Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2107-2111, June 1975 Beta-Adrenergic Stimulation of Pineal : Adenosine 3':5'-Cyclic Monophosphate Stimulates Both RNA and Protein Synthesis (actinomycin D/circadian rhythms/catecholamine neurotransmitters/arylamine acetyltransferase) JORGE A. ROMERO, MARTIN ZATZ, AND JULIUS AXELROD Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014 Contributed by Julius Axelrod, March 7, 1976 ABSTRACT The lag period in the induction of rat pineal N-acetyltransferase (arylamine acetyltransferase or acetyl-coa: arylamine N-acetyltransferase EC 2.3.1.5) by catecholamines via the beta-adrenergic receptor varies with the length of exposure of the rat to light or darkness. If rats have been exposed to light and reduced sympathetic nerve activity for more than 12 hr, this lag period is 1-2 hr long. Under these conditions, actinomycin D completely blocks the induction of N-acetyltransferase by isoproterenol and by dibutyryl adenosine 3': 5'-cyclic monophosphate (cyclic AMP). In contrast, if enzyme activity is caused to fall by brief exposure to light at night when N-acetyltransferase activity is high, reinduction by catecholamines occurs almost immediately. In this case, actinomycin D does not block the reinduction of N-acetyltransferase by isoproterenol or by dibutyryl cyclic AMP. Cycloheximide blocks N-acetyltransferase induction under all conditions tested. Thus, new protein synthesis is always required for N- acetyltransferase induction; however, the requirement for RNA synthesis is variable, and contributes to the length of the lag period for induction. It is postulated that both beta-adrenergic stimulation and dibutyryl cyclic AMP act intracellularly at two separate sites in the induction of pineal N-acetyltransferase. One site is in the stimulation of transcription, and the other is in the stimulation of post-transcriptional events. The activity of the enzyme serotonin N-acetyltransferase (arylamine acetyltransferase or acetyl-coa: arylamine N- acetyltransferase EC 2.3.1.5) in the rat pineal gland is 50- to 100-fold higher at night than during the day (1). This circadian change in enzyme activity is modulated by environmental lighting acting via norepinephrine-containing sympathetic nerves whose cell bodies lie in the superior cervical ganglion (2, 3). Exposure of rats to darkness stimulates the activity of these nerves (4) to release norepinephrine (5) which in turn activates the beta-adrenergic receptor on the pineal cell membrane and results in the induction of N-acetyltransferase activity (1, 3). There is compelling evidence that adenosine 3': 5'-cyclic monophosphate (cyclic AMP) acts as second messenger in the induction of N-acetyltransferase (6, 7), both in vivo and in cultured pineal organs. Exposure of rats to light during the night reduces nerve activity (4, 5) and causes N-acetyltransferase activity in the pineal gland to fall precipitously, with a half-life of less than ten minutes (3, 8). As long as the lights stay on, enzyme activity remains at its lowest level (1, 3). If animals that have been exposed to light are injected with isoproterenol (a betaadrenergic agonist), N-acetyltransferase activity in the pineal gland increases 50- to 100-fold after a variable lag period (3, 9). Abbreviation: cyclic AMP, adenosine 3': 5'-cyclic monophosphate. 2107 The duration of the exposure to light (which reduces nerve activity) modifies the characteristics of N-acetyltransferase induction in three ways (9). First, as the length of the light period increases, there is a gradual increase in sensitivity of N-acetyltransferase to induction by isoproterenol (shift to the left in the dose-response curve), and a progressive increase in the maximum enzyme activity achievable with high doses of isoproterenol (9). Second, the elevation of cyclic AMP levels by isoproterenol is also enhanced, indicating important changes in the properties of the system which regulates cyclic AMP levels (which includes the receptor adenylate cyclase complex and the phosphodiesterases) (9). Third, there is an increasing lag period in the induction of N-acetyltransferase, and a progressive delay in the attainment of peak activity after stimulation with isoproterenol (9). Thus, exposure of the gland to relatively low levels of neurotransmitter (e.g., when the lights are on) causes supersensitivity within a few hours (9) and a longer lag period prior to induction (3, 9); while prior exposure to higher levels of neurotransmitter (e.g., during periods of darkness) causes relative subsensitivity to further stimulation and shortens the lag period for the reinduction from baseline activity upon restimulation with isoproterenol (9) Ṫhis change in the lag period for enzyme induction as a function of the degree of prior exposure to neurotransmitter is consistent with a variation in the availability of a precursor necessary for the synthesis or activation of N-acetyltransferase. In other inducible enzyme systems, cyclic AMP has been shown to act either by stimulation of DNA dependent RNA synthesis (transcription) or by accelerating the processing of messenger RNA and enhancing protein synthesis at the ribosomal level (translation) (10). In those systems in which cyclic AMP acts at post-transcriptional sites, there is no lag between stimulation and appearance of active protein, and actinomycin D fails to block induction. On the other hand, in those systems in which cyclic AMP is believed to act by stimulating transcription, there is a lag period (1-2 hr) prior to the increase in activity, and actinomycin D effectively blocks induction (10). Because of the variations in lag period for induction of pineal N-acetyltransferase, the role of RNA synthesis in the induction by catecholamines was studied. The data presented show that N-acetyltransferase induction after activation of the beta-adrenergic receptor involves stimulation of both pretranscriptional and post-transcriptional events. Furthermore, cyclic AMP mediates either or both of these actions of the beta-adrenergic receptor, depending upon the length of exposure of the rat to light or darkness.
2108 Biochemistry: Romero et al. Proc. Nat. Ac(d. Sci. USA 72 (1975) ~w CO) W 1000 U. CO) z 500 T~~~~~ TABLE 1. Effect of actinomycin D on the induction of pineal N-acetyltrarsferase by isoproterenol after various periods of exposure to light (pmol/pineal per 10 min A S.E.) Reinduction Induction at midnight after after 20 Treatment 18 hr light min light 1 hr Isoproterenol 100 :1= 20* 1380 i 120 Isoproterenol plus actinomycin D 40 4 10*t 1030 i 80 3 hr Isoproterenol 980 ± 100 1140 160 Isoproterenol plus actinomycin D 150 ± 40*t 890 ± 140 MIDNIGHT LIGHT 15 MIN 30 MIN 60 MIN CONTROL 10 MIN l REINDUCTION BY ISOPROTERENOL FIG. 1. Animals were exposed to light for 20 min at the peak of the N-acetyltransferase cycle at midnight. When indicated, cycloheximide (20 mg/kg) was injected when the lights were turned on, and isoproterenol (5 mg/kg) 20 min later. Animals were killed 15, 30, and 60 min after isoproterenol injection and their pineal glands assayed for N-acetyltransferase activity. * P < 0.01 when compared with control by Student's 1-test. METHODS Chemicals. [1-14C]Acetyl coenzyme A (3.5-6.6 mci/mmol) was purchased from Amersham-Searle, Chicago. Tritiated uridine (20 Ci/mmol) was purchased from New England Nuclear Corp., Boston. 1-Propranolol was a gift from Ayerst Laboratories, and l-isoproterenol-d-bitartrate from Winthrop Laboratories. Cycloheximide, actinomycin D, and other chemicals were obtained from commercial sources. Animals. Male Sprague-Dawley rats (150-175 g) (obtained from Zivic-Miller, Allison Park, Pa.) were kept under diurnal lighting conditions in our facilities for 5 days before the experiments. Lights were on from 0600 to 1800 hr. Isoproterenol was injected subcutaneously in isotonic saline; cycloheximide and actinomycin D were administered intraperitoneally in 50% ethanol-saline solution. Groups of six to eight rats were killed by decapitation at the times indicated in each experiment. Pineal glands were denervated by bilateral superior cervical ganglionectomy performed 3 weeks before the experiments. Pineal Explant Culture. Pineal glands were placed in organ culture in plastic petri dishes (Falcon, 60 mm diameter) containing 2.5 ml of BGJb Fitton Jackson medium (Grand Island Biological Co.), supplemented with ascorbic acid (0.1 mg/ml), glutamine (2.0 mm), streptomycin (100;4g/ml), and penicillin (100 units/ml). Four to six pineals were incubated in each dish at 370 under 95% 02-5%o CO2. Isoproterenol, dibutyryl cyclic AMP, cycloheximide, and actinomycin D were added to the medium at the concentrations and times indicated. Assay for Activity. Immediately after decapitation or removal from culture, pineal glands were assayed for N-acetyltransferase activity by a method de- At midnight (t = 0) animals kept in diurnal lighting conditions (light 0600-1800 hr, dark 1800-0600 hr) were brought out into a lighted room and injected with actinomycin D (5 mg/kg) or vehicle. Simultaneously, animals exposed to light for 18 hr were injected with actinomycin D or vehicle. At t = 20 min, animals were injected with l-isoproterenol-d-bitartrate (5 mg/kg). Groups of six animals were killed 1 and 3 hr after isoproterenol injection and their pineal glands assayed for Ar-acetyltransferase activity. * P < 0.01 when compared with the corresponding 20 min light group by Student's t-test. t P < 0.01 when compared with the corresponding isoproterenol treated group. scribed previously (11), using 20 nmol of [14C]acetyl CoA instead of 3.4. Assay for Total RNA Synthesis. Total RNA synthesis was determined by measuring the incorporation of tritiated uridine into total RNA isolated by the technique of Perry et al. (12). RESULTS Rapid reinduction of N-acetyltransferase at midnight A short exposure of rats to light during the night, when pineal N-acetyltransferase is elevated, results in a rapid fall in enzyme activity (3, 8). To determine the time course of reinduction of N-acetyltransferase after turning the lights on at midnight, rats were injected with isoproterenol after the lights were on for 20 min. The rats were killed at 15, 30, and 60 min after the injection, and N-acetyltransferase activity in the pineal glands measured. Previous findings (3) were confirmed and extended, demonstrating that N-acetyltransferase activity increases within 15 min after injection, and levels of enzyme activity as high as the original darktime levels are achieved within 1 hr after the injection (Fig. 1, Table 1). In contrast, induction was much slower in animals which had been exposed to light for 18 hr (Table 1). In this case there was a low level of N-acetyltransferase measured 1 hr after injection, and a 50-fold increase in enzyme activity 3 hr after injection. Cycloheximide blocked the induction of N-acetyltransferase under all conditions tested (Fig. 1). of N-acetyltransferase after different periods of exposure to light To examine whether RNA synthesis is required for induction of N-acetyltransferase, the effect of actinomycin D at different
Proc. Nat. Acad. Sci. USA 72 (1975) Stimulation of Pineal 2,109 TABLE 2. Effect of actinomycin D on the induction of pineal N-acetyltransferaee in vitro TABLE 3. Induction of N-acetyltransferase by dibutyryl cyclic AMP (pmol/pineal per 10 min ± S.E.) Reinduction Induction at midnight after after 20 Treatment 18 hr light min light Control 25 ± 10 20 ± 5 Isoproterenol 1550 ± 100* 1620 i 350* Isoproterenol plus actinomycin D 220 ± 50*t 1510 i 400*t Isoproterenol plus cycloheximide 13 4± 2 50 i 20 Actinomycin D 8 ± 3 14 4 5 Cycloheximide 20 i 5 60 4-10 Pineal glands obtained from animals exposed to light for 18 hr or for 20 min at midnight were preincubated for 1 hr in media containing actinomycin D (10 1ug/ml) or cycloheximide (100,ug/ml). Isoproterenol was added to the media after the preincubation to a final concentration of 10-7 M. activity in the pineal organs was assayed 6 hr after the addition of isoproterenol (n = 6). * P < 0. 01 when compared with the control by Student's t-test. t P < 0.01 when compared with the corresponding isoproterenol treated group. t P < 0.01 when compared with the corresponding 20 min light-exposed group. times in the diurnal cycle was examined. Actinomycin D, which blocks RNA synthesis, did not block the reinduction of N-acetyltransferase at midnight after a short period of light exposure. High levels of enzyme activity were measured 1 and 3 hr after administration of isoproterenol (Table 1). However, in animals exposed to light for 18 hr, actinomycin D did block the induction by isoproterenol that occurred 3 hr after the administration of the catecholamine. These observations suggest that increased stimulation of the beta-adrenergic receptor during darkness results in the accumulation of a precursor RNA required for N-acetyltransferase induction. The observation that actinomycin D inhibited induction under one set of conditions and not the other makes it very unlikely that the effects noted are due to nonspecific, toxic effects of the antibiotic. of N-acetyltransferase in vitro The above results were confirmed by similar experiments in vitro. Pineal glands obtained from animals at midnight after 20 min of exposure to light, or after 18 hr of exposure to light, were incubated in the presence of isoproterenol and actinomycin D (Table 2). Again, actinomycin D was unable to block the reinduction of N-acetyltransferase in the glands from animals exposed to light for only 20 min, while it blocked almost completely the induction in pineal glands from animals exposed to light for 18 hr. The concentration of actinomycin D used (10 og/ml) inhibited the incorporation of tritiated uridine into total RNA by more than 90% (data not shown). Blockade of endogenous nighttime rise in N-acetyltransferase activity by actinomycin D On the basis of the above findings, it appeared likely that actinomycin D would inhibit the endogenous nighttime rise of N-acetyltransferase after 12 hr of exposure to light in a diurnal (percent of maximal induction i S.E.) Reinduction Induction Induction at midnight in after after 20 denervated Treatment 18 hr light min light glands Control 4 ± 1* 1 ± 0.5* 1 ± 0.5* AMP 100 ± 12 100 ± 20 100 ± 8 AMP plus actinomycin D 12± 3* 88± 26t 15 ± 2* AMP plus cycloheximide 1 i 0.5* 3 ± 1* Pineal glands were obtained from animals exposed to light for 18 hr or for 20 min at midnight. In addition, denervated glands from animals exposed to light for 20 min at 0600 hr were used. After 1 hr preincubation in media containing actinomycin D (10,g/ml) or cycloheximide (100,sg/ml), dibutyryl cyclic AMP (final concentration 10-i M) was added to the media. activity in the pineal glands was assayed 6 hr after the addition of dibutyryl cyclic AMP (n = 6). * P < 0.01 when compared with the corresponding dibutyryl cyclic group by Student's t-test. t P < 0. 01 when compared with the corresponding 18 hr light exposed group. cycle. Rats were injected with actinomycin D (5 mg/kg) or vehicle at 1800 hr, shortly before lights went out, and killed in the dark at 2300 hr. Actinomycin D blocked the nocturnal rise in N-acetyltransferase activity by more than 90% (data not shown). This indicates that the circadian rhythm of N- acetyltransferase activity is initiated by the stimulation of RNA synthesis in the pinealocytes as a result of increased release of norepinephrine with darkness. of N-acetyltransferase by dibutyryl cyclic AMP in vitro Activation of the beta-adrenergic receptor induces N-acetyltransferase activity via a cyclic AMP coupled mechanism (6, 7). Since the above results indicate that the beta-adrenergic receptor acts intracellularly to stimulate both RNA synthesis (transcription) and protein synthesis (translation), similar experiments were done to determine whether both of these actions are mediated by cyclic AMP. A synthetic nucleotide, dibutyryl cyclic AMP, which by-passes the beta-adrenergic receptor in the pineal (9), was used. Actinomycin D and cycloheximide blocked the induction of N-acetyltransferase by dibutyryl cyclic AMP in animals exposed to light for 18 hr (Table 3). However, at midnight, actinomycin D had no effect on the reinduction of N-acetyltransferase by dibutyryl cyclic AMP. At this time, inhibition of protein synthesis with cycloheximide still blocked N-acetyltransferase induction. The results obtained in denervated glands (from ganglionectomized animals killed after 12 hr of darkness) were identical to those obtained with glands from intact animals exposed to light for 18 hr. Thus, the effects of actinomycin D on the induction of N-acetyltransferase by isoproterenol or by dibutyryl cyclic AMP are similar and vary with the previous exposure of the gland to,-adrenergic stimulation.
2110 Biochemistry: Romero et al. DISCUSSION There is a variable lag period in the induction of N-acetyltransferase by isoproterenol. The duration of this lag period increases in direct relation to the length of exposure to light before the test stimulus (9). Since exposure of the animals to light decreases the turnover of norepinephrine at the sympathetic terminals in the pineal gland (5), the increase in the lag period is also correlated with this period of lower nerve activity. Conversely, the lag period is shortest when the enzyme is reinduced from baseline levels shortly after prior stimulation (9). This prior stimulation can be either physiologic induction by darkness as a result of increased sympathetic nerve activity, or induction by exogenously administered catecholamines (9). In the extreme case, isoproterenol reinduces N-acetyltransferase with essentially no lag period in animals whose high nocturnal N-acetyltransferase is reduced by brief exposure to light. In these animals, isoproterenol increases N-acetyltransferase activity immediately and restores it to the original darktime levels within less than 1 hr after injection (Fig. 1). In the daytime, similar high levels of enzyme activity are attained approximately 3 hr after isoproterenol administration (3, 9). In either case, the daytime induction or the nighttime reinduction by isoproterenol are blocked by cycloheximide, a compound which inhibits protein synthesis by interfering with translation of messenger RNA at the ribosomal level (13). This requirement for new protein synthesis, even for the rapid reinduction at midnight, argues against the reactivation of a reversibly inactivated form of the enzyme and suggests the requirement for synthesis of new enzyme protein. If the rapid reinduction with essentially no lag period is not the result of reactivation of an inactive form of the enzyme, then it is more likely to be due to the accumulation of an intermediate in the synthesis of N-acetyltransferase. The results obtained with actinomycin D support this possibility and further suggest that the precursor may be messenger RNA. Thus, actinomycin D blocked the induction of N-acetyltransferase in animals exposed to light for more than 12 hr, but failed to block the reinduction at midnight, either tn vivo or in vitro (Tables 1 and 2). This failure indicates that RNA synthesis is not required for the reinduction of N-acetyltransferase at midnight. A period of decreased activation of the,3-adrenergic receptor during longer exposure of the animals to light allows for the degradation of this RNA intermediate and accounts for the inhibition of N-acetyltransferase induction by actinomycin D in these animals. Although no direct evidence is presented for the formation of specific compatible with the proposal messenger RNA, the data are that synthesis of specific messenger RNA is involved in the induction of N-acetyltransferase by,3-adrenergic stimulation. These results shed new light on previous experiments that showed the effect of actinomycin D on the induction of N-acetyltransferase. In one series of experiments (14) data were presented to suggest that actinomycin D inhibits the response to norepinephrine in vitro, provided that pretreatment with the antibiotic before the test stimulus were sufficiently prolonged. In those experiments, however, the length of preincubation with actinomycin D (several hours) made it impossible to rule out nonspecific toxic effects of the antibiotic (14). Furthermore, the lighting conditions under which the animals were kept prior to the experiments is not specified, Proc. Nat. Acad. Sci. USA 72 (1975) and our results indicate that this is a critical parameter. In another series of experiments (3, 15), a dose of actinomycin D of 1 mg/kg failed to inhibit the induction of N-acetyltransferase in vivo. In the present experiments, in vivo doses of 5 mg/kg were used to show inhibition by actinomycin D. Despite the magnitude of this dose, toxic or nonspecific effects are unlikely to account for the effects described, since diametrically opposite results were obtained under different lighting conditions. The ability of isoproterenol and dibutyryl cyclic AMP to stimulate N-acetyltransferase activity at midnight, even in the face of almost total inhibition of RNA synthesis by actinomycin D, indicates that the activation of the betaadrenergic receptor affects intracellular post-transcriptional sites. Further, the ability of actinomycin D to block induction by isoproterenol and dibutyryl cyclic AMP in the lightexposed animals and in denervated pineals, indicates that under those conditions transcription is required. It also indicates that both isoproterenol and dibutyryl cyclic AMP affect pretranscriptional sites. Thus, a second site for the action of the beta-adrenergic receptor and cyclic AMP is likely to be the stimulation of transcription when required. The data are, therefore, indicative of two distinct intracellular sites of action for the pineal beta-adrenergic receptor: pretranscriptional and post-transcriptional. It appears likely that both of these actions are mediated by cyclic AMP. 1. Klein, D. C. & Weller, J. L. (1970) "Indole metabolism in the pineal gland: a circadian rhythm in N-acetyltransferase," Science 169, 1093-1095. 2. Klein, D. C., Weller, J. L. & Moore, R. Y. (1971) "Melatonin metabolism: neural regulation of pineal serotonin N- acetyltransferase activity," Proc. Nat. Acad. Sci. USA 68, 3107-3110. 3. Deguchi, T. & Axelrod, J. (1972) "Control of circadian change in serotonin N-acetyltransferase activity in the pineal organ by the beta-adrenergic receptor," Proc. Nat. Acad. Sci. USA 69, 2547-2550. 4. Taylor, A. N. & Wilson, R. W. (1970) "Electrophysiological evidence for the action of light on the pineal gland in the rat," Experientia 26, 267-269. 5. Brownstein, M. J. & Axelrod, J. (1974) "Pineal gland: 24- hour rhythm in norepinephrine turnover," Science 184, 163-165. 6. Klein, D. C. & Weller, J. L. (1973) "Adrenergic-adenosine 3 ',5'-monophosphate regulation of serotonin N-acetyltransferase and the temporal relationship of serotonin N- acetyltransferase activity to the synthesis of 3-H-N-acetylserotonin and 3-H melatonin in the cultured rat pineal gland," J. Pharmacol. Exp. Ther. 186, 516-527. 7. Deguchi, T. (1973) "Role of the beta-adrenergic receptor in the elevation of adenosine 3',5'-monophosphate and induction of N-acetyltransferase in rat pineal glands," Mol. Pharmacol. 9, 184-190. 8. Klein, D. C. & Weller, J. L. (1972) "Rapid light-induced decrease in pineal N-acetyltransferase activity," Science 177, 532-533. 9. Romero, J. A. & Axelrod, J. (1975) "Regulation of sensitivity to beta-adrenergic stimulation in the induction of pineal N-acetyltransferase," Proc. Nat. Acad. Sci. USA 72, 1661-1665. 10. Wicks, W. D. (1974) "Regulation of protein synthesis by cyclic AMP," in Advances in Cyclic Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 4, pp. 335-438. 11. Deguchi, T. & Axelrod, J. (1972) "Sensitive assay for N- acetyltransferase activity in rat pineal," Anal. Biochem. 50, 174-179. 12. Perry, R. P., La Torre, J., Kelley, D. E. & Greenberg, J. R. (1972) "On the lability of poly(a) sequences during ex-
Proc. Nat. Acad. Sci. USA 72 (1975) traction of messenger RNA from polyribosomes," Biochim. Biophys. Acta 262, 220-226. 13. Obrig, T. G., Culp, W. J., McKeehan, W. I. & Hardesty, B. (1971) "The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes," J. Biol. Chem. 246, 174-181. 14. Klein, D. C. & Berg, G. R. (1970) "Pineal gland: stimulation of melatonin production by norepinephrine involves cyclic Stimulation of Pineal 2111 AMP mediated stimulation of N-acetyltransferase," in Role of Cyclic AMP in Cell Function, eds. Greengard, P. & Costa, E. Advances in Biochemical Psychopharmacology (Raven Press, New York), Vol. 3, pp. 255-257. 15. Deguchi, T. & Axelrod, J. (1972) "Induction and superinduction of serotonin N-acetyltransferase by adrenergic drugs and denervation in the rat pineal organ," Proc. Nat. Acad. Sci. USA 69, 2208-2211.