Rat serum stimulates serotoninN-acetyltransferase activity in pineal monolayer cultures

Summary The effect of rat serum on serotoninN-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 bothN-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. This research was supported by NIH Grant HD12904, by a Basil O’Connor starter grant from the March of Dimes Birth Defects Foundation, and by the Veterans Administration. Vernon Rowe is the recipient of Teacher Investigator Award NS00439.     

The regulation of pineal serotonin N-acetyltransferase activity in newborn rats.

Rat pineal serotonin N-acetyltransferase activity increases 2–3 hr after birth and then decreases again. The activity at night is higher than that during the day in rats as young as 1 to 2 days old. Administration of the beta adrenergic blocker trimepranol to newborn or 2-day old rats at night lowers the elevated serotonin N-acetyltransferase activity. Hence, the activity is under adrenergic control even in the not yet innervated pineal gland of the newborn rats.     

Effects of acute ethanol administration on nocturnal pineal serotonin N-acetyltransferase activity

The effect of acute ethanol administration on pineal serotonin N-acetyltransferase (NAT) activity, norepinephrine and indoleamine content was examined in male rats. When ethanol was administered in two equal doses (2 g/kg body weight) over a 4 hour period during the light phase, the nocturnal rise in NAT activity was delayed by seven hours. The nocturnal pineal norepinephrine content was not altered by ethanol except for a delay in the reduction of NE with the onset of the following light phase. Although ethanol treatment led to a significant reduction in nocturnal levels of pineal serotonin content, there was no significant effect upon pineal content of 5-hydroxyindoleacetic acid (5-HIAA). The data indicate that ethanol delays the onset of the rise of nocturnal pineal NAT activity.     

Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland

Conclusion The circadian rhythm of melatonin synthesis in the pineal glands of various species has been summarized. The night-time elevation of melatonin content is in most if not all cases regulated by the change of N-acetyltransferase activity. In mammals, the N-acetyltransferase rhythm is controlled by the central nervous system, presumably by suprachiasmatic nuclei in hypothalamus through the superior cervical ganglion. In birds, the circadian oscillator that regulates the N-acetyltransferase rhythm is located in the pineal glands. The avian pineal gland may play a biological clock function to control the circadian rhythms in physiological, endocrinological and biochemical processes via pineal hormone melatonin.     

Atypical 24-hour rhythms of serotonin N-acetyltransferase activity in the rat pineal gland

Previous long-term studies have shown that in the pineal gland of rats melatonin synthesis is subject to infradian rhythms with periods between 4 and 7 days. Since in these studies melatonin-related parameters were measured at one timepoint of a 24-hr cycle only, the aim of the present investigation was to extend these experiments by more frequent sampling, to characterize the infradian rhythmicity in more detail. Male Sprague-Dawley rats kept under a light schedule of LD 12:12 (lights on at 0700) were killed at 6-hr intervals on 8 consecutive days. After decapitation the pineal gland was rapidly dissected out, followed by measurements of one of the melatonin-forming enzymes, serotonin N-acetyltransferase (NAT) activity. It was found that pineal NAT activity exhibited the well known day/night rhythm, i.e. low activity during daytime and strikingly enhanced activity at night, during the first 4 days of the experiment. On the fifth night (from Saturday to Sunday) an unusually high NAT peak occurred at 2400 hr, followed by two atypical 24-hr cycles. In the first cycle the midnight and 0600 hr values were equal and in the second cycle the 0600 hr value was significantly higher than the midnight value. To investigate whether the unusually high NAT peak was a single event or not, four additional short-term experiments were carried out at 2400 hr on 4 consecutive weekends, from Friday to Monday. In each of the four 4-day experiments a distinctly higher peak of NAT activity was found on Saturday, but with time the peaks became less prominent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Day-to-day variation in pineal serotonin N-acetyltransferase activity in stressed and non-stressed male Sprague-Dawley rats

Previous studies involving physical-immobilization stress in laboratory rats have yielded inconsistent results with respect to melatonin synthesis in the pineal gland. As melatonin formation undergoes circadian and infradian rhythms, the aim of the present study was to examine whether stress experiments exhibit day-to-day variation. Toward this end, groups of male Sprague-Dawley rats were stressed by physical immobilization on eight consecutive days, respectively, or left relatively undisturbed, and killed. The pineal gland was rapidly dissected out and serotonin N-acetyltransferase (NAT) activity and melatonin levels were measured. NAT activity was significantly depressed on experimental days 1, 3 and 5, and slightly depressed on day 7. In addition, both in control and experimental animals NAT activity exhibited statistically significant differences between experimental days. Pineal melatonin levels were less variable. On experimental days 3 and 6 immobilization led to a significant increase of pineal melatonin levels. These results show that day-to-day variation is an important factor that influences the outcome of stress experiments and represent another example that NAT activity and pineal melatonin levels do not always show corresponding changes.     

Injections of alpha-melanocyte stimulating hormone affect pineal serotonin, melatonin and N-acetyltransferase activity

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.     

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.     

Light-induced changes in frog pineal gland N-acetyltransferase activity

N-Acetyltransferase (NAT) activity was determined in the pineal gland of frogs (Rana tigrina) of different ages using 2-aminofluorene and p-aminobenzoic acid as substrates, and assayed by high-pressure liquid chromatography. Frogs of different ages were either killed during the light phase or exposed to darkness or light for 1 min during the dark phase of the lighting cycle, then returned to their cages in darkness for 30 min before being killed. The pineal gland NAT activity of 1-month-old frogs was inhibited when the animal was nocturnally exposed to 1 min of light. Nocturnal light exposure did not inhibit NAT activity in 1-month-old frogs, even though these animal displayed clear light-dark differences in pineal gland NAT activity. Nocturnal light exposure did not inhibit night-time levels of NAT activity in 1-month-old animals which had been bilaterally enucleated, thus suggesting that this effect is retinally mediated. Pretreatment of 1-month-old and 6-month-old animals with isoproterenol (a beta-adrenoceptor agonist drug) prevented the nocturnal light-induced inhibition of NAT activity. From the different sensitivity of 1-month-old and 6-month-old animals to different intensities or durations of nocturnal light exposure it was found that the duration or intensity of light exposure was not able to inhibit nocturnal NAT activity. The NAT activity was at least 4–5-fold greater in 1-month-old frogs than in 6-month-old frogs. This is the first demonstration of the retino-pineal gland pathway that appears to produce light-induced changes in pineal glands of frogs 1-month-old or older, but this pathway only functions in 1-month-old frogs, and does not appear to function in 6-month-old frogs.     

Meal feeding schedule and ventromedial hypothalamic lesions do not affect rhythm of pineal serotonin N-acetyltransferase activity in rats

Effects of meal feeding schedule and bilateral lesions of the ventromedial hypothalamus (VMH) on the circadian rhythm of pineal serotonin N-acetyltransferase (SNAT) activity were examined in rats, under LD (12:12) condition. Neither meal feeding nor VMH lesions affected the phase of the circadian rhythm of pineal SNAT activity, but the VMH lesions reduced the level. Meal feeding caused a shift of the phases of the daily rhythms of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase activities in the liver. These findings suggest that the circadian rhythm of pineal SNAT activity is not entrained by the food intake, and that the VMH does not function as a master oscillator of the rhythm.     

Arylamine N-acetyltransferase and arylalkylamine N-acetyltransferase in the mammalian pineal gland

Amine N-acetylation in the pineal gland is of special importance because it is the first step in the synthesis of melatonin from serotonin. In the present study the N-acetylation of arylamines and arylalkylamines by homogenates of rat and sheep pineal glands was investigated. The arylamines studied were p-phenetidine and aniline; the arylalkylamines studied were tryptamine, serotonin, 5-methoxytryptamine, 6-fluorotryptamine, and phenylethylamine. These amines were acetylated by pineal homogenates of both species, although marked interspecies differences in apparent Km and Vmax values were found. A series of observations in both species indicate that aromatic amine N-acetylation is catalyzed by two distinct enzymes; one preferentially acetylates arylamines and the other preferentially acetylates arylalkylamines. First, isoproterenol treatment of the rat increased arylalkylamine N-acetylation 100-fold without increasing arylamine N-acetylation. Second, cycloheximide treatment in sheep reduced arylalkylamine N-acetylation at night to one-tenth control values, without altering arylamine N-acetylation. Third, arylamine N-acetyltransferase and arylalkylamine N-acetyltransferase inactivated at different rates at 4 degrees C. Fourth, the two enzymes were resolved by size exclusion chromatography. These results clearly establish that the pineal gland contains an arylamine N-acetyltransferase and a second, independently regulated arylalkylamine N-acetyltransferase which appears to be primarily responsible for the physiological conversion of serotonin to melatonin via the intermediate N-acetylserotonin.     

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.