Serotonin N-acetyltransferase Activity Research Articles

Regulation of the diurnal cycle in activity of serotonin acetyltransferase in the chick pineal gland

When chick pineal glands were explanted into organ culture at midlight phase of a diurnal cycle of illumination and incubated in the dark, they developed marked increases in serotonin acetyltransferase (acetyl coA:arylamine N-acetyltransferase; EC 2.3.1.5) activity. Either this increase in activity was inhibited or its onset was retarded in glands incubated under constant illumination. Supplements of theophylline, isobutylmethylxanthine, quinidine, and compound Ro 20-1724 (4-(3-butoxyl-4-methoxybenzyl)-2-imidazolidinone) elicited very marked increases in serotonin acetyltransferase activity in glands cultured in the dark. Levels of activity attained after 6 h in culture approached or exceeded the maximum levels attained at middark phase of the diurnal cycle in vivo. Effects of theophylline and compound Ro 20-1724 were additive. Supplements of dibutryl cAMP had little or no effect upon levels of serotonin acetyltransferase activity when tested alone or in combination with theophylline but further enhanced the increase in the level of enzyme activity elicited by Ro 20-1724. Adenosine and cAMP had little or no effect upon levels of serotonin acetyltransferase activity. It is concluded that levels of serotonin acetyltransferase activity in the chick pineal gland are regulated by a repressive, negative-control mechanism, which probably involves a membranous adenosine receptor.

Sensitivity and cyclic nucleotides in the rat pineal gland.

beta-Adrenergic stimulation induces serotonin N-acetyltransferase (SNAT) activity in the rat pineal gland. The magnitude and some of the characteristics of this response vary as a function of the gland's previous exposure to stimulation. A period of stimulation results in a subsensitive response to subsequent stimulation. A period without stimulation provides a supersensitive response to subsequent stimulation. Investigations concerned with the mechanisms regulating the rat pineal's sensitivity to beta-adrenergic stimulation are described. These have focused on the regulation of cyclic AMP metabolism. Several of the components involved in the induction of SNAT activity appear to participate in the regulation of sensitivity. These include the beta-adrenergic binding sites, the catecholamine-sensitive adenylate cyclase, the cyclic nucleotide phosphodiesterase, and the cyclic AMP-dependent protein kinase. Thus, the rat pineal's sensitivity to beta-adrenergic stimulation appears to be regulated at multiple sites. Other investigations have focused on the regulation of pineal cyclic GMP metabolism. Unlike cyclic AMP, the stimulation of cyclic GMP synthesis requires the presence of intact nerve endings and of extracellular calcium. Some of the characteristics of pineal cyclic GMP regulation are described.

Physiological control of melatonin synthesis and secretion: mechanisms, generating rhythms in melatonin, methoxytryptophol, and arginine vasotocin levels and effects on the pineal of endogenous catecholamines, the estrous cycle, and environmental lighting.

Daily rhythms in pineal methoxyindole metabolism have been described in rodents and humans: serotonin levels in rat pineals are highest during the daylight hours and fall markedly soon after the onset of darkness, coincident with increases in the levels of pineal melatonin and 5-methoxy-tryptophol and the activities of pineal serotonin-N--acetyltransferase (SNAT) and hydroxyindole-O-methyltransferase (HIOMT). The fact that the levels of melatonin and 5-methoxytryptophol vary in parallel suggests that the major factor generating the methoxyindole rhythms is not SNAT activity, as has been suggested, but a proximal step, perhaps a change in the availability (for metabolism) of "stored" serotonin. Melatonin levels in human serum and urine exhibit rhythms similar to those observed in rats, i.e., they rise sharply during the daily dark period. When the onset of darkness is delayed by 12 hours, human melatonin rhythms usually require 3 or 4 days to adjust to the new lighting regimen. Environmental factors, other than light, that activate the sympathetic nervous system or cause epinephrine to be secreted from the adrenal medulla (e.g., the stress of immobilization; insulin-induced hypoglycemia) can override the inhibitory effects of light and accelerate melatonin synthesis. Melatonin levels in rat blood and urine are lowest during the proestrous and estrous phases of the estrous cycle. Although this effect of the ovarian steroid hormones is accompanied by a reduction in urinary norepinephrine levels, it is not caused simply by a decrease in the quantity of norepinephrine acting on the pineal but also involves a direct action of the hormones. Ovariectomy increases serum melatonin levels, whereas the administration of estradiol plus progesterone (to ovariectomized animals) lowers melatonin levels. The spectral and intensity-response characteristics of the photic inhibition of melatonin synthesis have been established for the rat. Rhythms in melatonin synthesis apparently persist among animals placed in environments of continuous darkness; the source of the cyclic signal (mediated by the pineal sympathetic nerves) has not yet been identified. Preliminary evidence suggests that levels of a peptide hormone, arginine vasotocin, in rat pineals and sera also exhibit daily rhythms and are increased by norepinephrine.

Ontogenesis of circadian rhythm of melatonin synthesis in pineal gland of rat.

Serotonin N-acetyltransferase (NAT) activity regulates the circadian rhythm of melatonin synthesis in pineal gland. NAT rhythm persisted in continuous darkness or in blinded rats. Development of the circadian rhythm of NAT activity was followed in the infant rats raised under various lighting schedules. The pups born and raised in continuous darkness or under constant illumination showed a rhythmic change indicating that the biological clock for NAT is generated inborn independent of environmental light-dark schedules. In the absence of light, the rhythms of pups synchronized with mother's rhythm. When rats were maintained under a ultradian light-dark schedule (LD 6:6), NAT activity showed a circadian change increasing once a day. When pups were born and raised under the ultradian lighting condition, NAT activity rhythm coincided with mother's rhythm. When pups were raised by a foster mother who had an inverted rhythm from that of an original mother, the rhythm of the pups synchronized with that of the foster mother. Whe pups were separated from their mother daily for 12 hours, their NAT rhythms reappeared when they were separated from their mother. These observations indicated a novel mechanism that in the absence of light-dark schedule, mothers taught the circadian rhythm to the pups as they raised them.

Effects of calcium-free medium on the induction of serotonin N-acetyltransferase in the rat pineal

Incubation of rat pineal glands in calcium-free medium containing ethyleneglycol-bis-(β-aminoethyl ether) N,N'-tetra-acetic acid (EGTA) inhibited the induction of serotonin N-acetyltransferase activity caused by l-isoproterenol or dibutyryl cyclic AMP. Although changes in the dose-response relationship between l-isoproterenol and N-acetyltransferase activity were observed, the most prominent effect of calcium-free medium containing EGTA was to delay and prolong the increase in N-acetyltransferase activity. There was also a reduction in the maximal enzyme activity attained. Differences between supersensitive and subsensitive glands in the rate and extent of induction were retained in calcium-free medium containing EGTA. Other results indicate that chelation of extracellular calcium indirectly reduced the rate of the protein synthesis which is required for the induction of increased N-acetyltransferase activity.

Pineal serotonin N-acetyltransferase activity and melatonin concentrations in prepubertal and adult Syrian hamsters exposed to short daily photoperiods.

Pineal serotonin N-acetyltransferase activity (NAT) and melatonin concentrations were determined at various intervals in prepubertal (35 days old) and adult male hamsters (74 day old) throughout a 24 hour period with the animals kept in a light:dark cycle of 6:18 (lights on at 0600 h and off at 1200 h). In prepubertal animals, daytime pineal NAT activity of 0.20-0.28 nmoles 14C-N-acetyltryptamine/pineal/hour was maintained for 8 hours after the initiation of darkness. Peak pineal NAT activity of 0.46 +/- 0.64 nmoles 14C-N-acetyltryptamine/pineal/hour occurred 13 hours after the onset of darkness and remained significantly elevated until 0400 h (p less than 0.001). Daytime pineal melatonin concentrations of 78-194 pg/pineal gland also were maintained for 8 hours after the initiation of darkness. At 13 hours into the dark period, pineal melatonin concentrations rose to 788 +/- 150 pg/pineal gland (p less than 0.01 vs all other time points except 0230 h and 0400 h). At one hour before the onset of light both the pineal NAT activity and pineal melatonin concentrations returned to daytime values. Adult male hamsters had diurnal pineal NAT and melatonin rhythms which are indistinguishable from those found in the prepubertal animals.

The 'suprachiasmatic syndrome': endocrine and behavioural abnormalities following lesions of the suprachiasmatic nuclei in the female rat.

Lesions of the suprachiasmatic nuclei that caused failure of spontaneous ovulation in female rats consistently produced abnormalities in other functions that are normally influenced by the light-dark cycle. In such animals morning plasma corticosterone concentrations were abnormally high and evening values abnormally low though the response to stress was unaffected. Pineal serotonin N -acetyl transferase activity was abnormally high in animals killed during the day and abnormally low in those killed at night. Although the animals were in persistent be­havioural oestrus, total voluntary wheel-running activity was not con­sistently altered but was distributed evenly between the light and dark periods rather than being confined principally to the dark periods as in normal animals. Similarly the proportion of the daily water and food intake that occurred during the dark period was reduced. The incidence of these associated abnormalities was low in lesioned rats that continued to ovulate spontaneously.

Regulation of pineal rhythms in chickens: photoperiod and dark-time sensitivity

Pineal serotonin N-acetyltransferase activity and melatonin content exhibit marked daily rhythms in chickens: Peak values occur during the period of low locomotor activity which coincides with dark in a 24-hr light-dark regimen. Manipulation of the light-dark ratio (photoperiod) in 24-hr light-dark schedules modifies the shape of the oscillation in N-acetyltransferase activity so that the enzyme activity is always low in the light; total dark-time enzyme activity increases (17%) while average dark-time activity decreases linearly (35%) in response to a doubling of the length of the dark period. Dark-time pineal N-acetyltransferase activity decreases rapidly in response to a light signal during the dark; sensitivity of the dark-time enzyme activity to 10-min light signals is higher during the first half of the dark period and lower during the second half of the dark period in a light-dark cycle (LD12:12). N-acetyltransferase activity is regulated by light in such a way as to keep account of photoperiod and changes in photoperiod which could be of physiological utility. Photoperiod has a long-term effect on the N-acetyltransferase activity rhythm; it modifies the shape of the oscillation. Changes in photoperiod are also translated into modifications of the N-acetyltransferase activity rhythm: Rapid responses to light during the dark-time permit detection of an instantaneous change in the daily light-dark ratio.

Circadian rhythms of enzyme and running activity under ultradian lighting schedule.

Serotonin N-acetyltransferase activity in the pineal gland and running acitvity of rats were measured under an ultradian lighting schedule (light/dark 6:6). When rats were moved from a diurnal lighting condition to the ultradian conditions, N-acetyltransferase activity showed a circadian rhythm, increasing once a day. N-acetyltransferase activity in the pups born and raised under the ultradian lighting conditions also exhibited a circadian change, the phase of which coincided with that of their mothers. When pups were raised by a foster mother with an inverted rhythmic phase from that of the original mother, the phase of the rhythm in N-acetyltransferase activity of the pups synchronized with that of the foster mother. When pups were separated from their mothers for 12 h/day, the circadian increase of N-acetyltransferase activity appeared during the dark period when they were separated from their mothers. The circadian rhythms of running acitvity were in phase with those of N-acetyltransferase activity in the pineal gland.

Pineal gland cells in culture: Morphology, biochemistry, differentiation, and co-culture with sympathetic neurons

We present here the initial report of a method for reproducibly obtaining primary cell cultures from pineal glands of 2-day-old rats. During culture, the putative pinealocytes became associated with each other in “nests”. Cells in these nests displayed vesicle-crowned rodlets and cilia, which are fine structural features in vivo of pinealocytes from neonatal rats. Treatment of the cultured cells with either norepinephrine or dibutyryl-cyclic AMP (db-cAMP) resulted in an increase in the activity of serotonin N-acetyltransferase, a marker activity for pineal function. This stimulation could be blocked by either cycloheximide or actinomycin D, and norepinephrine stimulation was also blocked by L-propranolol. Further, the pineal cell cultures were able to support the growth of dispersed cells of rat superior cervical ganglia and to allow neurite outgrowth in these co-cultures, though the presence of nerve growth factor (NGF) in the medium of these cultures could not be detected.

On GABA function and physiology in the pineal gland

Pineal gamma-aminobutyric acid (GABA) content and glutamic acid decar☐ylase (GAD) activity were found not to be influenced by environmental light, catecholamines, sympathetic innervation, or input via the pineal stalk. The observation that GAD activity did not disappear after pineal stalk section, ganglionectomy, or 48 h of organ culture leads us to suggest that GAD activity is not located in nerve processes entering the pineal gland. Treatment in organ culture with an inhibitor of protein synthesis did not greatly influence the slow rate of decrease of GAD activity. This finding is consistent with the conclusion that GAD turnover is slow. Treatment of denervated glands or glands containing functional sympathetic nerve structures with GABA, amino-oxyacetic acid (AOAA) or bicuculline in organ culture did not alter unstimulated levels, or significantly block the adrenergic stimulation of the activity of pineal serotonin N-acetyl transferase (NAT). It is clear from our studies that GABA does not influence or modulate the adrenergic regulation of pineal NAT activity, and that GABA content and synthesis are not regulated by an adrenergic mechanism. The role of GABA in the pineal gland remains to be discovered.

Sympathetic nerve endings in the pineal gland protect against acute stress-induced increase in N-acetyltransferase (EC 2.3.1.5.) activity.

Injection of the antidepressant desmyethylimipramine (DMI, 10 mg/kg) into intact rats or into rats in which the superior cervical ganglia had been decentralized caused a marked enhancement of the swimming stress-induced increase in pineal gland acetyl-CoA:serotonin N-acetyltransferase (N-acetyltransferase, EC 2.3.1.5) activity. DMI is known to block uptake, the transport of catecholamines by nerve endings. It was found that DMI had no effect on enzyme activity in superior cervical ganglionectomized (SCGX) rats which were swimming stressed. The pineal glands of these animals are devoid of nerve endings. In unstressed intact or unstressed surgically altered rats, injection of DMI caused only a minor increase in N-acetyltransferase activity, which was much smaller than that seen after stress. After 5 h in organ culture sympathetic nerve endings within the pineal gland are still intact. At this time DMI treatment of pineal glands taken from intact rats shifted the dose-response curve for epinephrine (EPI) stimulation of N-acetyltransferase activity by two orders of magnitude, but caused only a slight change in the dose-response curve for isoproterenol, which is not taken up into nerve endings. In contrast, DMI treatment in organ culture had no effect on the dose-response curve for EPI in denervated pineal glands. These results support the hypothesis that the response of pineal N-acetyltransferase activity to stimulation by stress is influenced by uptake. It would appear that in addition to terminating neuronal adrenergic transmission, this transport process physiologically protects the pineal gland against nontranssynaptic adrenergic stimulation.

Beta adrenergic-blockers decrease adrenergically stimulated N-acetyltransferase activity in pineal glands in organ culture

Treatment with 10 μM l-propranolol caused a rapid decrease in the high level of acetyl coenzyme A: serotonin N-acetyltransferase (EC 2.3.1.5, N-acetyltransferase) activity in pineal glands cultured in the presence of 1 μM norepinephrine. d-Propranolol was ineffective except at high concentration (1 mM). The same stereospecificity was also observed in organ culture when d- or l-propranolol was tested as a blocker of the norepinephrine-induced increase in N-acetyltransferase activity. Practolol (10–300 μM), a relatively specific β 1,-blocker, also initiated a decrease and inhibited the norepinephrineinduced increase in N-acetyltransferase. Butoxamine (10–300 μM), a relatively specific β 2-blocker, had no significant effect in either experimental situation. The percentage decrease in N-acetyltransferase activity after 40 min of treatment with l-propranolol was strongly dependent on temperature. At 40° C enzyme activity decreased by 92% but at 34° C enzyme activity decreased by only 50%. l-Propranolol (20 μM) did not inhibit the increase or initiate a decrease in N-acetyltransferase activity in pineal glands treated with 1 mM N 6,O 2 -dibutyryl adenosine cyclic 3',5'-monophosphate. The fast response observed when l-propranolol was added to the culture medium of norepinephrinetreated glands was not blocked by cycloheximide treatment. Concentrations of ouabain or K +, which are known to completely inhibit the norepinephrine-stimulated increase in N-acetyltransferase activity, caused only a small decrease in induced N-acetyltransferase activity, similar to that due to cycloheximide treatment.

Rat liver N-acetyltransferase: inhibition by melatonin

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.

Decrease of serotonin N-acetyltransferase activity in rat pineal organs after treatment with prostaglandin synthesis inhibitor indomethacin

Administration of indomethacin to rats abolished the cyclic AMP dependent, dark induced rise in serotonin N-acetyltransferase, presumably by inhibiting prostaglandin synthesis.

The influence of lighting conditions upon the level and course of increase in specific activity of serotonin N-acetyltransferase in the developing chick pineal gland.

A diurnal cycle in level of serotonin acetyltransferase (acetyl-C0A:arylamine N-acety-transferase (EC 2.3.1.5) activity was found in the pineal gland of chicks aged 16 to 20 days maintained under lighting conditions. Diurnal variation in levels of activity was markedly reduced in the pineal gland of birds kept in constant darkness, and suppressed in the gland of chicks under constant illumination. High levels of activity attained during the dark phase of the normal cycle rapidly declined when the birds were transferred to the light. The light phase level of serotonin acetyltransferase of the pineal increased progressively from the 11th day of incubation to about 1 week post-hatch. This course of increase in enzyme activity was largely unaffected by lightning conditions. Under conditions for assay of serotonin acetyltransferase activity in the chick pineal gland and brain, radioactive serotonin gave rise to N-acetylserotonin, 5-hydroxy-indoleacetic acid and a further unidentified metabolite, which was quantitatively the major product.

Pineal and associated neuroendocrine rhythms

(1) The mammalian pineal gland exhibits a number of biosynthetic rhythms. (2) Pineal serotonin levels are highest during the day and lowest at night. (3) Conversely, the activity of serotonin N-acetyltransferase, which converts serotonin to N-acetylserotonin, is greatly increased at night over daytime values. (4) As a consequence, N-acetylserotonin content increases in the pineal gland at night as does the product of its metabolism, melatonin. (5) Either indoleamines, such as melatonin, or polypeptides, such as arginine vasotocin, may be the pineal substances which influence the secretion of hormones from other endocrine glands. (6) Besides the obvious circadian rhythms within the pineal gland there are indications that the activity of this organ also changes on a seasonal basis, being more active during the winter than during the summer. (7) One physiological niche of the pineal gland may be to act as an intermediary between seasonal photoperiodic changes and the neuroendocrin reproductive axis. (8) In long day breeders, the pineal is believed tobe most active during the winter and, as a result, its hormonal products suppress sexual physiology. (9) This ensures that animals can only breed during restricted periods of the year.

Diurnal Rhythms in Pineal N-Acetyltransferase and Hippocampal Norepinephrine: Effects of Water Deprivation, Blinding and Hypothalamic Lesions

The pineal gland in the rat exhibits a diurnal rhythm in activity of the enzyme serotonin N-acetyltransferase (N-AT) with peak values during the dark period of a diurnal lighting schedule approximately 100-fold those during the light period. After blinding the rhythm becomes free-running. It is abolished by partial hypothalamic deafferentation with a knife cut made caudal to the optic chiasm. Water deprivation for 23 h daily has no effect on the pineal rhythm in either intact, blinded or deafferented animals. In contrast to this, there is a diurnal rhythm in hippocampal formation in norepinephrine content which can be entrained by a water deprivation schedule in both intact and blinded animals. These observations indicate that in the same animals 1 diurnal rhythm may remain entrained to the light-dark cycle while another rhythm is entrained to a secondary synchronizer, the water deprivation schedule.

Regulation of pineal rhythms in chickens: effects of blinding, constant light, constant dark, and superior cervical ganglionectomy.

Pineal serotonin N-acetyltransferase activity and melatonin content exhibit marked daily changes in chickens; peak values occur during the period of low locomotor activity which coincides with dark in a 24-hour light-dark cycle. The photic and neural regulation of these daily changes were studied by measuring pineal serotonin N-acetyl-transferase activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin content in experiments in which chickens were subjected to light-dark cycles, constant light, and constant dark and were surgically blinded or superior cervical ganglionectomized. It was found that: 1) The daily changes in N-acetyltransferase activity and melatonin content appear to persist in constant dark, and they disappear in constant light. 2) The eyes are not necessary for photic control of the daily changes, and the effect of constant light on N-acetyltransferase activity and melatonin content may be non-visual, that is, the eyes not being necessary. 3) The occurrence of the daily change in N-acetyltransferase activity and melatonin content does not require the superior cervical ganglia; the persistence of the changes in constant dark, however, may require the ganglia. 4) HIOMT activity was lower in constant light than in light-dark cycles and lower still in constant dark than in constant light. Neither the presence of the eyes nor the superior cervical ganglia affected HIOMT activity, as previously reported.

Pineal enzymes in chickens: Development of daily rhythmicity

Pineal serotonin N-acetyltransferase activity and HIOMT (hydroxyindole- O-methyl-transferase) activity were measured in chicks (White Leghorn Gallus domesticus) over a period of 8 weeks from the day of hatching. The measurements were made at two time points on selected days: one time point was taken during the light portion of the birds' environmental lighting regime, the other point was taken during the dark portion (1000 and 1600, respectively, in an LD12 : 12 cycle with lights-off at 1200 and lights-on at 2400). There were no dramatic differences in HIOMT activity between the light and dark measurements on any day, though the enzyme activity rose steadily with age. Changes were always seen in N-acetyltransferase activity in light versus dark. Dark-time N-acetyltransferase activity was higher than light-time activity. This includes the initial exposure of the chicks to dark on day 2 after hatching. N-acetyltransferase activity was also measured in the pineal glands of chick embryos kept in LD12 : 12. In embryos incubated for 17 and 19 days the dark-time values for enzyme activity were almost twice the light-time values.