Levels of serotonin N-acetyltransferase (NAT: acetul CoA:arylamine N-acetyltransferase; EC 2.1.1.5.) activity in the chick pineal gland exhibit a marked diurnal variation in birds kept under a diurnal cycle of ilumination. Activity begins to rise rapidly at the start of the dark phase of the cycle and reaches maximum levels at mid-dark phase about 25-fold greater than the minimum basal level at mid-light phase. Thereafter, the level of activity declines to the basal level about the start of the light phase. This diurnal cycle in chick pineal NAT activity found in vivo has recently been reproduced in vitro with intact glands incubated in organ culture. The mechanism of the 'biological clock' which regulates these variations in level of chick pineal NAT activity is unknown. However, I now report that chick pineal glands cultured under a diurnal cycle of illumination exhibit a diurnal cycle in content of cyclic GMP which roughly parallels the cycles in NAT activity. In contrast, there was no correlation between variations in pineal content of cyclic AMP and in level of NAT activity.

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.

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.

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.

1981)The rddionship between variations in levels of serotonin wtyltransferme activity and cQMP content in cultured chick pineal gbnds.Can. J. Bioclaem. 59,[593][594][595][596][597][598][599][600][601] Chick pineal glands cultwed in continuous darkness exhibited a circadian rhythm in 39951cyclic GMP (cGMP) content. cGMP content increased earlier and feu later than in glands incubated under diurnal lighting. A 66spike9e of further increase in c@MP content preceded s t a t of decline in acetyl-Col8:aqkHnine N-aeetyltransfemse (NAT) activity.Theophyuine or 4-(3-butoxy-4-methoxy~nzyl)-2-imdzldinne (c~mpoupnd Ro 20.1724) alone markedly stimulated increase of NAT activity in the dark, and retarded subsequent loss of activity either in the dark or in the photoperiod of a diurnal cycle of illurnhation. In constant darkness a combination of the two agents elicited greater increases of NAT than either alone and suppressed subsequent loss of activity for several hours. Under diurnal lighting conditions the combination was only slightly more effective than either alone. Compound Wo 20.1724 suppressed the precipitous loss of NAT activity seen when ghnds are transferred prematurely to the light. However, neither the rate or extent of loss of activity was greatly aEected by f Reophy Kne.Effects of theophylline and compound Wo 28.1724 upon levels of NAT activity appeared to be due primarily to effects upon the prowss(es) by which enzyme activity is lost. 'They could not be attributed solely to eaects upon pineal contents of cGMP and (or) 3',5kyclis AMP (CAMP).Tbeophylline alone was as effective as the drug combination in eliciting sand maintaining elevated pineal content of cGMP, but had little effect upon CAMP content. Compound Wo 20.1724 alone elicited large increases in pineal contents of both nucleotides but did not sustain them. Wainwright, S. D. & Wainwright, L. K. (8981) The relationship between variations in levels of serotonin acetyltransferase activity and cGMP content in cultured chick pineal glands.Can. 9. Bbchem. 59, 593-601 Dans les glandes pinCsales de poussins cultivCes dans une noirceur continue, Ia teneur en cGMP suit un rythme circadien. La teneur en cGMP augmente gllss t6t et tombe plus tard que dans les glandes incubCes B la lurmikre diurme. Un nouveaa "pic" d'augmentation du cGMP prkkde ie debut du d&Iin de 19activitC de l'adtyl-CoA:arylamine WP-acktyltransf6rase (NAT).