Effect of Anticonvulsant Drugs In Vivo On Rat Pineal N-Acetyltransferase (EC 2.3.1.5) and Hydroxyindole-O-Methyltransferase (EC 2.1.1.4)

The effect of anticonvulsant drugs on daytime and nighttime N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) levels was investigated. Some of the drugs caused a significant reduction in daytime NAT and HIOMT levels, possibly owing to beta-receptor blockade although other mechanisms cannot be excluded. Sulthiame caused an increase in daytime NAT levels, and this may represent a compensatory mechanism by the pineal gland to allow normal melatonin production. None of the drugs affected nocturnal HIOMT levels, and only phenobarbitone affected NAT levels, apparently causing a shift in the nocturnal peak later in the dark phase.     

Adrenalectomy Prevents Changes in Rat Pineal Melatonin Content N-Acetyltransferase Activity Induced by Acute Insulin Stress

The activity of N-acetyltransferase (NAT) the content of melatonin (MEL) in the rat pineal have been shown to be sensitive to several types of stressors. This study was designed to assess the role of the adrenals in mediating the effect of one such stressor, insulin-induced hypoglycemia, on pineal synthetic activity. Intact bilaterally adrenalectomized (ADX) adult male rats were kept under light:dark cycles of 14:10 (lights on 0600 h) injected intraperitoneally with 10 IU insulin at 1300 h, groups (n = 8) were killed 2, 3, or 4 h postinjection. Plasma catecholamines were assayed by means of high performance liquid chromatography radioimmunoassay was used to assess pineal NAT activity MEL content. All injected groups were rendered hypoglycemic by insulin administration. Compared to uninjected controls, plasma epinephrine in hypoglycemic intact rats rose after 2 h, whereas epinephrine did not change in hypoglycemic ADX animals. The increase in epinephrine in intact animals was correlated with a rise in NAT activity at 2 h. Moreover, pineal MEL content at 2, 3, 4 h was significantly greater than control values. In contrast, no changes in pineal biosynthetic function were found in ADX rats. This differential response by intact ADX rats suggests that an adrenal product (possibly epinephrine) is responsible for mediating the stimulatory effects of acute insulin-induced hypoglycemic stress on the rat pineal.     

Effect of Various Cations on the Activity of Pineal Gland N-Acetyltransferase (EC 2.3.1.5) and Hydroxyindole-O-Methyl Transferase (EC2.1.1.4) In Vitro

The effect of various cations on activity of hydroxyindole-O-methyltransferase (HIOMT) and N-acetyltransferase (NAT) from pineal gland was studied. Copper inhibited both enzymes while calcium and magnesium inhibited HIOMT; the mechanisms of these inhibitions are unknown at this stage. Calcium, magnesium, potassium, and sodium initially stimulated NAT activity, and as the cation concentration increased inhibitory activity was observed. These differential effects on NAT activity may in some way be involved in pineal regulation of cation levels.     

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.     

Improved Method for the Measurement of Chicken and Rat Pineal Serotonin N-Acetyltransferase Activity

EGTA, a nonspecific ion chelator, showed very good potential for preventing the loss of serotonin N-acetyl transferase activity (arylamine: acetyl-CoA:N-acetyltransferase, EC 2.3.1.5.) (SNAT) from rat and chicken pineal glands during preincubation at 37 degrees C before substrate addition. SNAT activity was higher than that of the controls when EGTA was present in the homogenization buffer due to the prevention of the loss of activity probably not only during the preincubation period but also during incubation with its substrates. This characteristic of EGTA suggests that its use is suitable in the improved SNAT assay described in this paper since higher SNAT activities were found in both rat and chicken pineal glands than the activities recorded with the usual methods.     

Responsiveness of Pineal N-Acetyltransferase and Melatonin in the Cotton Rat Exposed to Either Artificial or Natural Light at Night

In three separate experiments, the effect of acute exposure to either artificial or natural light during darkness of pineal N-acetyltransferase (NAT) activity and melatonin content was studied in the cotton rat (Sigmodon hispidus). The exposure of animals to an artificial-light irradiance of 160,000 microW/cm2 during darkness for either 1 s, 5 s, or 30 min was followed by a precipitous decline in pineal NAT activity and melatonin content when measured at either 15 or 30 min after light onset. When cotton rats were acutely exposed to light at night for 5 s, irradiances of either 3.2, 32, 320, and 3,200 did not suppress either pineal NAT or melatonin 30 min later; however, if the 5-s exposure had an irradiance of either 32,000 or 160,000 microW/cm2, the pineal enzyme activity and indole content were depressed. Moonlight, which had a maximal irradiance of 0.32 microW/cm2, was unable to suppress pineal NAT activity and melatonin content even when the animals were exposed to the moonlight for 30 min. The treatment of cotton rats with either norepinephrine or its agonist, isoproterenol, before their exposure to light at night retarded slightly the suppressive effect of light on the pineal constituents measured. Also, these drug treatments suppressed the pre-exposure levels of both NAT activity and melatonin content in the cotton rat pineal gland.     

Entrainment of the Circadian Rhythm in Rat Pineal N-Acetyltransferase Activity Under Extremely Long and Short Photoperiods

Entrainment of a pacemaker driving the circadian rhythm in rat pineal N-acetyltransferase activity was studied under extremely long and short photoperiods. Adult male rats maintained under the light-dark regime (LD) 18:6 or under the regime LD 6:18 were exposed to a 1-min light pulse at different times at night, then they were released into darkness, and the next night phase-shifts of the evening N-acetyltransferase rise and of the morning N-acetyltransferase decline caused by light pulses were determined. The evening rise was phase-delayed by at most 0.5 h under LD 18:6, but by as much as 2.8 h under LD 6:18. The morning decline was phase-advanced by at most 1.9 h under LD 18:6, but by as much as 3.5 h under LD 6:18. Hence, the magnitude of phase-shifts and consequently patterns of phase-response curves, which show possibilities of discrete entrainment, depend on the photoperiods under which animals are maintained. A 1-min light pulse applied within 1 h before the end of the dark period phase-advanced the morning N-acetyltransferase decline under LD 18:6 as well as under LD 6:18, while a pulse applied within 1 h after the beginning of the dark period phase-delayed the evening N-acetyltransferase rise only in rats maintained under LD 18:6, but not in those kept under LD 6:18. It seems that under very long photoperiods, the N-acetyltransferase rhythm may be entrained by evening as well as by the morning light, while under very short photoperiods the rhythm may be synchronized by morning light only.     

Production of Methoxyindoles In Vitro From Methoxytryptophan by Rat Pineal Gland

Pineal glands were incubated in the presence of [3H] methoxytryptophan with and without methoxamine, epinephrine, and norepinephrine. The beta-adrenoceptor-stimulated pineal glands were capable of converting methoxytryptophan to methoxytryptamine, melatonin, methoxyindole acetic acid, and methoxytryptophol, albeit in small quantities. Only methoxyindole acetic acid was detectable after incubation of unstimulated and alpha-adrenergic-agonist-treated pineal glands. These results support the proposal that melatonin can be formed from methoxytryptophan although this is a minor synthetic pathway, and the classic pathway from serotonin via N-acetylserotonin should be considered to be responsible for the majority of pineal melatonin production.     

Effects of Some Fatty Acids on the Serotonin N-Acetyltransferase Activity in Cultured Chick Pineal Glands

Low concentrations of arachidonate, oleate, or palmitate significantly affected the cycle of NAT activity of cultured chick pineal glands in different ways. The effects observed were altered by change of the lighting conditions of culture. Effects of arachidonate were also shown to be altered by change of the serum component of the culture medium. Effects of premature exposure to light of glands cultured under diurnal conditions of illumination were changed markedly by substitution of the serum component of the medium or a supplement of ionophore A23187 and less markedly by supplements of fatty acids.     

Opposite Effects of EGTA and Neutral Surfactants on the Loss of Chicken Pineal Serotonin N-Acetyltransferase Activity

The effects of some general purpose drugs on the deactivation and activity measurement of the chicken pineal gland enzyme serotonin N-acetyl transferase (EC 2.3.1.5.) were studied. The drugs used were EGTA and two neutral surfactants, Nonidet P40 and Triton X-100. Enzyme activity showed significant variations ranging from 2.8 +/- 1.3 nmol/gland/h when Nonidet P40 was added to the homogenate buffer, to 31.8 +/- 1.7 nmol/gland/h when EGTA was present. This striking variation seemed to be caused by the ability of these compounds to modify the rate of NAT deactivation acting either as accelerating agents, as in the case of the detergents or as braking agent, as in the case of EGTA.     

Interdependent Effects of the Ionophore A23187 and Serum on the Serotonin N-Acetyltransferase Activity of Cultured Chick Pineal Glands

Marked effects of the ionophore A23187 on the cycle of N-acetyltransferase (NAT) activity in cultured chick pineal glands were observed under three conditions of illumination. However, the effects were qualitatively and quantitatively dependent on the batch of fetal calf serum used in the medium and time of explanation into culture. Ionophore increased the level of NAT activity remaining in glands exposed prematurely to light regardless of the serum used. The ionophore suppressed the "spike" in cyclic GMP content of glands cultured in the dark, and extended the period of maximum cyclic GMP content of glands under diurnal illumination.     

Pharmacological Studies on the Regulation of N-Acetyltransferase Activity and Melatonin Content of the Pineal Gland of the Syrian Hamster

Thus far, all attempts to stimulate melatonin synthesis by beta-adrenergic receptor agonists in the Syrian hamster pineal gland have failed. Neither a wide range of dosages of isoproterenol (0.5 mg/kg to 24 mg/kg), nor prolonged treatment with norepinephrine, the natural neurotransmitter, increased N-acetyltransferase (NAT) activity or melatonin production. In the present study, the administration of isoproterenol at night was likewise ineffective in advancing or enhancing the normal nightly melatonin peak. Also, we did not find a delayed effect 7 or 8 h after the administration of the drug. Furthermore, we tested the idea of coneurotransmitters such as octopamine or dopamine being possibly necessary for stimulation, but could not find any effect of these substances on melatonin synthesis. In addition, a parasympatholytic agent, atropine, did not increase the responsiveness to sympathomimetic agents. Administration of a phosphodiesterase inhibitor was also ineffective in stimulating NAT activity. On the other hand, isoproterenol did retard the drop in NAT and melatonin after lights-on at night, indicating that beta-receptors are involved in maintaining elevated melatonin levels.     

Phase Shift of Daily Profiles of N-Acetyltransferase in the Rat Pineal Gland

Pineal N-acetyltransferase activity (NAT) has a circadian rhythm with peak values in the dark time and low values in the light time. NAT time profiles were measured in rats exposed to LD 14:10, to constant dark, and to acute (less than 48 hr) light-dark treatments. In all experiments, imposition of light suppressed NAT. The phase of the dark time NAT cycle was altered 2 hr or less by the following treatments: 3 hr light in the early subjective night, 3 hr light in the late subjective night, 2 hr or 6 hr light in the early subjective day, 4 hr early lights-on, 1 day of constant dark, or 1 day of constant light. When light was extended 4 hr into the dark time, NAT rose at lights out but fell again as the time of "expected" dawn approached. In contrast, the phase of the NAT cycle was shifted 12 hr (180 degrees) within 72 hr by reversing the phase of the light-dark cycle. NAT did not rise in the first dark period (coincident with the time of the subjective light time). The amplitude of the first shifted cycle was less than four control NAT profiles measured in rats kept in the original (unshifted) light-dark cycle.     

Status of Dopamine in Bovine Pineal Glands and the Stimulation of N-Acetyltransferase Activity by D2-Dopaminergic Receptor Agonists in the Rat Pineal Glands in Culture

In a previous study, we identified in the bovine pineal gland two [3H]spiroperidol-binding sites with KD values of 0.18 and 2.1 nM and Bmax values of 37 and 630 fmol/mg protein, respectively. In this study, the status of dopamine in the bovine pineal glands was delineated further by measuring the relative concentrations of dopamine and norepinephrine and the relative concentrations of serotonin and melatonin. Furthermore, the presence of 4.0 +/- 0.6 micrograms/dopamine/gm tissue encouraged us to delineate the effects of select dopaminergic receptor agonists and antagonists on the synthesis of melatonin in vivo and on the activity of N-acetyltransferase in the rat pineal gland in culture. The acute administration of haloperidol (3 mg/kg intraperitoneally [ip]) or sulpiride (200 mg/kg ip) increased the concentration of melatonin in the pineal gland from 160.6 +/- 8.18 to 327.6 +/- 45.43 and 306.5 +/- 40.53 pg/gland, respectively. Dopamine exhibited dual effects on the activity of N-acetyltransferase, inhibiting the basal activity at 0.1 microM and stimulating it at 10 microM, and the later effect was blocked by propranolol. D2-dopaminergic receptor agonists such as bromocriptine (4.0 microM) or LY-171555 (10.0 microM) partially attenuated the norepinephrine-induced stimulation of N-acetyltransferase, and these attenuating effects were reversed by D2-dopaminergic antagonists such as haloperidol (10 microM) or domperidone (10 microM). The results of these studies are interpreted to indicate that for the synthesis of melatonin, the pineal D2-dopaminergic receptors may function independently from those of the beta 1-adrenergic receptor sites. Furthermore, the said D2-dopaminergic receptor are amenable to down regulation since the activity of N-acetyltransferase remained unaltered (0.0717 vs. 0.0729 nmol/gland/h) following chronic treatment (4 mg/kg ip/day for 30 days) with bromocriptine.     

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.     

Comparison of the Effects of β-Adrenergic Agents on Pineal Serotonin N-Acetyltransferase Activity and Melatonin Content in Two Species of Hamsters

The nighttime rise in pineal melatonin levels can be blocked by administration of the β-adrenergic receptor antagonist, propranolol, in both Syrian hamsters and rats. Although the administration of β-adrenergic receptor agonists such as norepinephrine or isoproterenol stimulates pineal melatonin production in the rat, these drugs are without apparent effect on indole production in the Syrian hamster. To determine whether this lack of stimulatory effect in the Syrian hamster is characteristic of this species, a comparison of the effects of norepinephrine and isoproterenol on pineal serotonin N-acetyltransferase activity and melatonin content was conducted. In contrast to their lack of effect in the Syrian hamster, norepinephrine and isoproterenol stimulated pineal serotonin N-acetyltransferase activity and melatonin content in the Djungarian hamster. Hourly injection of norepinephrine during a continuation of light into the normal dark period stimulated increases in the activity of serotonin N-acetyltransferase and melatonin content in the Djungarian hamster but was without effect on these pineal parameters in the Syrian hamster.     

Investigation of Circadian Rhythms for Pineal 5-Hydroxytryptophol and Other Indoles in the Rat

5-hydroxytryptophol (5HTL) occurs in the pineal gland of the rat at levels comparable to those of melatonin, yet few studies have been conducted to investigate 5HTL as a potential alternative pineal hormone. In this study the pineals of 90-day-old male Sprague Dawley rats have been assayed by high performance liquid chromatography coupled with electrochemical detection. Significant (P less than .0001) circadian variation was measured in 5HTL levels, and a fivefold plateau elevation occurred during the middle of the light period. By comparison with the timing of the variations in N-acetyl serotonin and melatonin levels, it is suggested that 5HTL may not be regulated by simple competition with N-acetyl transferase for the common substrate 5HT but may, in fact, be regulated independently. Literature supporting such a suggestion, and a model incorporating it, are presented for discussion.     

Effects of Chemical and Surgical Ganglionectomy on Electrical Activity of the Pineal Gland of Male Rats

In order to elucidate further the role of sympathetic innervation for pineal function, the influence of sympathectomy on the spontaneous electrical activity of single cells in the pineal gland of adult male rats was investigated. Extracellular single-unit recordings were made during nighttime in the pineal gland of urethane-anesthetized, blinded adult male rats that had been treated neonatally with 6-hydroxydopamine, or that were ganglionectomized either during, or 12-16 h or 36-40 h, prior to the recording experiment. These experiments revealed that the excitatory influence of the sympathetic system on pineal nocturnal electrical activity can be abolished by either chemical sympathectomy of neonatal rats or surgical superior cervical ganglionectomy in adult animals.     

Immunoreactive Levels of Pineal Arginine Vasopressin Change During the Rat Estrous Cycle

It has been reported that mammalian pineal activity is influenced by reproductive hormones. However, even though a relationship between serum estrogen levels and pineal indole metabolism is well documented, little is known about other pineal components. Among peptides, arginine vasopressin (AVP) has been identified in the pineal gland of several species. In the present work, variations of immunoreactive (IR)-AVP in the rat pineal during the estrous cycle were studied by radioimmunoassay. Pineal IR-AVP levels increased significantly at the proestrus afternoon, and returned to basal levels by the end of estrus. These variations were partially related to those of serum estradiol concentration. Although the functional role of AVP in the rat pineal has not been clearly elucidated, these findings provide an additional indication that it might be involved in the reproductive function of the female rat.     

The Effect of Copper on (3H)-Tryptophan Metabolism in Organ Cultures of Rat Pineal Glands

Copper toxicity has been implicated in various neurodegenerative disorders such as Wilson's disease and Alzheimer's disease. Free copper in the brain is toxic and leads to neuronal and cellular damage, through free radical generation. Melatonin has been investigated as a possible copper ion chelator. Melatonin could prevent copper-induced neuronal and cellular damage through binding with copper and preventing copper-induced free radical generation. The effect of copper on pineal indolamine synthesis has not been studied extensively. In the present study, copper (2 mg/kg) and melatonin (12 mg/kg) were administered daily to Wistar rats for a 2-week and 6-week period. Pineal organ culture was utilized to monitor pineal indolamine synthesis. The pineals from the 2-week copper/melatonin-treated group showed a statistically significant decrease in 5-methoxytryptophol synthesis (p < 0.01), compared to the pineals from the copper-treated group. Conversly, in the 6-week experiment, 5-methoxytryptophol synthesis was increased in both the copper- and copper/melatonin-treated groups. There was a statistically significant decrease in the N-acetyl serotonin level in the pineals from the 6-week copper-treated animals, as compared to the control- and copper/melatonin-treated group (p < 0.01). These results imply that copper reduces N-acetyltransferase activity, which results in a decrease in N-acetyl serotonin synthesis. Melatonin when coadministered with copper appears to prevent the N-acetyltransferase inhibition by copper. Copper exerts contradictory effects on 5-methoxytryptophol synthesis. Further investigations need to be carried out to examine the effects of copper on the pineal enzymes.