Chick pineal serotonin acetyltransferase: a diurnal cycle maintained in vitro and its regulation by light.

We have reproduced in vitro the diurnal cycles in levels of serotonin acetyltransferase activity found in the chick pineal gland in vivo. The more closely the lighting conditions of culture matched those under which the birds were raised, the closer was the similarity between cycles in levels of enzyme activity in vitro and in vivo. Repetitive cycles in levels of acetyltransferase activity persisted in culture for at least 4 days under a diurnal cycle of illumination, and at least 2 days in continuous darkness. When glands were explanted into culture in the light phase of a cycle, short periods of further exposure to light markedly stimulated subsequent increase of acetyltransferase in the dark (after a short lag). Prolonged exposure to light in culture markedly inhibited increase of enzyme activity. Cycles in the levels of enzyme activity in glands cultured under altered light cycles were regulated primarily by changes in illumination. However, the endogenous biological 'clock' remained at least partly entrained to the original light cycle. Increase of acetyltransferase activity in vitro was markedly stimulated by theophylline plus compound Ro. 20.1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) under all lighting conditions. Kinetics (to the time of attaining maximum levels in situ) of the increase under diurnal lighting and in constant darkness were indistinguishable from those in vivo. A high concentration of dl-propranolol markedly stimulated an increase in acetyltransferase activity in glands cultured in constant darkness but had little effect on glands under diurnal lighting or continuous illumination.     

Metabolism of tryptopan and serotonin by the chick pineal gland in organ culture.

Marked differences were seen between the metabolism of L-[3-14C] tryptophan and of [2-14C]serotonin by the intact chick pineal gland in organ culture. The major metabolite of tryptophan recovered by our procedures was melatonin, which accounted for about half the radioactivity recovered as metabolic products. In contrast, the principal product of serotonin metabolism recovered was hydroxyindoleacetic acid, and the yield of products derived through monoamine oxidase (EC 1.4.3.4) activity vastly exceeded that of melatonin. Metabolism of tryptophan yielded a much larger proportion of methlated metabolites among the products recovered than did metabolism of serotonin. However, the yield of methoxyindoleacetic acid from serotonin was greater than that from tryptophan. Serotonin formed endogenously and serotonin supplied exogenously appear to enter two or more largely distinct metabolic pools.     

Dehydrogenase activity of the pineal gland during the estrous cycle

An investigation of the relationship of succinic dehydrogenase (SDH) and lactate dehydrogenase (LDH) in the pineal gland of the adult female albino rats was carried out during seven definitive stages of the estrous cycle: Early Estrus (E1), Late Estrus (E2), Metestrus (ME), Early Diestrus (D1), Late Diestrus (D2), Preproestrus (PPE) and Proestrus (PE). Controlled housing conditions were maintained on 7:00 A.M. to 7:00 P.M. light-dark cycle. The animals were necropsied at precise times after the onset of estrus and subsequent stages of the cycle. Comparative histochemical and biochemical analysis revealed the following for SDH activity: E2 greater than E1 greater than D2 greater than D1 greater than PPE greater than PE greater than ME; and for LDH activity: PE greater than E2 greater than ME greater than D1 greater than PPE greater than E1 greater than D2. Interrelationships are postulated between pineal SDH, GOT, GPT, and estrogen concentrations in the rat.     

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.     

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.     

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 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.     

Hormone-induced increase of hydroxyindole. O. methyltransferase activity in the embryonic chick pineal gland in organ culture.

The technique of organ culture was used to investigate the regulation of the increase of hydroxyindole.O.methyltransferase activity of the pineal gland during embryonic development. Glands of 15- and 17-day chick embryos developed small increases of enzyme activity when incubated in organ culture with a standard tissue culture medium. The increase of enzyme activity was markedly stimulated by a combination of hydrocortisone, somatotropin and thyroxine, and simulated the pre-hatch increase found $\text{in ovo}$. All three hormones appeared to be required for consistent development of maximal increases in enzyme activity but none was essential for some increase in activity.     

Iodothyronine 5'-deiodinating activity in the pineal gland.

1. The presence of an iodothyronine 5'-deiodinating activity has been described in the pineal gland of various rodents, and it has been identified as a type II 5'-deiodinase isoenzyme since it is relatively insensitive to inhibition by propylthiouracil and its activity increases during hypothyroidism. 2. 5'-Deiodinase activity in the rat pineal gland follows a nyctohemeral profile, exhibiting basal values during the day and maximal values at night. The nocturnal increase is dependent on the noradrenergic input from the superior cervical ganglia, and both in vivo and in vitro studies show that beta-adrenergic receptors are primarily involved in the activation of the enzyme. 3. Day-night differences in rat pineal 5'-deiodinase activity are found beginning at 2 weeks of age, with rhythms increasing in amplitude until maximal differences are reached in adult animals. During the maturation of the rhythm, changes in regulation of enzyme activation are observed. Thus, during the first 2-3 weeks of age, alpha-adrenergic receptors appear to be as important as beta-adrenergic receptors in regulating the deiodinating activity of the pineal. However, in adults, no role of alpha-adrenergic receptors has been described. 4. Although regulation of 5'-deiodinase activity in the pineal gland is well established, few data are available concerning the physiological significance of the enzyme in the gland. Of the studies that have been performed, those attempting to demonstrate a relationship between pineal 5'-deiodinase activity and other pineal rhythms, e.g. those of melatonin production and N-acetyltransferase activity, indicates that the latter rhythms do not rely on the cyclic production of T3. The alternate possibility that the 5'D rhythm depends on the cyclic production of melatonin remains to be examined.     

Effects of white light on the pineal gland of the chick embryo.

Chick embryos were directly exposed to a source of white light during incubation and sacrificed before hatching. The light caused a number of teratological effects such as high mortality, delay in development, celosomy, hepatomegaly, auricular dilation and micrognatia. The pineal gland of the illuminated embryos showed an increase in number and size of the intracytoplasmic lipid droplets of the follicular pinealocytes. These findings suggest that the pineal gland of the chick embryo is sensitive to light.     

Monoamino-oxidase activity in rat pineal gland. Histochemical studies

Monoamine-oxidase (MAO) activity was detected in rat pineal gland with dopamine, 5-hydroxytryptamine (5-HT), norepinephrine and tryptamine as substrates, and nitroblue tetrazolium salt as electron acceptor. Pinealocytes stained deeply when 5-HT was the substrate. Dopamine and tryptamine substrates gave similar patterns, with moderate activity in the pinealocytes. Norepinephrine reactivity was detected in the nerve-endings.