Evidence that interferon-gamma alters pineal metabolism both indirectly via sympathetic nerves and directly on the pinealocytes.

1. Interferon-gamma (IFN-gamma) has been shown to suppress N-acetyltransferase (NAT) activity in cultured rat pineal glands when stimulated with isoproterenol (ISO). 2. Conversely, IFN-gamma has also been shown to increase the melatonin content of the rat pineal gland in organ culture. 3. Circumstantial evidence leads to a hypothesis that the NAT suppressive effect may be due to the action of IFN-gamma on the sympathetic nerve terminals. 4. To test this hypothesis, pineal glands from intact (INT) and superior cervical ganglionectomized (SCGX) rats, which had been operated 5 days earlier, were cultured with either ISO or ISO + IFN-gamma. 5. The concentration of ISO was 10(-8) M and that of IFN-gamma was 300 antiviral units/ml. 6. The pineals were incubated for a total period of 5.5 hr, after which the activities of NAT and hydroxyindole-O-methyltransferase (HIOMT) and the levels of melatonin and cAMP were estimated. 7. Suppression of NAT by IFN-gamma was observed in the pineals from INT rats, but not in those from SCGX animals. 8. IFN-gamma significantly enhanced melatonin levels over those in ISO-stimulated pineals and culture media from the SCGX animals, but not from the INT animals. 9. IFN-gamma treatment had no effect on either the HIOMT activity or cAMP levels. 10. The results indicate that the IFN-gamma-induced NAT suppression requires the integrity of the sympathetic nerve terminals and the IFN-gamma-induced enhancement of melatonin production is accomplished through its direct action on pinealocytes.     

A specific and sensitive enzymatic-isotopic microassay of coenzyme A in pineal gland

The relationship between the concentration of CoASH and the activity of serotonin N-acetyltransferase (NAT) was studied in rat pineal glands in culture. A technique for microdetermination of CoASH was developed by utilizing acetyl CoA synthetase and partially purified rat liver NAT. Initially CoASH was acetylated with [1–³H] acetate using acetyl CoA synthetase. Subsequently, the labelled acetyl group was transferred from [1–³H] acetyl CoA to tryptamine forming [1–³H acetyl-tryptamine which was then extracted into chloroform and measured by scintillation spectrometry. A direct relationship appeared to exist between the concentrations of CoASH and [1–³H] acetyltryptamine. This method is sensitive and specific since it can detect as low as 10–15 pmoles of CoASH but not structurally related substances such as acetyl CoA, ADP, cysteamine, or D-pantothenic acid. After treating the rat pineal glands in culture with 10 μM norepinephrine for six hours, the concentration of CoASH was found to decrease significantly from 31.96 ± 0.68 to 24.44 ± 0.37 pmoles/gland, while the activity of NAT increased 68 fold. This inverse relationship indicates that CoASH does not play a direct role in NAT induction although it does protect darktime NAT activity in pineal homogenates against thermal inactivation. The sensitivity and the adaptability of this method can be utilized to measure CoASH in discrete regions of rat brain and in experimental conditions where the micromeasurement of CoASH may be required.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

Changes in pineal indoleamine metabolism in vitamin A deficient rats

Weanling, male rats were fed a vitamin A deficient (VAD) diet from 20 to 77 days of age. The circadian rhythms of the precursors and metabolites of pineal melatonin were measured along with the activity of N-acetyltransferase (NAT). Significant decreases in peak melatonin levels (0100 hours) and in nightime NAT activity (0100 and 0300 hours) were found in the pineals of the VAD rats. In contrast, the contents of serotonin, 5-hydroxytryptophan and 5-hydroxyindole acetic acid were only moderately affected by the deficiency. Daily administration of 25 micrograms melatonin from 20 to 74 days of age markedly reduced NAT activity in control and VAD rats. These data suggest that NAT activity is more sensitive to chronic VAD than any other parameters of melatonin metabolism.     

Rapid and simple measurement of serotonin N-acetyltransferase activity by liquid biphasic diffusion assay

We report here a rapid, simple, and accurate method to assay for serotonin N-acetyltransferase (NAT) activity. This assay relies on the selective diffusion of radiolabeled acetyltryptamine into a water-immiscible scintillation fluid. Unlike organic solvent extraction, thin-layer chromatography, or high performance liquid chromatography, the separation of acetyltryptamine from acetyl CoA and tryptamine is not required in the method. Moreover, the limit of sensitivity is less than 4 pmol of N-acetyltryptamine formed per sample. Enhancement of NAT activity upon β-adrenergic receptor stimulation in the rat pineal gland was clearly detected with this method. In addition, the NAT activity measurements obtained with this method agreed quantitatively in the pineal gland and other brain tissues with the conventional organic solvent extraction method. The results suggest that this liquid biphasic diffusion assay is applicable to the detection of NAT activity in tissues and cells.     

Rat serum stimulates serotonin N-acetyltransferase activity in pineal monolayer cultures

The effects of rat serum on serotonin N-acetyltransferase (NAT) activity and indole synthesis in monolayer cultures of neonatal rat pineal glands was examined. The addition of 5% rat serum to these cultures resulted in stimulation of NAT activity equal to that obtained with optimal concentrations of the adrenergic agonist norepinephrine (NE). Rat serum also increased the synthesis of both N-acetylserotonin and melatonin from tryptophan. Stimulation of NAT activity by rat serum was partially blocked by metoprolol and propranolol, but not by phentolamine or butoxamine. The serum factor responsible for the stimulation was stable to both freezing and boiling. No significant amounts of epinephrine, norepinephrine, or dopamine were detected in the serum.     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.     

Ontogeny of N-acetyltransferase activity rhythm in pineal gland of chick embryo

1. N-acetyltransferase was present in pineal glands of 14-day-old chick embryos though no rhythm either in LL, DD or LD 12:12 was observed in this age. 2. Daily rhythm in pineal NAT activity was found in 18-day-old embryos incubated under LD 12:12 and LD 16:8 but no NAT rhythm was detected in DD or LL. 3. NAT rhythm persists for 2 days in constant darkness and it may be circadian in nature. 4. Presence of melatonin (85 +/- 8 pg/mg tissue) was detected in pineals of 18-day-old chick embryos.     

Vasoactive intestinal peptide stimulation of pineal serotonin-N-acetyltransferase activity: general characteristics

Vasoactive intestinal polypeptide (VIP) stimulates basal serotonin-N-acetyltransferase (NAT) activity in pineals in organ culture and enhances the effects of catecholamines in inducing the enzyme. VIP appears to act postsynaptically; its action is independent of the beta receptor and is dependent upon protein synthesis. Its effects may be mediated by a receptor. The magnitude of the pineal response to VIP varies with age, is greater in pineals maintained in 48-h organ culture than in those in acute culture, and can be detected in pineals from newborns after 48-h organ culture. Intravenous administration of VIP can increase pineal NAT activity in vivo.     

Neonatal Hormone Treatment and Maturation of the Pineal Noradrenergic System: Hydrocortisone and Thyroxine

Abstract The circadian release of norepinephrine from nerve terminals in the pineal gland drives acetyl-CoA:serotonin N -acetyltransferase (NAT; EC 2.3.1.5) activity in the adult pineal from a daytime low to a nighttime high. In the newborn, enzyme activity is intermediate between the adult's daily extremes and has only a small circadian fluctuation. With age, these fluctuations increase in amplitude until the adult pattern is attained at about days 10–12. Treatment of neonates with thyroxine for the first 3 days of life accelerated, whereas administration of hydrocortisone acetate at birth retarded the developmental decline in daytime serotonin-N-acetyltransferase activity. Maximal differences in daytime enzyme activity of controls and thyroxine-treated animals were seen at day 4 and between controls and steroid-treated pups at day 8. Desipramine treatment increased NAT activity in 8-day-old animals; hydrocortisone-treated animals were least affected. Freshly cultured pineals from steroid-treated animals were more responsive to low, and less responsive to high, concentrations of norepinephrine than glands from thyroxine-treated or control animals. They were also less responsive to isoproterenol both in acute and 48-h organ culture. Pineals from hydrocortisone-treated animals in culture accumulated less exogeneous norepinephrine than glands from controls but released a greater fraction of their content on transfer to fresh medium. Normal and steroid-treated animals released the same fraction of their norepinephrine contents into the medium when reuptake was blocked by desipramine (DMI).     

Influence of 4-day long treatment with vasoactive intestinal peptide on ultrastructure and function of the rat pinealocytes in organ culture

Vasoactive intestinal peptide (VIP) is one of neuropeptides involved in the regulation of the pineal gland function. The acute treatment of rat pinealocytes with VIP caused changes in their biochemical parameters. The present study concerns the effects of the chronic treatment with VIP on ultrastructure and function of the rat pinealocytes in organ culture. The pineals of adult male rats were assigned to one of three groups and placed in organ culture for four consecutive days. The pineals of the first group were incubated in the control medium, the pineals of the second group--12 hrs in control medium and 12 hrs in medium with 1 microM VIP (between 20.00 and 8.00) during each day, the pineals of the third group--24 hrs per day in medium with 1 microM VIP. The melatonin concentration was measured using RIA and activity of enzymes using radiochemical methods. Point count method was used in quantitative ultrastructural analysis. Both modes of chronic treatment with VIP increased significantly the level of melatonin secretion during four days of the culture and the content of this hormone in the pineal explants at the end of the experiment. Treatment with the neuropeptide for 12 hrs and 24 hrs per day elevated also the activity of arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. On the other hand, VIP had no effect on the activity of arylamine-N-acetyltransferase. VIP increased the relative volume of rough endoplasmic reticulum, Golgi apparatus and mitochondria and did not influence the relative volume of lysosomes and lipid droplets as well as the numerical density of dense core vesicles in the examined rat pinealocytes. The obtained results indicate stimulatory effect of chronic treatment with VIP on the synthesis and secretion of melatonin in the rat pinealocytes in vitro. The results of morphological study are in agreement with the obtained biochemical data and point to the increase in secretory and metabolic activity of the rat pinealocytes in response to VIP.     

Adrenergic Regulation of the Reduction in Acetyl Coenzyme A: Arylamine 7V-Acetyltransferase Activity in the Rat Pineal

Abstract: Pharmacologically active agents were employed to study the mechanisms that control the reduction in levels of acetyl-coA: arylamine N-acetyltransferase activity (NAT) (EC 2.3.1.5) in the rat pineal. Pretreatment of rats with phenoxybenzamine or phentolamine prevented the rapid light-mediated decrease in NAT activity, although pretreatment with yohimbine or atropine did not alter this effect of light. Administration of mecamylamine resulted in a rapid reduction in enzyme activity prior to light exposure. When clonidine was administered intraperitoneally to animals with elevated NAT levels, there was a rapid decrease in enzyme activity, mimicking the effects of light. However, intraperitoneal injections of norepinephrine, methoxamine and phenylephrine into similar groups of animals had no significant effect on enzyme acitivity. When clonidine and norepinephrine were administered intraventricularly, there was a rapid reduction in enzyme activity. On the other hand, intraventricular administration of phenylephrine did not result in reduced enzyme activity. Pretreatment of animals with phenoxybenzamine failed to block the reduction in NAT activity precipitated by low doses of clonidine. This clonidine-mediated reduction in enzyme activity was, however, blocked by yohimbine. When animals were simultaneously exposed to light and administered clonidine, the rapid reduction in NAT activity was affected only when animals were pretreated with both yohimbine and phenoxybenzamine. In contrast to the decrease in pineal NAT activity observed in in vivo preparations, incubation of pineals with clonidine in an organ culture system produced a moderate, but consistent, rise in enzyme activity. These results suggest that stimulation of a receptor with α-adrenergic characteristics mediates the reduction in NAT activity produced by light. Stimulation of yet a second adrenergic-like receptor appears to mediate a reduction in pineal NAT activity precipitated by clonidine. Our evidence suggests that one or both of these receptors are located within the central nervous system.     

Stimulatory effects of LH on release of melatonin and activities of its synthesizing enzymes NAT and HIOMT in organ-cultured pineal glands of female rats.

The pineal hormone melatonin (N-acetyl-5-methoxytryptamine) exerts antigonadotropic effects in some mammalian species. To evaluate the effect of luteinizing hormone (LH) on melatonin release and its synthesizing enzyme activities in pineal glands, pineals of adult female rats undergoing diestrus were organ-cultured in a medium containing 10(-12), 10(-10) or 10(-8) M LH for 6 h. Melatonin release increased significantly in pineals cultured with 10(-12) and 10(-10) M LH, as compared to control values. Similarly, the activity of arylalkylamine N-acetyltransferase (NAT), the key regulatory enzyme in melatonin biosynthesis, was significantly higher in pineals cultured with 10(-12) and 10(-10) M LH for 6 h, while LH at 10(-8) M had no effect. Although LH at 10(-10) M increased pineal hydroxyindole-O-methyltransferase (HIOMT) activity, which catalyzes the final step of melatonin biosynthesis, LH at 10(-12) and 10(-8) M had no effect. These results demonstrate that at relatively low physiological levels, LH stimulates pineal melatonin synthesis and release, mainly by increasing NAT activity.     

Phorbol Esters Mimic α-Adrenergic Potentiation of Serotonin N-Acetyltransferase Induction in the Rat Pineal

alpha-Adrenergic stimulation of the rat pineal gland is known to stimulate phosphatidylinositol turnover and to potentiate the induction of serotonin N-acetyltransferase (SNAT) activity evoked by submaximal beta-adrenergic stimulation. In some (other) systems tumor-promoting phorbol esters are known to mimic physiologic stimulation and to enhance specifically the activity of protein kinase C. Here it is shown that phorbol esters specifically mimic the potentiating effect of alpha-adrenergic stimulation on SNAT activity in the rat pineal. These effects contribute to the argument for a role for phosphatidylinositol turnover and protein kinase C in mediating alpha-adrenergic stimulation.     

Effects of luteolin on arylamine N-acetyltransferase activity in rat blood and liver.

In this study we investigated inhibition of Arylamine N-acetyltransferase (NAT) activity in rat blood and liver tissue cytosols by luteolin. Using high-performance liquid chromatography, NAT activity for acetylation of 2-aminofluorene and remaining unacetylated 2-aminofluorene were examined. The NAT activity in rat blood and liver tissue was inhibited by luteolin in a dose-dependent manner: higher concentrations of luteolin in the reaction resulted in greater inhibition of NAT activities in both examined tissues. The data also indicated that luteolin decreased apparent Km and Vmax of NAT enzymes from rat blood and liver tissue cytosols. This report is the first demonstration that luteolin can affect rat blood and liver tissue NAT activity.     

Interaction Between Adrenergic and Peptide Stimulation in the Rat Pineal: Pituitary Adenylate Cyclase-Activating Peptide

Abstract: The 27 amino acid peptide, pituitary adenylate cyclase-activating polypeptide (PACAP-27), and its 38 amino acid analogue, PACAP-38, stimulate serotonin- N -acetyltransferase (NAT) activity and N -acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP-38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N-terminal histidine C-terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP-38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP-38 reduces the magnitude of subsequent stimulation with PACAP-38 or forskolin. Concurrent stimulation of α-receptors or treatment with active phorbol ester augments rat pineal response to PACAP-38 stimulation just as it increases the response to VIP, PHI, and β-receptor stimulation. Pineals from newborn rats respond to PACAP-38 with an increase in NAT activity and the increase is augmented by concomitant α₁-adrenergic stimulation. The putative PACAP inhibitor PACAP (6–38) and the putative VIP inhibitor (Ac-Tyr, d -Phe)-GRF 1–29 amide, in 100–1,000-fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.     

Vitamin C Inhibited DNA Adduct Formation and Arylamine N-Acetyltransferase Activity and Gene Expression in Rat Glial Tumor Cells

Studies have been demonstrated that vitamin C (ascorbic acid) exhibit the protective role of vin in certain types of cancer. Rat glial tumor cells also have been shown have N-acetyltransferase activity. In this study, we reported the effects of vitamin C on arylamine N-acetyltransferase (NAT) activity and DNA adduct formation in rat glial tumor cell line (C6 glioma). The activity of NAT was measured by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene and p-aminobenzoic acid and nonacetylated 2-aminofluorene and p-amonibenzoic acid. Rat C6 glioma cells were used for examining NAT activity and gene expression and 2-aminofluorene-DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene-DNA adduct formation in C6 glioma cells were inhibited and decreased by vitamin C in a dose-dependent manner. But vitamin C did not affect NAT gene expression in examined cells. The apparent kinetic parameters (apparent values of Km and Vmax) from C6 glioma cells were also determined with or without vitamin C cotreatment. The data also indicated that vitamin C decreased the apparent values of Km and Vmax from C6 glioma cells.     

STIMULATION OF SEROTONIN N-ACETYLTRANSFERASE IN PINEAL ORGAN CULTURE BY DRUGS

Abstract— Drugs such as cocaine, procaine, pheniprazine (Catron) and veratridine, which have actions on sympathetic nerves and nerve terminals, were examined for their ability to increase serotonin N-acetyltransferase (EC 2.3.1.5; NAT) in pineal organ culture. The absence of potassium (0 KCl) was also examined. NAT is known to respond to β-adrenergic stimulation. It was found that these drugs and 0 KCl increased the enzyme activity 100 to 2000-fold in innervated pineals but had virtually no effect in denervated pineals. The effects on innervated pineals were blocked by the β-blocker propranolol but not by the α-blocker, phentolamine. These drugs and 0 KCl inhibited to varying degrees [³H] 1-norepinephrine uptake in pineals. It is concluded that these agents activated the β-adrenergic receptor on pineal cells by causing an accumulation of extraneuronal norepinephrine. The accumulation of norepinephrine is due, at least in part, to the blockade of norepinephrine reuptake by nerve terminals. The ability of veratridine to stimulate NAT and to inhibit norepinephrine uptake was reversed by tetrodotoxin, a blocker of sodium permeability in excitable tissue, thus veratridine acts by increasing sodium permeability in nerve terminals. This adds support to the theory that catecholamine uptake is a process that requires a sodium gradient across the nerve terminal membrane.     

Effects of Steroids on Serotonin-N-Acetyltransferase Activity of Pineals in Organ Culture

Treatment of neonatal, but not adult, rats with glucocorticoids decreases the rise in pineal serotonin N-acetyltransferase activity upon stimulation with beta agonists. Pineals in organ culture and exposed to steroids also show a dose-dependent decrement in response to beta agonists which increases with steroid exposure time. Pineals from neonatal and adult animals are equally sensitive. The effects of steroids on pineals in organ culture appear to be reversible, and the order of potency of different steroids differs from that observed when steroids are administered in vivo. Both in vitro and in vivo steroids appear to act at a site after cyclic AMP generation. Hydroxyindole-O-methyltransferase activity in the adult pineal does not appear affected by steroid exposure.     

Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Spermophilus richardsonii)

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.