on Melatonin Synthesis in the Rat Pineal Gland in Vivo.

The effect of diazepam (DZP) on melatonin synthesis in rat pineal gland was investigated in vivo. Subcutaneous injection of DZP (3 mg/kg) 1 h before the start of darkness significantly suppressed nocturnal elevations of pineal N-acetylserotonin (NAS) and melatonin contents in rats, and caused a 2-h delay in reaching the maximum melatonin level in the dark phase. DZP treatment also markedly suppressed the dark-induced increase of pineal N-acetyltransferase activity, which catalyzes the rate-limiting step in melatonin synthesis, but had no effect on hydroxyindole-O-methyltransferase activity, which catalyzes the final step of melatonin formation. Pineal norepinephrine and dopamine contents, in contrast, were not altered by DZP injection. The distribution rate of DZP to the brain reached the highest level 30 min after a single injection, while that to the pineal gland was observed 5 h later (i.e., 4 h after the start of darkness). It is clear that the inhibitory effect of DZP on melatonin synthesis in rat pineal gland appears concomitantly with the increase in the distribution volume of DZP into this gland. These results suggest that the inhibitory effect of DZP on melatonin synthesis results from the drug's direct action on the rat pineal gland.

N-Methyl-<i>D</i>-Aspartate Receptor Participates in Neuronal Transmission of Photic Information through the Retinohypothalamic Tract

In order to assess the hypothesis that excitatory amino acids (EAA) are involved in the transmission of light information from retina to suprachiasmatic nucleus (SCN) and pineal via the retinohypothalamic tract (RHT), we have determined whether injections of EAA agonist into SCN could mimic the suppressive effects of light pulse on pineal melatonin production, and whether pretreatment with antagonists could block effects of light pulse in the intact rat. Injection of the EAA agonist N-methyl-D-aspartate (NMDA: 1.0 mM; 0.5 microliter) into the SCN suppressed plasma melatonin level and pineal N-acetyltransferase activity. The pretreatment with D-aminophosphonovalerate (D-APV: 2.5 or 10 mM; 2.0 microliters) or N-[1-(2-thienyl)-cyclohexyl]-piperidine (10 mM; 2.0 microliters) which are NMDA type receptor antagonists blocked the suppressive effect of the light pulse (3.0 Ix for 2 min), while the pretreatment with neither vehicle nor L-APV (optic isomer APV: 10 mM; 2.0 microliters) could block the effect of light. Alpha-D-glutamyl-amino-methylsulfonate (10 mM; 2.0 microliters or 25 mM; 2.0 microliters), which is a relative antagonist for non-NMDA type receptor, had no effect, either. These results suggest that EAA is involved in the transmission of light information through RHT and that in rat SCN EAA operates at the NMDA type receptor on the SCN.

Effects of Adrenergic Blockers or Bicuculline on Diazepam Induced Changes in Rat Pineal Melatonin Synthesis in Vivo and in Vitro.

The effect of propranolol (PPL), phenoxybenzamine (PBZ) or bicuculline (BCL) on the diazepam (DZP)-induced changes of pineal melatonin synthesis in male rats was examined in vivo and in vitro. Administration of PBZ did not affect the inhibitory action of DZP on pineal melatonin synthesis in vivo. A single injection of PPL inhibited the pineal melatonin synthesis similarly to the administration of DZP alone, but the two drugs together did not exhibit additive or synergistic effects on the melatonin synthesis. Significant decreases in the N-acetyltransferase (NAT) activity and the N-acetylserotonin (NAS) and melatonin contents were observed in the BCL-injected group, being greater than those in the DZP-treated group. Unexpectedly, however, the combination treatment of DZP and BCL causes an increase in the NAT activity and melatonin content compared with the BCL-alone group. Incubation with DZP at higher concentrations resulted in an increase of pineal NAT activity in vitro, but this increase was inhibited by preincubation with PPL, PBZ or BCL. DZP treatment thus appeared to have different effects on pineal NAT activity in vivo and in vitro. These results suggest that both a GABAergic mechanism and peripheral benzodiazepine (BZP) receptors in rat pineal gland may be involved in the modulation of melatonin synthesis by DZP.

Rate of re-entrainment of circadian rhythms to advances of light-dark cycles may depend on ways of shifting the cycles

When an 8-h advance of a light-dark (LD) cycle was accomplished by shortening of one dark period by 8 h, the rat pineal N-acetyltransferase rhythm was abolished during the first 3 subsequent cycles and reappeared in its original waveform during the fifth cycle only. When the 8-h advance shift was accomplished by lengthening of two consecutive light periods by 8 h, the N-acetyltransferase rhythm persisted and attained its original waveform by 2 days earlier than under the former shift. Re-entrainment is thus more rapid when the advance shift is accomplished by twice delaying than by once advancing of the LD cycle by 8 h.

Calcium influx through voltage-sensitive calcium channels regulates in vivo serotonin N-acetyltransferase (NAT) activity in hen retina and pineal gland

The involvement of transmembrane transport of Ca 2+ in regulation of retinal and pineal serotonin N-acetyltransferase (NAT) activity was studied in vivo in hens. Intramuscular administration to hens of organic antagonists of voltage-sensitive calcium channels (VSCC), nimodipine, nifedipine and verapamil, resulted in a marked inhibition of the nocturnal increase of NAT activity in retina and pineal gland. In both tissues the inhibitory effect of nimodipine on the night-time rise of NAT activity was abolished by Bay K 8644, a VSCC agonist. Bay K 8644 administered to hens at the end of the light phase of the light-dark cycle significantly enhanced the dark-dependent increase in retinal and pineal NAT activity. It is suggested that the nocturnal increase of NAT activity in hen retina and pineal gland is similarly regulated in vivo by Ca 2+ influx through the VSCC.

Type II Thyroxine 5′‐Deiodinase Activity in the Rat Brown Adipose Tissue, Pineal Gland, Harderian Gland, and Cerebral Cortex: Effect of Acute Cold Exposure and Lack of Relationship to Pineal Melatonin Synthesis

The effect of acute cold exposure for 6 hours on nocturnal type II thyroxine 5'-deiodinase (5'-D) activity was studied in brown adipose tissue (BAT), Harderian gland, cerebral cortex, and pineal gland of the rat. Moreover, the effect of iopanoic acid (IOP), a potent inhibitor of 5'-D activity, on both pineal N-acetyltransferase (NAT) activity and melatonin content in rats maintained in a cold environment was also examined. Results show that acute cold exposure significantly increases 5'-D activity in BAT but not in either the pineal gland, Harderian gland, or cerebral cortex. In all tissues, the injection of IOP reduced dramatically 5'-D activity, while exposure of the animals to light at night reduced 5'-D activity in pineal gland but not in either the Harderian gland or BAT while light exposure at night increased cerebrocortical 5'-D activity. Cold exposure did not change either pineal NAT activity or the melatonin content of the gland. Finally, when pineal 5'-D activity was inhibited by IOP treatment, neither nocturnal pineal NAT activity nor melatonin content was affected.

MRNA transcription determines the lag period for the induction of pineal melatonin synthesis in the Syrian hamster pineal gland

The nocturnal pattern of Syrian hamster pineal melatonin synthesis is characterized by a 6-8 h lag period, followed by a late-night, short-duration peak in both N-acetyltransferase (NAT) activity and melatonin content. Administration of cycloheximide (20 mg/kg body weight) given either at the time of lights out or 4 h into the dark phase to Syrian hamsters blocked the nocturnal increase in both pineal NAT activity and melatonin content. Actinomycin D (5 mg/kg body weight) prevented the nocturnal increase in both constituents only when it was administered at darkness onset, being significantly less effective when injected after 4 h of dark exposure. Reinduction of melatonin production by isoproterenol (2 mg/kg body weight) administration to acutely light-exposed animals during late darkness was prevented by cycloheximide, but not by actinomycin D administration. The results suggest that whereas Syrian hamster pineal melatonin production requires protein synthesis both early and late in the dark phase, the transcription of a putative NAT-related mRNA, which occurs only during the early night, seems to determine the lag period in melatonin synthesis and pineal responsiveness to beta-adrenergic receptor agonist stimulation.

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.

Nocturnal increase in pineal melatonin production in two lemming species, Dicrostonyx hudsonius and D. groenlandicus

Pineal N-acetyltransferase (NAT) activity and melatonin levels and thyroxine 5′-deiodinase (5′D) activity in brown adipose tissue (BAT) were compared during the day and night in two species of lemmings, Dicrostonyx hudsonius and D. groenlandicus. In both species, NAT and melatonin exhibited significant night-time increases in the pineal gland. Thyroxine 5′D activity in BAT did not show a statistically significant day-night difference in either species of lemming, suggesting the night-time rise in melatonin does not induce a similar increase in BAT 5′D.

Posthatch Day/Night Differences in Synaptic Ribbon Populations of the Chick Pineal

Pineal synaptic ribbon (SR) populations of the early posthatch white leghorn chick were counted to determine if they demonstrate a rhythm that is in accordance with the light/dark cycle. SRs were counted between day 7 and day 10 and on day 14 of posthatch development, with samples at midlight, middark (14L:10D), and constant darkness. SR populations did not exhibit significant changes on days 7 and 8 under cycled lighting conditions nor on days 9 and 10 under constant darkness. A second experiment demonstrated that the dark:light ratio of SR populations of day 14 chicks, under cycled lighting, was 3.4:1.0, indicating SR rhythmicity by that stage of development. In that a preliminary experiment had demonstrated a 4.2:1.0 dark:light ratio in SR populations in a predominantly day-10 population of chicks, we believe that SR rhythmicity begins on, or near, day 10 of posthatch development. To determine if the invasion of sympathetic fibers from the superior cervical ganglion (SCG) correlates with the initiation of SR light/dark population differences, we employed tyrosine hydroxylase immunofluorescence to reveal the distribution of catecholaminergic fibers in chick pineal follicles. Follicular innervation doubled over the day 7 to day 14 period, during which time light/dark differences in SR populations were established. There is a correlation, in time, between the invasion of the pineal by the sympathetic fibers and the initiation of SR light/dark differences. The circadian rhythm of pineal N-acetyltransferase (NAT) activity, the rate-limiting enzyme in the melatonin pathway, is established earlier (day 2) than the light/dark differences in SR populations (day 10). It is possible that SR rhythmicity is influenced by the ingrowth of the pineal sympathetic innervation, and that SRs respond to an extrapineal oscillator rather than the independent oscillators of the chick pineal responsible for the rhythm of NAT activity and melatonin synthesis.

The rat pineal N-acetyltransferase rhythm persists after a five-hour, but disappears temporarily after a seven-hour advance of the light-dark cycle: A six-hour shift may be a turning point

After a 5-h advance of a lighting regime with 12 h of light and 12 h of darkness by shortening of one dark period, the rat pineal N-acetyltransferase (NAT) rhythm was retained, though at the beginning in a changed waveform, and re-entrained to the shift within 5 to 6 cycles. After a 7-h advance of the regime, the NAT rhythm faded away between the 2nd and 4th cycle and re-entrained to the shift within 7 cycles. A 6-h advance of the lighting regime might thus be a turning point for the persistence or temporal disappearance of the NAT rhythm.

The role of the hypothalamic paraventricular nuclei for the regulation of pineal melatonin synthesis: New aspects derived from the vasopressin-deficient Brattleboro rat

There is evidence for an involvement of the hypothalamic paraventricular nuclei (PVN) in the regulation of pineal melatonin synthesis in rats. Since electrical stimulation of the PVN or the systemic administration of arginine-vasopressin (AVP) result in a depression of the nocturnal melatonin surge, this neuropeptide appears to be pivotal for the transduction of PVN-efferent, pinealopetal signals. We therefore used an AVP-deficient animal model, the Brattleboro rat, to further investigate the mechanisms responsible for pineal regulation. Anesthetized adult male animals received 2 min of bilateral electrical stimulation of the PVN either during the day or at night. Thirty min later, pineal glands were removed and pineal N-acetyltransferase (NAT) activities and melatonin contents were determined. Stimulation resulted neither during the day nor at night in any significant alterations of pineal NAT activity or melatonin content when compared to control or sham-stimulated animals. These data further support the proposed modulatory role of AVP for the regulation of melatonin synthesis in the Epiphysis cerebri of genetically intact rats.

Light-Induced Changes in Pineal Gland N-Acetyltransferase Activity: Developmental Aspects

In adult rats, light acting via a retino-pineal gland neural pathway influences pineal gland biochemistry in two ways: (1) it entrains endogenous circadian rhythms in melatonin biosynthesis to the environmental photoperiod and (2) exposure to even very brief periods of light during the nighttime rapidly suppresses the high levels of nocturnal melatonin production. The present studies were undertaken to determine precisely when photic stimulation first influences the enzymic activity of N-acetyltransferase (NAT), the pineal gland enzyme which rate-limits the overall biosynthesis of the hormone melatonin, and to examine some of the cellular mechanisms which might mediate light-induced effects in neonatal animals. Rats of different ages were either killed during the light phase or were exposed to darkness or light for 1 min during the dark phase of the lighting cycle, returned to their litters in darkness for 30 min and then killed. Pineal gland NAT activity in animals nocturnally exposed to 1 min of light was suppressed in animals 6 days of age or older. Nocturnal light exposure did not suppress enzyme activity in 3- to 5-day-old rats, even though these animals displayed clear light:dark differences in pineal gland NAT activity. Nocturnal light exposure also did not suppress nighttime levels of NAT activity in 7-day-old animals who had been bilaterally enucleated, suggesting that this effect is retinally mediated. Pretreatment of 7-day-old animals with the beta-noradrenoceptor agonist drug, isoproterenol, prevented the nocturnal light-induced suppression of NAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute cold exposure at night on pineal N-acetyltransferase activity and melatonin content in white-footed mice, Peromyscus leucopus

1. 1. Pineal N-acetyltransferase (NAT) activity in long-day white-footed mice ( Peromyscus leucopus) exposed to cold soon after onset of darkness was depressed relative to controls, whereas mice exposed to cold later at night had slightly elevated enzyme activity. 2. 2. NAT activity in short-day mice exposed to cold soon after lights off did not differ from controls. Pineal melatonin in these mice, however, did not rise, as it did in controls. 3. 3. These results suggest that acute cold exposure may modulate NAT activity, which is controlled primarily by the L:D cycle.

Effect of 6‐Methoxy‐2‐Benzoxazolinone on the Activities of Rat Pineal N‐Acetyltransferase and Hydroxyindole‐O‐Methyltransferase and on Melatonin Production

A naturally occurring compound, 6-methoxybenzoxazolinone (6-MBOA), present in grasses, has been shown to induce sexual maturation in a number of rodent species. The structural similarity of 6-MBOA and melatonin has led researchers to suspect that 6-MBOA might induce its progonadal effects by directly altering pineal function. Previous studies have shown that 6-MBOA has the properties of a weak beta-adrenergic agonist capable of stimulating rat pineal N-acetyltransferase (NAT) activity at pharmacological concentrations of 10(-3) M. In the present study we have examined the effect of 6-MBOA on both the pineal melatonin synthesizing enzymes, namely, NAT and hydroxyindole-O-methyltransferase (HIOMT) as well as on melatonin production, in organ cultured rat pineal glands. In addition, we have also examined the ability of 6-MBOA to displace a ligand from rat brain alpha- and beta-adrenoceptors. Our results confirm that 6-MBOA stimulates NAT activity and melatonin production at the high concentration of 10(-3) M. It appears to have no effect on HIOMT activity. A competition study shows that 6-MBOA is able of displacing ligands at the alpha- and beta-adrenoceptors but only at concentrations greater than 10(-4) M. Whether such high concentrations of 6-MBOA reach the pineal of rodent in their natural habitat is unknown. However, if 6-MBOA does mediate progonadal effects by altering pineal function it would be expected that 6-MBOA would ultimately inhibit the effects of melatonin. The possibilities are that the high melatonin levels induced by 6-MBOA cause desensitization of melatonin receptors or that 6-MBOA is an antagonist at the level of the melatonin receptor, thus restricting the inhibitory effects of melatonin on the reproductive system.

Melatonin Entrains the Circadian Rhythm in the Rat Pineal N-Acetyltransferase Activity

It has been recently suggested that in mammals the pineal hormone melatonin may be involved not only in transduction of a photoperiodic information, but in a subtle modulation of a phase of the circadian system as well. This suggestion has been based mainly on data about entrainment of the locomotor activity rhythm by melatonin. The present study was undertaken to check whether melatonin may phase-shift also other circadian rhythms, namely the rhythm in pineal N-acetyltransferase activity which is supposed to drive the rhythmic melatonin production in the rat. 50-day-old male Wistar rats were maintained on a regimen of 10 h of light and 14 h of darkness per day. Vehicle or melatonin (1mg/kg rat weight) was injected subcutaneously just before the dark onset. After a single melatonin injection or after administration of melatonin for 5 successive days or after a 4-day treatment with melatonin and a 1-day withdrawal, the evening N-acetyltransferase rise was phase-advanced relative to the rise in rats treated with vehicle only; the phase shift was larger after a repeated than after a single melatonin injection. Under all above-mentioned paradigms of melatonin administration, the morning N-acetyltransferase decline was less phase-advanced than the evening rise. Persistence of the phase advance of the N-acetyltransferase rise after withdrawal of melatonin treatment and a larger phase shift after a repeated than after a single melatonin administration suggest that melatonin may entrain directly a circadian pacemaker controlling the N-acetyltransferase rhythm and affect thus its own rhythmic production.

Aging alters resynchronization of the circadian system in rats after a shift of the light-dark cycle

Four days following an 8-h advance of the light-dark cycle, the circadian rhythms in the pineal N-acetyltransferase activity and melatonin content reappeared in 7-week-old rats, but were still abolished in 24-month-old animals.