Serotonin N-acetyltransferase Activity Research Articles

Effects of EGTA and calmodulin, neutral thiol proteinases and protein kinase C inhibitors on loss of chicken pineal serotoninn-acetyltransferase activity

This paper shows that the loss of chicken pineal serotonin N-acetyltransferase activity in crude homogenates of the pineal gland during preincubation at 37 degrees C is a complex process which seems to involve protein kinase C, calmodulin and calcium-activated neutral protease. All three compounds are strongly related to free calcium levels, and hence EGTA effectively prevents this loss of activity. It is proposed that the loss of serotonin N-acetyltransferase activity in crude chicken pineal homogenates is due to a series of molecular events, probably triggered by loss of the calcium gradient present in the intact gland by the homogenization process, leading to rapid serotonin N-acetyltransferase deactivation. In these homogenates two calmodulin inhibitors, a protein kinase C inhibitor and a neutral thiol proteinase inhibitor, and EGTA were found to markedly reduce the rate of serotonin N-acetyltransferase deactivation.

Serotonin N-acetyltransferase (NAT) activity in hen retina and pineal gland: pharmacological induction at noon and antagonism of the light-evoked suppression at night

In vivo effective pharmacological procedures are described which markedly increase activity of serotonin N-acetyltransferase (NAT), the key regulatory enzyme in melatonin biosynthesis, during the daytime (in light) and counteract suppressive effects of light on NAI activity at night in the hen retina and pineal gland. Of the tested compounds, and their combinations, the most effective were: "aminophylline + spiroperidol + alpha-methyl-p-tyrosine" for the retina, and "aminophylline + yohimbine (+ alpha-methyl-p-tyrosine)" for the pineal gland. The results give strong support to the concept that the dopaminergic (C(2)-receptor) and noradrenergic (alpha(2)-adrenergic receptor) mechanisms control NAT activity, and melatonin synthesis, in the hen retina and pineal gland, respectively.

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.

Forskolin, an activator of adenylate cyclase activity, promotes large increases in N-acetyl transferase activity and melatonin production in the Syrian hamster pineal gland only during the late dark period

The exposure of organ cultured pineal glands of Syrian hamsters to forskolin, an adenylate cyclase activator, caused marked increases in serotonin N-acetyltransferase activity and melatonin content in a dose-related manner (1-100 microM) when glands were collected in the second half of the dark period. However, addition of forskolin to glands collected anytime during the light period or at the beginning of the dark period failed or only modestly stimulated either pineal N-acetyltransferase activity or melatonin levels. Similar results were obtained with isoproterenol. The results suggest that intrapinealocyte regulatory mechanisms may determine the nocturnal rise in the Syrian hamster pineal gland.

Purification and characterization of DSIP-like material from ovine pineal glands: possible peptide-protein interaction.

The nonapeptide delta-sleep-inducing peptide (DSIP) has been isolated from venous blood of rabbits induced to sleep. Numerous reports have described sleep as well as extra-sleep effects. Radiochemical and immunochemical data suggest a relationship of DSIP with the pineal gland supported by interactions of this peptide with pineal functions such as the serotonin N-acetyltransferase activity. In order to demonstrate the natural occurrence of DSIP-like material associated with high Mr proteins in the ovine pineal, organs were water-extracted and fractionated by ultrafiltration and gel filtration. Radioimmunoassay (RIA) for DSIP-like fragments of the fractions revealed considerable amounts of pineal DSIP-like immunoreactivity (DSIP-LI) apparently existing in small as well as large molecular forms. Acidification of large DSIP-LI forms resulted in the elution from Sephadex G-50 of Mr less than or equal to 1,000 DSIP-like material. This free DSIP-LI form coeluted with the synthetic DSIP nonapeptide from microBondapak C18 on high-performance liquid chromatography. The results, therefore, appear to indicate the presence of a (biospecific) noncovalent intermolecular interaction of DSIP (1-9) with proteins (Mr greater than or equal to 10,000) of the ovine pineal gland.

Response of pineal serotonin N-acetyltransferase activity in male guinea pigs exposed to light pulses at night.

Serotonin N-acetyltransferase (NAT), which is crucial for the formation of melatonin, undergoes a typical day/night rhythm in the pineal gland with low levels during daytime and high levels at night. Short pulses of light given at night have been shown to rapidly depress NAT activity in some species, but not in others, the reasons for this difference being unclear. As diurnality and nocturnality of the experimental animals may play a role and since diurnally active animals have been little investigated in this respect, in the present study the diurnally active guinea pig was investigated. Male guinea pigs kept under a lighting regimen of LD 12:12 (lights off at 1700 hrs) were killed between 1200 or 1300 hrs and between 0000 and 0200 hrs, at night in the dark or after exposure to 10 or 45 min of light. The results obtained show that the day/night difference of NAT activity is about 2-fold. 10 min or 45 min of light given at night significantly depress pineal NAT activity. Re-exposure to darkness for 1 hr of animals previously given light for 10 min leads to restoration of NAT activity. These findings together with data from the literature suggest that it does not appear to be the activity pattern (diurnality versus nocturnality) of an animal nor the amplitude of the day/night difference of pineal NAT activity that account for the suppressibility or non-suppressibility of pineal NAT activity by light at night.

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.

Effect of Selective Monoamine Oxidase Inhibitors on Rat Pineal Melatonin Synthesis In Vitro

Freshly cultured pineal glands respond to the monoamine oxidase A (MAO A) inhibitor clorgyline and to high concentrations of the MAO B inhibitor, deprenyl, with an increase in serotonin N-acetyltransferase activity and N-acetylated indoles and a fall in 5-hydroxylated serotonin degradation products. Glands cultured for 48 hours before challenge respond less. Response is absent in glands cultured for 72 hours and in glands from ganglionectomized animals cultured for 48 hours before challenge. These data are consistent with the hypothesis that these MAO inhibitors stimulate melatonin synthesis by protecting norepinephrine from degradation.

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)

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.

Pineal transplants in oculo: limitations on the ability of collateral sprouts of foreign neurons to establish normal function

The pineal gland is innervated by sympathetic neurons whose cell bodies are located in the superior cervical ganglia. This pathway, which carries information concerning environmental lighting to the gland, is important for maintaining a circadian rhythm in the activity of the pineal enzyme serotonin N-acetyltransferase (NAT). Lesioning this pathway blocks the normal nighttime increase in NAT activity. Following transplantation of the pineal gland to the anterior chamber of the eye, the gland becomes reinnervated by collateral sprouts of sympathetic neurons that innervate the iris. In addition, a day-night rhythm in NAT activity is restored to the transplanted glands (Moore, 1975; Bäckström et al., 1976). These findings raise the possibility that the neural regulation of pineal function may be restored to normal by collateral sprouts of foreign neurons. To determine whether this is the case, other aspects of the neural regulation of NAT activity were examined in transplanted and in situ pineal glands. When the dark period was extended into the normal light period, NAT activity decreased to daytime values in both groups, suggesting that, in both cases, darkness is only effective in maintaining high levels of NAT activity at certain times during the day-night cycle. In contrast to their similar responses to extended darkness, the 2 groups of pineal glands responded differently to a brief pulse of light during the dark period. While the light exposure caused a dramatic decrease in nighttime NAT activity in in situ pineal glands, it produced no change in this enzyme activity in transplanted glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-2 adrenergic regulation of melatonin release in chick pineal cell cultures

The chick pineal gland expresses a circadian rhythm of melatonin biosynthesis, with elevated levels at night and low levels during the day. The rhythm of melatonin is regulated both by circadian oscillators located within the gland itself and by adrenergic input from the sympathetic nervous system. Previous work has shown that norepinephrine administration inhibits melatonin biosynthesis, as measured by the activity of the enzyme serotonin N-acetyltransferase. As a first step toward understanding the mechanisms by which norepinephrine regulates melatonin production in the chick pineal, we have identified the adrenergic receptor involved. Dissociated chick pineal cell cultures were prepared and melatonin release was measured on days 5 and 6 of culture using radioimmunoassay. The effects of adrenergic agonists and antagonists on the nocturnal increase of melatonin release during the 12 hr dark portion of a LD12:12 light cycle were determined. Norepinephrine inhibited melatonin release in a dose-dependent manner, with an average EC50 of 19.7 nM +/- 2.23 (SEM). Melatonin release values ranged from 100 to 4% of the level seen in control cultures, depending on the dose of norepinephrine. The physiological response to epinephrine, norepinephrine, and isoproterenol was stereospecific. The (-) stereoisomer was 6, 8, and 37 times more potent than the (+) stereoisomer, respectively. EC50 values (in nM) for adrenergic agonists were as follows: alpha-methyl-(-)-norepinephrine, 2.46; tramazoline, 3.06; guanabenz, 3.31; clonidine, 3.70; oxymetazoline, 4.29; (-)-epinephrine, 7.44; (-)-norepinephrine, 19.7; (-)-isoproterenol, 463; and (-)-phenylephrine, 659. Schild analysis was used to determine the relative potency of adrenergic antagonists. pA2 values for adrenergic antagonists were as follows: rauwolscine, 9.55; RX78 1094, 8.32; yohimbine, 8.14; phentolamine, 7.11; prazosin, 5.93; and (-)-propranolol, less than 6. The relative potencies of both adrenergic agonists and antagonists demonstrate that alpha-2 receptors mediate norepinephrine-induced inhibition of melatonin release in chick pineal cell cultures. The identification of alpha-2 receptors in chick pineal cells should aid in our understanding of the biochemical events initiated by receptor activation that regulate melatonin synthesis.

Dopamine mediates the light-evoked suppression of serotonin N-acetyltransferase activity in retina

The possible role of dopamine in the light-induced suppression of serotonin N-acetyltransferase (NAT) activity in retinas of the African clawed frog ( Xenopus laevis) was investigated using an in vitro eye cup preparation. The nocturnal increase of retinal NAT activity was significantly inhibited by either light exposure or exogenous dopamine. Spiperone, a dopamine receptor blocker, antagonized this inhibitory effect of light on NAT activity, but had no effect in darkness. The effect of spiperone required the presence of cyclic nucleotide phosphodiesterase inhibitors, 3-isobutylmethylxanthine (IBMX), papaverine, or Ro 20-1724. Under the conditions employed in this study, neither spiperone nor the phosphodiesterase inhibitors significantly affected NAT activity when added alone. This observation suggests a synergistic interaction between the dopaminergic antagonists and the phosphodiesterase inhibitors. Other dopamine receptor blockers, including haloperidol, cis-flupenthixol, clozapine and metoclopramide, increased NAT activity of light-exposed retinas incubated in the presence of IBMX. SCH 23390, a D 1-selective dopamine receptor antagonist, did not increase NAT activity, nor did the α- and β-adrenergic receptor antagonists tested. The effect of spiperone and IBMX on NAT activity was blocked by apomorphine and by the D 2-dopamine receptor agonist LY 171555, but not by the D 1-receptor agonist SKF 38393-A. The concentration of 3,4-dihydroxyphenylacetic acid was higher in light-exposed retinas than in dark-adapted retinas, suggesting that light exposure increases dopamine metabolism in Xenopus retina. The results presented in this paper suggest that dopamine, released in response to light exposure and acting on D 2-dopamine receptors, is partially responsible for the light-induced suppression of the nocturnal increase in retinal NAT activity.

Pineal indole metabolism in the mouse

The activity of two pineal enzymes serotonin N-acetyltransferase (SNAT) and hydroxyindole- O-methyltransferase, (HIOMT) and the pineal content of serotonin (5-HT) and N-acetylserotonin (NAS) were measured in several strains of mice ( Mus domesticus) in order to compare melatonin synthetic pathways among them. Of the strains we examined, C57BL/6J, AKR/J, BALB/c, NZB/BLNJ and wild mice, only wild mice synthesize pineal melatonin. Wild mice had high activity of both SNAT and HIOMT; NZB had SNAT activity but did not have HIOMT; the three other strains had neither SNAT nor HIOMT; the activity of SNAT in wild mice showed a clear daily rhythm but HIOMT activity did not show any significant daily changes. The pineal content of 5-HT in wild and NZB mice was higher during the day than during the night, however in C57BL and AKR, 5-HT levels were higher during the night than during the day. As expected NAS was found in wild and NZB mice.

Multiple forms of arylalkylamine N-acetyltransferases in the rat pineal gland: purification of one molecular form.

Rat pineal serotonin N-acetyltransferase (EC 2.3.1.87) activity is isolated in two molecular forms (Mr approximately equal to 10,000 and 95,000) by high performance size exclusion liquid chromatography in the presence of ammonium acetate (0.1 M, pH 6.5). In the presence of sodium citrate (0.1 M, pH 6.5), however, it is eluted as a single peak of intermediate size (Mr approximately equal to 30,000). A highly enriched preparation of one of the molecular forms has been obtained by a two-step purification procedure involving disulfide-exchange and anion-exchange chromatography. The N-acetyltransferase in 250 pineal glands obtained from isoproterenol-treated rats can be purified about 80-fold in 1 day; recovery is about 3%. Polyacrylamide gel electrophoresis of the final preparation indicates that a single major band (Mr approximately equal to 11,000) is present; this appears to be serotonin N-acetyltransferase.

A histochemical study of the adrenergic innervation of the rat pineal gland: Evidence for overlap of the innervation from the two superior cervical ganglia and for sprouting following unilateral denervation

The rat pineal gland receives sympathetic innervation, via the right and left internal carotid nerves, from neurons whose cell bodies are located in the two superior cervical ganglia. Using fluorescence microscopy, we have examined the density and distribution of fluorescent profiles in the pineal gland after lesioning of the internal carotid nerves. Thirty-two hours after sectioning both internal carotid nerves, the density of the fluorescent profiles was 3% of that seen in sham-operated controls, indicating that the lesioned fibers had degenerated. Thirty-two hours after sectioning one internal carotid nerve, the density of the processes was decreased to approximately 50% of the control value. The magnitude of the decrease following a unilateral lesion was similar on both the right and left halves of the gland. Thus, the results suggest that each internal carotid nerve innervates both sides of the pineal gland. The implications of this overlap in the innervation from the two internal carotid nerves for recovery of pineal function after a unilateral lesion are discussed. Sections of pineal glands were also analyzed at later time points after a unilateral lesion. Two weeks after cutting one internal carotid nerve, the density of the fluorescent profiles had increased to greater than 80% of the control value. When the contralateral internal carotid nerve was cut 2 weeks after a unilateral lesion and the pineal gland was examined 32 h later, the density of the fluorescent profiles had decreased to 2% of the sham value. This suggests that all of the compensatory increase in adrenergic processes that takes place following the unilateral lesion is due to sprouting of the contralateral internal carotid nerve rather than to regeneration of the lesioned internal carotid nerve or to sprouting and ingrowth of other adrenergic neurons. It remains to be determined what, if any, functional significance this sprouting has, since the neurally dependent circadian rhythm in serotonin N-acetyltransferase activity in the pineal gland is restored to normal within 32 h after a unilateral lesion; that is, before significant sprouting has occurred.

The effects of short pulses of light at night on numbers of pineal "synaptic" ribbons and serotonin N-acetyltransferase activity in male Sprague-Dawley rats.

To characterize further the functionally enigmatic "synaptic" ribbons (SR) of the mammalian pineal gland and to study possible relationships to melatonin synthesis, in the present investigation rats were exposed to short pulses of light at night when both SR numbers and serotonin N-acetyltransferase (NAT) activity are high in comparison to day-time values. Male Sprague-Dawley rats were killed at 13:00 and 01:00 h, respectively, and at 01:10 and 02:00 h after exposure to light for 10 and 60 min, respectively. The pineals were rapidly taken out and cut sagittally in half. One half was processed for electron-microscopic quantitation of SR numbers and the other half for NAT determinations. It was found that both SR numbers and NAT activity decreased significantly when the animals were exposed to light at night. Although both parameters showed corresponding changes, there was no clear-cut correlation between SR numbers and NAT activity in individual animals within a group, except after exposure to light for 60 min when a positive correlation (R = 0.939; p less than 0.05) existed. After exposure to light the electron-lucent vesicles of the SR decreased in number, but the length of the SR was unchanged. These results show that numbers of pineal SR can be easily and quickly manipulated and that the presently used model may be ideal in studying the poorly understood mode in which degradation of SR occurs.

Studies on High-Affinity [3H] Substance P Binding Sites in Bovine Pineal Gland

The presence of substance P in numerous mammalian pineal glands prompted us to search for its binding sites in the bovine pineal gland. The binding assays to pineal membrane were carried out in polypropylene microcentrifuge tubes in a final volume of 500 microliters of 50 mM Tris-HCl buffer (pH 7.4) containing aliquots of 200-500 micrograms protein, 0.02% BSA, 6 micrograms/ml chymostatin, 4 micrograms/ml leupeptin, 40 micrograms/ml bacitracin, 5 mM MnCl2, and 50 microliters of [3H]substance P (3H-SP, 45.7 Ci/mmol to yield a final concentration of 0.02-20 nM) to start the reactions, which were incubated for 20 min at 20 degrees C. The reactions were terminated by centrifugation in a Fisher Microcentrifuge Model 235A for 30 seconds at 13,000 X g, and the pellets were washed twice with 1 ml of ice-cold 50 mM Tris-HCl buffer containing 0.02% BSA, 6 micrograms/ml chymostatin, 4 micrograms/ml leupeptin, 40 micrograms/ml bacitracin, 5 mM MnCl2, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2. The bottoms of the tubes were cut, the membrane pellets were dissolved in 5 ml of Triton X-100/toluene fluor (1:3) scintillation fluid, and the radioactivity was counted. The specific [3H]-substance P binding at 1-2 nM was 40-50% of the total binding, and the non-specific binding was assessed by using 2 microM of unlabelled substance P. These studies identified in bovine pineal gland a high affinity receptor site for [3H]SP with a KD value of 0.43 nM and a Bmax value of 71.14 fmol/mg protein. The relative affinity of various substance P analogues or fragments was: substance P greater than physalaemin greater than SP2-11 greater than SP3-11 greater than SP4-11 greater than SP6-11 greater than substance K = eledoisin greater than kassinin greater than SP7-11 greater than SP free acid. Substance P did not alter the basal or the norepinephrine-induced stimulation of the activity of serotonin N-acetyltransferase in rat pineal gland in culture.

Relationship of putative nicotinic cholinergic receptors in the suprachiasmatic nucleus to levels of pineal serotonin N-acetyltransferase activity in the normally cycling female, the male, and the ovariectomized rat.

We have examined the nocturnal increase in pineal serotonin N-acetyltransferase (SNAT) activity in the rat and the decrease in activity of this enzyme as a result of exposure of the rats to light during the nocturnal period. In females with normal ovulatory cycles and ovariectomized females, levels of the enzyme were similar during the dark (695 +/- 70 pmol/min/gland vs. 590 +/- 68) or after 20 min of lights on at night (107 +/- 5 vs. 105 +/- 8). Carbachol, a cholinergic agonist, was injected at 2400 hr (EST) into the lateral ventricle of orbitally enucleated rats. The agonist simulated the lights on effect in both intact and ovariectomized rats. Highly purified fractions of alpha-bungarotoxin, a nicotinic cholinergic antagonist were injected into either the third ventricle or bilaterally adjacent to the SCN in intact females, intact males, or ovariectomized rats. These injections had no effect on either the nighttime increase or the lights on inhibition of SNAT. This study makes the following observations: 1) As was previously demonstrated [Illnerova, H. Endocrinol. Exp. 9:141-148, 1975], ovariectomy affects neither the daytime (light period) nor nighttime (dark period) activity of pineal SNAT. This study extends previous work by demonstrating that ovariectomy also has no effect on the lights on at night decrease of SNAT activity. 2) This study confirms that intraventricular infusion of carbachol decreases the nocturnal pineal SNAT activity [Zatz, M., and Brownstein, M.J. Brain Res. 213:438-442, 1981]. 3) This study does not confirm the report that the nicotinic cholinergic antagonist alpha-bungarotoxin prevents the loss of nocturnal SNAT activity induced by turning lights on at night [Zatz, M., and Brownstein, M.J. Brain Res. 213:438-442, 1981].

Effects of some inhibitors of DNA synthesis and repair upon the cycle of serotonin N-acetyltransferase activity in cultured chick pineal glands

When chick pineal glands were cultured in the dark with aphidicolin from midphotoperiod, the increase of serotonin N-acetyltransferase (NAT) activity was stimulated and the time of peak NAT activity was advanced. The peak level of NAT activity was also reached sooner on the 2nd day of culture. The increase of NAT activity was also stimulated in glands cultured under diurnal illumination, but the time of peak activity was not advanced. Effects with glands explanted into culture in the dark at other times were smaller and the time of peak NAT activity was not changed. Cytosine arabinoside and dideoxythymidine also stimulated the increase of NAT activity and advanced the time of peak activity with glands cultured in the dark from midphotoperiod. 3-Aminobenzamide markedly stimulated the increase of NAT activity both in the dark and under diurnal lighting when pineal glands were explanted into culture at mid- or late photoperiod. In contrast, with glands in culture from earlier in the photoperiod, aminobenzamide had no effect upon the increase of NAT activity up to the peak level found with control glands. Thereafter results were variable. Effects of cordycepin upon development of NAT activity were similar to those of 3-aminobenzamide but less marked. Incorporation of thymidine into acid-insoluble material in the dark was very markedly inhibited by aphidicolin, cytosine arabinoside, and dideoxythymidine, but only slightly by cordycepin. Aminobenzamide strongly inhibited incorporation by glands cultured from midphotoperiod, but had little effect with glands in culture from near the end of the photoperiod. We adopt the working hypothesis that excision repair of DNA may be a major component in the mechanism of the chick pineal clock.