Reprinted from 28 June 1974, Volume 184, pp. 1341-1348 SCIENCE The Pineal Gland: A Neurochemical Transducer Chemical signals from nerves regulate synthesis of melatonin and convey information about internal clocks. The pineal gland has become the subject of considerable investigation during the past decade because it pro- vides a productive experimental model for studying circadian rhythms and reg- ulation of end organs by nerves. In the mammal, the pineal gland rests between the two cerebral hemispheres and weighs about 100 milligrams in man and 1 mg in the rat (1). The pineal gland originates in the brain of the developing mammalian embryo, but it loses direct nerve connection with the brain soon after birth. The pineal paren- chymal cells are innervated by sympa- thetic nerves (noradrenaline-containing) whose cell bodies lie in the superior cervical ganglia (2). Amphibian pineals have photoreceptive cells that can gen- erate nerve impulses in direct response to environmental light (3). Photorecep- tor elements, however, are not found in the mammalian pineal cells. The beginning of the modern era in pineal research stemmed from the iso- lation and identification of the indole- amine melatonin (5-methoxy-N-acetyl- The author is chief of the pharmacology section, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014. Julius Axelrod _tryptamine) from bovine pineals by Lerner et al. (4). It then becamie possi- ble to examine its localization, physio- logic properties, formation, and metab- olism: Melatonin is the most potent agent for causing contractions of me- lanophores in frog and fish skin. When treated with melatonin at concentrations of 10-18 gram per milliliter, the skin of many fish and amphibians rapidly blanches (5). The amphibian pineal contains melatonin and the enzymes that make it (6). These results indicate that melatonin causes changes in skin pigmentation in fish and amphibians when it is released from pineal organs. In the mammal, melatonin is synthe- sized mainly in the pineal (1), and it exerts inhibitory effects on gonads. When injected into birds, it causes a decrease in weight of the ovaries, testes, and oviduct (7). It delays vaginal opening and reduces ovary weight in young Tats (7). When melatonin is implanted in the median eminence, the elevation in the content of leutinizing hormone (LH) in the pituitary following castra- tion is blocked, and plasma LH con- centration is lowered (8). Blinding of male hamsters causes a fall in the weight of testes, but when pineals are removed or when nerves to the pineal are cut the reduction in testicular weight is prevented. During proestrus in rats, melatonin inhibits ovulation by preventing the release of LH (9). The early morning elevation in plasma pro- lactin in male rats is mediated by in- creased release of a pineal hormone (10). In the sparrow, the pineal serves as a time-measuring system (//). The physiological aspects of the pineal have been reviewed recently (2). Melatonin is synthesized almost exclu- sively within the pineal cell as follows (Fig. 1): tryptophan — 5-hydroxytrypto- phan — serotonin — N acetylserotonin > melatonin. Tryptophan is hydroxyl- ated to 5-hydroxytrytophan by trypto- phan hydroxylase (1/3). The_ latter amino acid is then decarboxylated by l-aromatic amino acid decarboxylase to form the biogenic amine serotonin. Serotonin then undergoes a complex fate. One portion is deaminated to 5- hydroxyindoleacetic acid by mono- amine oxidase, and another portion leaves the pineal cell and is taken up by the sympathetic nerve terminal and stored together with the neurotrans- mitter noradrenaline (J) (Fig. 1). A third portion is acetylated to N-acetyl- serotonin by the enzyme serotonin N- acetyltransferase (14). This is a critical regulatory step, as will be shown later. N-Acetylserotonin is then O-methylated by hydroxyindole O-methyltransferase to form melatonin, S-adenosylmethio- nine serving as the methyl donor (/5). Hydroxyindole O-methyltransferase is highly. localized in the pineal glands of mammals and birds. Small amounts of the enzyme are also present in the retina of the rat. In other classes (rep- tiles, amphibia, and fish), hydroxy- indole O-methyltransferase is also found in ‘the eye and brain as well as the pineal region (J6). Although in- direct, the evidence that the frog pineal blanches skin by secreting melatonin is compelling. Copyright® 1974 by the American Association for the Advancement of Science Reprinted by the U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE National Institutes of Health Light and the Melatonin-Forming Enzyme Rats exposed to constant illumination remain in persistent estrus. This condi- tion could be reduced or prevented when rats were injected with extracts of bovine pineal glands (/7). As a re- sult of these findings Wurtman er al. (17) concluded that the pineal gland releases a substance that inhibits the gonads and that the formation and release of this substance is reduced when animals are kept in constant illu- mination. Fiske et al. (18) also found that pineal glands of rats exposed to continuous light weighed less. Then my colleagues and I (7) found that mela- tonin reduces the incidence of estrus in rats exposed to continuous light. It be- came apparent that environmental light- ing might effect the melatonin-forming enzyme, hydroxyindole O-methyltrans- ferase. Rats kept in continuous light for about 7 days showed a marked re- duction in enzyme activity as compared to those kept in constant darkness (19). Thus, constant light decreased the ac- tivity of hydroxyindole O-methyltrans- ferase, which in turn reduced the production of the gonad-inhibiting compound, melatonin. This reduction of melatonin synthesis in constant light would result in persistent estrus. The question then arose as to how messages about environmental lighting could reach the pineal, which lies deep between the two cerebral hemispheres. The most likely possibility was a neural pathway. The mammalian pineal is heavily innervated by sympathetic nerve terminals, which are highly branched and contain swellings or vari- cosities that are in close juxtaposition with pineal parenchymal cells (Fig. 1). These varicosities contain numerous granulated vesicles that are the site of storage of the neurotransmitter nor- adrenaline (20). The nerve terminals that innervate the pineal can readily be destroyed by bilateral removal of the superior cervical ganglia. When rats with denervated pineals were kept in constant darkness or light, there was no longer a difference in hydroxyindole O-methyltransferase activity in the pin- -eal (21). In blinded rats, continuous darkness or light had no effect on hy- droxyindole O-methyltransferase activi- ty, which suggests that the retina is necessary for transmission of light mes- sages to the pineal. Bilateral lesions of the medial forebrain bundle, which contains noradrenergic and serotonergic nerves, also abolished the effects of Nerve terminal vartcosity Pineal cell Tryptophan J TROH 5-OH-Tryptophen AAD Sut NAT \une AcHT ° HIAA tiomr Melatonin ‘ Fig. 1. The pineal cell, sympathetic nerve, and melatonin synthesis. Abbreviations: TROH, tryptophan hydroxylase; AAD, aromatic amino acid decarboxylase; 5 HT, serotonin; NAT, serotonin N-acetyltrans- ferase; MAO, monoamine oxidase; AcHT, N-acetylserotonin; HIAA, 5-hydroxyin- doleacetic acid; HIOMT, hydroxyindole O-methyltransferase; and NA, noradrena- line. environmental lighting on pineal hy- droxyindole O-methyltransferase (22). In a series of experiments (23) it was shown that information about en- vironmental lighting reaches the rat pineal as follows: retina — inferior ac- cessory optic tract > medial forebrai:f bundle — medial terminal nucleus of the accessory optic system — pregangli- onic sympathetic tract in the spinal cord — superior cervical ganglia — post- ganglionic sympathetic fibers paren- chymal cells of the pineal. Circadian Rhythms of the Pineal Soon after melatonin was discovered, relatively large amounts of its precursor serotonin were found in the pineal (24); the serotonin was evenly dis- tributed between the parenchymal cells and the sympathetic nerve terminals (25). Quay (26) then found a marked, 24-hour cycle in the serotonin content of the rat pineal. Peak levels of sero- tonin were reached at about midday (Fig. 2). Soon after nightfall, there was a rapid fall in serotonin content (27). The day-night rhythm of sero- tonin content in the pineal persisted unchanged in continuous darkness, but was abolished in rats that were kept in continuous light (27) (Fig. 2). Reversal of the lighting schedule (light kept on during the night and off during the daytime) changed the pineal serotonin rhythm in the pineal by 180° within 6 days (28). All of these experiments indicated that the daily rhythm in pineal serotonin is endogenous (circa- dian) but is synchronized by environ- mental lighting. Denervation of the pineal by removal of the superior cervical ganglia abolished the serotonin rhythm (27). Interruption of the nerve impulses from the central nervous sys- tem to the superior cervical ganglia and depletion of brain noradrenaline and serotonin with reserpine (29) also suppressed the pineal serotonin rhythm. These observations indicated that the circadian rhythm of serotonin is gen- erated by sympathetic nerve terminals innervating the pineal, presumably by changes in the release of the neuro- transmitter noradrenaline. The circa- dian serotonin rhythm appeared to be generated by a “clock” in the brain. The circadian rhythms in serotonin content in the rat pineal appear as early as 6 days after birth (30). When lights were left on during the night, the nocturnal decline in serotonin con- tent was prevented in adult and new- born animals. Lights left on during the night prevented the fall in serotonin in blinded 12-day-old rats. When the head of the 12-day-old rat was covered with a hood and the lights were on, the serotonin content fell at night. After the hooded rats were 27 days old, additional lighting no longer pre- vented the decline of serotonin at night (30). Thus, environmental lighting can reach the pineal gland by an extra- retinal pathway in the newborn but not in the adult rat, Extraretinal pineal re- sponses have also been found in birds (11). There is a marked circadian rhythm in pineal N-acetyltransferase (31) (Fig. 2), which is 180° out of phase with that for serotonin. One hour after the onset of darkness, there is a 30- to 50-fold rise in the enzyme activity. A circadian rhythm in the melatonin con- tent of the rat pineal has the same phasing as that of N-acetyltransferase. Like the serotonin rhythm, the N-ace- tyltransferase rhythm is abolished by denervating the sympathetic nerves to the pineal or by interrupting nerve im- pulses from the brain (32). Bilateral lesions in the suprachiasmatic nucleus (present in the hypothalamus) abol- ish the circadian rhythm of N-ace- tyltransferase in the pineal (33). This suggests that a biological clock in the brain sends fibers through, innervates, or is localized in the suprachiasmatic nucleus. The observation that the circadian rhythms in pineal serotonin and N-ace- tyltransferase are abolished by cutting sympathetic innervation to the pineal indicated that there might be differ- ences in the release of noradrenaline from these nerves during the day and at night. Brownstein and I (34) found a 24-hour rhythm in the turnover of noradrenaline in the sympathetic nerves innervating the pineal. More nor- adrenaline is utilized (presumably by release from nerve terminals onto the pineal cell) at night than during the day. This rhythm in turnover persisted in blinded rats and was abolished in continuous light. This strongly sug- gested that the circadian rhythm in the pineal cell is generated by diurnal release of the neurotransmitter nor- adrenaline. The circadian rhythm in sympathetic nerve activity is presum- ably driven by a biological clock aris- ing in or transmitted via the supra- chiasmatic nucleus in the hypothalamus. Control of Pineal Indoleamine Metabolism in Organ Culture The sympathetic nerves in the pineal contain both noradrenaline and sero- tonin, and it was not certain whether these compounds released from the nerve terminals exert their effects on pineal indoleamines. If, as appeared likely, the neurotransmitter noradrena- line stimulates the pineal cell, does it act on an a- or #-adrenergic receptor, and is adenylate cyclase involved? Also, which is the critical step in the syn- thesis of melatonin from tryptophan? The rat pineal in organ culture proved ‘to be a productive experimental tool to answer these questions. In such a system, the effects of biogenic amines, adrener- gic blocking agents, and cyclic AMP (adenosine 3’,5’-monophosphate) could be examined directly, free of the com- plexities in vivo. In studies on pineal organ culture initiated independently by Shein and collaborators (35) and Klein et al. (36), (4C]tryptophan was added to the culture media and the formation of radioactive serotonin, 5- hydroxyindoleacetic acid (the deamin- ated metabolite of serotonin), and mela- tonin was measured. The pathway of synthesis, of melatonin in pineal organ culture was found to be the same as in tle innervated pineal (Fig. 1). The addition of cycloheximide, an inhibitor of protein synthesis, to the incubation media completely inhibited the forma- tion of [4C]serotonin and [!4C]mela- tonin from [!4C]tryptophan, which in- dicates that synthesis of new enzyme Fig. 2. Circadian | thythms in pineal N - acetyltransferase, serotonin, and mela- tonin. 0000 0600 1200 protein was obligatory for the forma- tion of melatonin (37). The addition of [noradrenaline to the pineal culture caused a marked increase in the formation of radioac- tive melatonin from tryptophan during 2 days of incubation (35-37). The stimulation of melatonin synthesis in the pineal organ culture by /-noradren- aline was prevented by the addition of /-propanolol, a f-adrenergic block- ing agent (37a). e-Adrenergic blocking agents had no effect on the increased formation of melatonin in the presence of /-noradrenaline. When added to or- gan cultures, a variety of sympathomi- metic amines such as [/-adrenaline, dopamine, octopamine, and tyramine also stimulated the formation of mela- tonin from tryptophan (37). Serotonin or melatonin was without effect. Many actions of noradrenaline are mediated by cyclic AMP. Evidence that noradrenaline might be acting via cyclic AMP in the pineal came from the obser- vation that the catecholamine stimulatés adenylate cyclase activity in homoge- nates of rat pineal (38). The addition “of cyclic AMP to pineal organ culture, however, was without effect in stimu- lating melatonin synthesis. Dibutyryl! cyclic AMP, a compound that, unlike cyclic AMP, is not metabolized by phosphodiesterase, markedly stimulated the formation of {?4C]melatonin from ['4C]tryptophan (39). These studies in organ culture indicated that noradrena- line released from sympathetic nerves increases melatonin synthesis by stimu- lating a 8-adrenergic receptor on the membrane of the pineal cell. This stimulation results in activation of adenylate cyclase inside the cell to make cyclic AMP. Klein and Berg (40) examined the effect of I-noradrenaline stimulation of hydroxyindole O-methyltransferase and N-acetyltransferase, the enzyme that converts serotonin to N-acetylserotonin. Noradrenaline caused only a small in- om NeAcety!> transferase on Melatonin Serotonin f Serres 1800 2400 0600 1200 1800 2400 0600 1200 Clock hours ’ Dar Light crease in hydroxyindole O-methyltrans- ferase activity—not enough.to account for the large increase in melatonin formation. On the other hand, the cate- cholamine caused about a 20-fold in- crease in N-acetyltransferase activity and melatonin formation after incuba- tion of pineal organ culture for 18 hours. Dibutyryl cyclic AMP also markedly stimulated the N-acetyltrans- ferase activity. The increase in N-acetyl- transferase activity by either /-nor- adrenaline or dibutyryl cyclic AMP was blocked by the addition of protein syn- thesis inhibitors to the culture media. These observations suggested that the regulation of N-acetyltransferase syn- thesis by the f-adrenergic receptor is the critical step“in the control of pineal melatonin synthesis. Regulation of Pineal Circadian Rhythms N-Acetyltransferase (32), serotonin (26, 27), N-acetylserotonin (41), and melatonin (42) undergo marked circa- dian rhythms (Fig. 2). In view of the diurnal rhythms in turnover of nor- adrenaline in sympathetic nerves inner- vating the rat pineal, it appeared likeiy that the circadian rhythms in the in- doleamines and N-acetyltransferase are generated by day-night changes in the release of the neurotransmitter (34). Such a mechanism of regulation of pineal circadian rhythms was estab- lished by a series of experiments in vivo (43). After the onset of darkness at 1800 hours, N-acetyltransferase un- derwent -a 30- to 50-fold increase in activity (Fig. 3). In the first hour after the lights were turned off at 1800 hours, only a small increase in enzyme activity was observed. Beginning at 1900 hours there was a sharp increase in enzyme activity, reaching a maxi- mum at 2200 hours (Fig. 3). When lights were kept on after 1800 hours, Lights on or we propanolol in dark 400 F- Z Ss g 5 a £ 200 - $ < Propanolol z Reserpine Denervation Decentralization Cycloheximide 10 , ; SS -=— : i i i 1200 $ 1500 1800 si a6 } 0300 6600 Isoproterenol Clock hours Isoproterenol Light Dark Light Fig. 3. Induction and suppression of serotonin N -acetyltransferase in the pineal. however, there was no increase in N- acetyltransferase activity. Rats kept in darkness during the day from 1000 to 1600 hours also showed no daytime elevation in enzyme activity. These findings showed that both darkness and the proper setting of an internal clock are necessary for the nighttime increase in N-acetyltransferase activity. Inter- rupting sympathetic nerve impulses by bilateral removal of the superior cer- vical ganglia or preganglionic decen- tralization completely prevented the nocturnal elevation of N-acetyltrans- ferase (32) (Fig. 3). When Lpropanolol, a #-adrenergic blocking agent, was administered to tats before the onset of darkness, the nighttime rise of enzyme was also blocked (43) (Fig. 3). Injection of an a- adrenergic blocking agent did not pre- vent the elevation. Reserpine, a drug that depletes nerves of both catecholamines and serotonin, also prevented the elevation of N-acetyltransferase. p- Chlorophenylalanine, a compound that depletes nerves. of serotonin, had no effect on the circadian rhythm of N- acetyltransferase, which indicates that noradrenaline but not serontonin is in- volved in inducing the enzyme. Cyclo- heximide injected immediately before the onset of darkness blocked the rise of N-acetyltransferase, whereas actinomy- cin D, an inhibitor of RNA synthesis, had no such effect. These experiments clearly showed that noradrenaline re- leased from sympathetic nerves stimu- lated the B-adrenergic receptor which in turn initiated events that lead to the synthesis of new N-acetyltransferase molecules. Administration of the catecholamine l-isoproterenol during the daytime when both N-acetyltransferase activity and N-acetylserotonin content are low and serotonin content is high results in ele- vation of the enzyme (Fig. 3) (44) and its product N-acetylserotonin (41), and a fall of serotonin (45). The day- time rise in N-acetyltransferase and N-acetylserotonin and fall in serotonin induced by isoproterenol are blocked by prior administration of a #-adre- nergic blocking agent. These observa- tions indicate that circadian rhythms of pineal indoles are generated by changes in N-acetyltransferase activity which in turn are controlled by the @-adrenergic receptor. Thus, the rise in N-acetyltratis- ferase at night causes a fall in sero- tonin and a rise in N-acetylserotonin, the precursor of melatonin (Fig. 2). The reverse occurs during the daytime. When rats are exposed to light during the night, there is a precipitous fall in pineal N-acetyltransferase (43, 46) (Figs. 2 and 3). Isoproterenol injected before the rats are exposed to light prevents the light-induced decrease in N-acetyltransferase activity (43). When rats are returned to darkness after 10 minutes of light, N-acetyltransferase activity begins to rise immediately and” attains its initial level after 3 hours. Exposure to light during the night causes a rise in serotonin content as enzyme activity falls (45). Thus, main- tenance of N-acetyltransferase activity requires continuous occupation of the f-adrenergic receptor. A brief expo- sure to light reduces or shuts off the release of nonadrenaline from sympa- thetic nerves, and a rapid fall in enzyme activity ensues (Fig. 3). The action of light on the retina is a stimulatory event, yet it inhibits the release of the neurotransmitter from sympathetic nerves in the pineal. This would indi- cate that, somewhere in the brain be- tween the retina and the superior cervi- cal ganglia, a stimulatory signal is con- verted to an inhibitory signal. When the @-adrenergic blocking drug propanolol is injected into rats at night, N-acetyltransferase activity is decreased to less than 15 percent of its initial value within 10 minutes (43), a reduction similar to that caused by exposure to light (Fig. 3). Cyclohexi- mide, given at night at a dose that im- mediately inhibits protein synthesis in vivo, results in a gradual decrease in N-acetyltransferase activity with a half- life of about 60 minutes (43). The rapid decrease in pineal N-acetyltrans- ferase activity after light exposure or blockage of the 8-adrenergic receptor (half-life of 5 minutes) and the slower fall in enzyme activity when protein synthesis is inhibited indicate at least two mechanisms for the degradation of N-acetyltransferase in vivo. The rap- id decrease might result from the con- version of an active to an inactive form of the enzyme or from a dis- aggregation of subunits of the enzyme molecule. The slower decrease after in- hibition of protein synthesis probably represents normal degradation of the enzyme. The character of the N-acetyltrans- ferase induction by catecholamines de- pends on the previous exposure to light. When rats are given isoproterenol after exposure to light for 6 hours, there is no increase in N-acetyltransferase for the first hour (Fig. 3). Then, between 1 and 3 hours after isoproterenol ad- ministration, enzyme activity rises, reaching a maximum after 2 hours -(43) (Fig. 3). When rats are kept in darkness from 1800 hours to 2400 hours and then placed in light for 10 minutes, there is the expected rapid decrease in N-acetyltransferase activity. After a brief exposure to light follow- ing a prolonged period of darkness, isoproterenol injection causes an imme- diate increase in N-acetyltransferase activity without the 1-hour lag period (43) (Fig. 3). Cycloheximide, admin- istered just before isoproterenol, blocks both the immediate and delayed in- creases in enzyme activity after isopro- terenol injection. The delayed elevation of enzyme activity in the absence of §-adrenergic stimulation for long peri- ods of time might indicate de novo synthesis of new enzyme molecules, The immediate elevation of N-acetyl- transferase after a prolonged period of stimulation of the §-adrenergic re- ceptor in darkness indicates an accumu- lation of messenger RNA or precursors necessary for the synthesis of N-acetyl- transferase. In pineal organ culture, -noradrena- line causes a rapid increase in cyclic AMP (47, 48) as well as N-acetyl- transferase and melatonin (40). Theo- phylline, a phosphodiesterase inhibitor enhances the effect of noradrenaline on pineal cyclic AMP, while f-adrenergic blocking agents prevent this effect. Thus noradrenaline stimulates N-acetyl- transferase and melatonin production via $-adrenergic receptors linked to adenylate cyclase and cyclic AMP for- mation. The temporal relationships between stimulation of the pineal -adrenergic receptor, cyclic AMP content, and formation of N-acetyltransferase in vivo were reported by Deguchi (49). Injection of J-isoproterenol during the daytime (arrow-A in Fig. 4) resulted in a 15-fold increase in pineal cyclic AMP content within 2 minutes which was maintained for 10 minutes; cyclic AMP content then fell to baseline lev- els 30 minutes after administration of the catecholamine. One hour after iso- proterenol injection there was no change in N-acetyltransferase activity; during this time cyclic AMP content reached a peak and then returned to its initial level. Thirty minutes after cyclic AMP content returned to baseline, N-acetyltransferase activity rose, reach- ing a maximum 3 hours after injection of isoproterenol, and then returned to the low daytime value after 5 hours (Fig. 4). i-Propanolol injected before isoproterenol (arrow A in Fig. 4) pre- vented the rise of both cyclic AMP content and of N-acetyltransferase ac- tivity. When propanolol was injected 60 minutes after isoproterenol at a time when cyclic AMP content had already reached a peak and then re- turned to its initial level (arrow B in Fig. 4), the increase in N-acetyltrans- ferase was still prevented. The admin- istration of cycloheximide before iso- proterenol (arrow A in Fig. 4) did not affect the rise and fall of cyclic AMP content, but it prevented the elevation of N-acetyltransferase activity. When the protein synthesis inhibitor was given after the rise and fall of cyclic AMP (arrow B in Fig. 4), the formation of N-acetyltransferase was still blocked. It can be concluded that stimulation of e i i 4 600 |- a) £ - \ £ 7] * 7 500 |- / \ 4500 2 é s s = ‘ \ a @ é 3s : = 4 400 = 400 / \ 40 2 s \ . ° ‘ 7 3 400F 3 y 1 300 o 5 é ‘ s & , ‘. E a ? & _ - 200 = 200 A \. g 2 Fd \ 5 $ ° ‘qo 2 100 + : ” t 5 : ’ 2 ., . z 0 Boro y i —* ! } 0 = { | 2 3 4 5 { Time (hours) A 8 Fig. 4. Relation between cyclic AMP, N-acetyltransferase, and f-adrenergic receptors. “Isoproterenol hydrochloride (5 mg per kilogram of body weight) was given intra- venously to rats. Cyclic AMP and N-acetyltransferase activity was measured at times indicated. Arrows A and B are the times at which [-propanolo! (20 mg/kg) was in- jected intravenously. Vertical bars indicate standard error of the mean. {From Deguchi (49), with permission of Academic Press] the 8-adrenergic receptor activates the formation of cyclic AMP via adenylate cyclase. This in turn induces the syn- thesis of new N-acetyltransferase mole- cules and thus more melatonin. Cyclic AMP may act on the translational process in protein synthesis, since cy- clohexamide (but not actinomycin D) interferes with the induction of N-ace- tyltransferase. It also appears that there is an additional B-adrenergic receptor that is not involved in cyclic AMP formation but is concerned with the formation of N-acetyltransferase. Supersensitivity and Subsensitivity in Pineal Response An important and puzzling cellular phenomenon is the increase in respon- siveness of the end organ after denerva- tion, decentralization, or administra- tion of drugs. Prior administration of cocaine or denervation of sympathetic nerves results in a markedly enhanced physiologic response to noradrenaline (50). This increased response is due mainly jo a presynaptic event: the capacity of cocaine to block reuptake of noradrenaline into sympathetic nerves (52), a major mechanism of in- activation of the neurotransmitter (52). Denervation of sympathetic nerves also causes supersensitivity by destroying the catecholamine uptake mechanism and also by affecting the postsynaptic sites (50). Recent work has suggested a hypothesis for the changes in respon- siveness of postsynaptic sites on the pineal cell after a variety of manipula- tions. N-Acetyltransferase activity in the pineal is low during the daytime (32, 43). Various drugs and physiological manipulations were studied for their capacity to induce this enzyme during the daytime (44). Adrenaline, L-dopa, noradrenaline, and isoproterenol (Fig. 3) caused a marked increase in pineal N-acetyltransferase activity when in- jected during the daytime. The f-adre- nergic blocking agent /-propanolol pre- vented the elevation of pineal N- acetyltransferase activity caused by the drugs or stress, while an a-adrenergic blocking agent did not. To examine whether these com- pounds induced N-acetyltransferase di- rectly or acted by the release of nor- adrenaline from the nerves innervating the pineal, the pineal was denervated by removal of the superior cervical ganglia or by chemical sympathectomy with 6-hydroxydopamine (44). When the pineal was denervated, administra- tion of L-dopa increased N-acetyltrans- ferase activity 100-fold as compared to 20- to 30-fold when the gland was innervated. Thus, denervation caused an increased responsiveness (supersen- Sitivity) to catecholamines. Supersensi- tivity of denervated pineal with respect to N-acetyltransferase was also ob- served after administration of isopro- terenol (48) or insulin or after stress Table 1. Supersensitivity and subsensitivity in cultured pineals. Rats were denervated by bi- lateral removal of the superior cervical ganglia 7 days before they were killed. Isoproterenol- treated rats received the drug (2.0 mg/kg) 8, 16, and 24 hours before they were killed, Pineals were cultured for 10 hours with indicated concentrations of isoproterenol, and N.-acetyl- transferase activity was measured. !-Isoproterenol N-Acetyltransferase (units) Intact Denervated l-Isoproterenol- treated 1x 10° 1322 330 + 90* 5 x« 10-* 330 + 40 1330 + 210* 26 + 7* 2x 10° 680 + 160 2190 + 330° 70 + 24* 1x 10’ 940 + 80 1180 + 150 320 + 50* 1 x 10° 1720 + 180 1490 + 170 1380 + 110 * P< .01 compared to intact rats at the same isoproterenol concentration. (53). The increased formation of N- acetyltransferase was prevented by £- adrenergic receptor blocking agents or by inhibitors of protein synthesis. These experiments demonstrated that stimula- tion of the pineal B-adrenergic receptors results in enhanced synthesis of the pro- tein N-acetyltransferase (superinduc- tion) after nerve denervation. Superinduction of N-acetyltransferase appeared within 24 hours after removal of the ganglia innervating the pineal (44). It had been reported that pineal adenylate cyclase became more sensi- tive to noradrenaline, but not until 4 weeks after denervation (54). Thus, the appearance of adenylate cyclase supersensitivity does not explain the rapid appearance of superinduction of N-acetyltransferase. The f-adrenergic agonist J-isoproterendi was used to examine whether the changes in re- sponsivity occurred on the postsynaptic membrane. This compound, unlike noradrenaline, is not taken up into sympathetic nerve terminals (55); any presynaptic effects are thus eliminated. Injection of small amounts of isopro-' terenol into rats resulted in tenfold greater induction of N-acetyltransferase in denervated pineals as compared to normally innervated pineals (48). When large doses of isoproterenol were in- jected, the same maximum induction was achieved in the intact and de- nervated pineals (48, 56). Innervated and denervated pineals were then cultured in varying amounts of isoproterenol (48, 56). In the pres- ence of the catecholamine, N-acetyl- transferase activity increased gradually and reached a maximum after 6 to 10 hours in culture. Protein synthesis in- hibitors or -adrenergic blocking agents prevented this rise. Maximum induction of N-acetyltransferase in de- nervated pineals in organ culture occurred with an isoproterenol concen- tration of 5 x 10-®M (Table 1), while enzyme activity in the innervated pineal increased slightly at this concentration. Maximum induction in the innervated pineal was reached at an isoproterenol concentration of 1 x 10—*M (Table 1). Thus, the maximum response in the de- nervated pineal occurred at a dose of f-adrenergic agonist about 1 percent of that in the innervated gland. The maximum enzyme activity achieved, however, was the same in both cases. To examine whether the increase in mT I 200 fF N-Acetyltransferase (% of change) Th Th —100 Ss 3 ~ ° es 3 se <= = - 6 = go » € Cy e 3 « = iy en fe 9 e 2 2 en + (x) se <@£s é 2s e es ag e 33 2 = esa 8 >2 3 ao a so & oe 2? 8 a ec a Fig. 5. Supersensitivity and subsensitivity of pineal N-acetyltransferase. Reserpine (1.5 mg/kg) was injected subcutaneously into rats 24 hours before they were killed. A group of reserpine-treated rats received Lisoproterenol (1.5 mg/kg) 8 and 16 hours before they were killed. Rats were denervated by bilateral removal of the superior cervical ganglia 48 hours before death. One group of denervated rats re- ceived Lisoproterenol (0.5 mg/kg) 8, 16, 24, and 32 hours before they were killed. One group of rats was exposed to light for 7 days. sensitivity of the pineal response was due to absence of the neurotransmitter or to the nerve itself, rats were treated with reserpine to deplete the sympa- thetic nerve terminal of noradrenaline (56). Injection of a small amount of isoproterenol during the daytime re- sulted in a greater increase in pineal N- acetyltransferase activity in reserpine- treated rats as compared to nontreated animals (Fig. 5). Again, when higher doses of isoproterenol were injected, there was no difference in the maxi- mum increase of N-acetyltransferase activity in normal and drug-treated ani- mals. Pineal glands of rats with intact nerves that were depleted of noradrena- ‘line by reserpine also showed a marked sensitivity in organ culture. The super- sensitivity evoked by reserpine treat- ment developed less than 24 hours after the drug was given. Light reduces the release of noradrenaline from nerve terminals innervating the pineal. As predicted, cultured pineals of rats - exposed to continuous lighting were many times more responsive to induc- tion of N-acetyltransferase by isopro- terenol than were pineals from rats under diurnal lighting conditions (56) (Fig. 5). Pineals were about ten times more responsive to catecholamines at the end of the light period (1800 hours) than at the end of the dark period, 0600 hours ($7). The large (30-fold) elevation in pineal N-acetyl- transferase at night could be explained by the reduced release of noradrenaline during the daytime causing supersensi- tivity to the increased neurotransmitter discharged with the onset of darkness. Exposure to long periods of light also resulted in increased elevation of pineal cyclic AMP content after noradrena- line injection (47). These findings raised the possibility that supersensitivity after depletion of noradrenaline by reserpine or denerva- tion might be prevented by the admin- istration of the catecholamine. When isoproterenol was injected twice in a 24-hour period to denervated or reserpine-treated rats, not only was the superinduction of pineal N-acetyltrans- ferase suppressed, but there was a markedly reduced induction of the en- zyme when the pineal was later re- moved and exposed to isoproterenol in organ culture (56) (Fig. 5). The induction of N-acetyltransferase in organ culture by low concentrations of isoproterenol was considerably re- duced in the pineals of isoproterenol- treated rats (Table 1). However, the maximum response at high concentra- tions of the catecholamine was about the same in pineals in organ culture in both the isoproterenol-treated and un- treated rats. Thus, continuous exposure of the -adrenergic receptor to its agonist catecholamine results in a re- duced responsiveness, or tolerance. The effect of various doses of isopro- terenol on stimulation of cyclic AMP in the pineal was examined 3 days after pineal denervation (56). Injection of small doses of isoproterenol resulted in more than twice the cyclic AMP in- crease in the denervated gland as in the innervated pineal. Large doses of isoproterenol gave the same maximum increase of cyclic AMP content in in- nervated and denervated pineals. Es- sentially the same cyclic AMP increases were obtained in innervated and de- nervated pineal glands in organ culture. There was no difference between the intact and denervated pineals with regard to induction of N-acetyltrans- ferase activity when the organs were cultured in the presence of dibutyryl cyclic AMP (56), a compound that acts intracellularly. This indicated that denervation supersensitivity is due to changes proximal to the site of action of cyclic AMP, presumably on the B- -adrenergic receptor on the outer cell membrane. The increase in cyclic AMP in pine- als from reserpine-treated rats was two to three times greater than in those from untreated rats (56). Tre&ting rats with the protein synthesis inhibitor cycloheximide did not block the in- creased cyclic AMP response in re- serpine-treated pineals. This experiment suggests/but does not prove that new synthesis of receptor or of the adenyl- ate cyclase system is not involved in the development of supersensitivity. These experiments indicate that the responsiveness of the postsynaptic B-adrenergic receptor and the forma- tion of the pineal hormone depend on previous exposure of the receptor to its neurotransmitter. When the nor- adrenaline discharged from sympathetic nerves is decreased or eliminated by denervation, decentralization, reserpine, or light, the responsiveness of the B-ad- renergic receptor of the pineal to cate- cholamines and N-acetyltransferase in- duction and, ultimately, to melatonin formation is greatly increased (Fig. 6). The same magnitude of induction of N- acetyltransferase could be achieved with a much smaller amount of catechola- mine in noradrenaline-depleted pineals as compared to pineals exposed to a. complement of neurotransmitter nor- mally discharged from sympathetic nerve terminals. If the number of catecholamine molecules impinging on the f-adrenergic receptor is increased, the pineal cell jbecomes less responsive to the catecholamine (Fig. 6). Thus, larger amounts of catecholamine are re- quired to produce the same increase of N-acetyltransferase in these glands as compared to pineals receiving the normal amount of noradrenaline. This phenomenon is essentially the same as tolerance. The shift in dose response in the induction of N-acetyltransferase by catecholamine in supersensitive and subsensitive pineals (Table 1) indi- cates a change in the affinity of the receptor for its agonist isoproterenol. Such changes in affinity suggest that the conformation of the #-adrenergic receptor has been changed by prior exposure to a larger or lesser amount Normal response Cyclic Protein AMP synthesis © © N-Acetyl- ATP transferase Supersensitive response Cyclicgayy, Protein MPa, ". 4s N- ‘. transferase Subsensitive response Cyclic Protein ® “ AMP synthesis *, , of ATP —sN-Acetyl- transferase Pineal cel! Nerve termina! Fig. 6. Possible mechanisms for the de- velopment of supersensitivity and sub- sensitivity., After exposure to a reduced number of catecholamine molecules, there is an increase in the coupling of the p- adrenergic; receptor on the outside of the pineal cell! membrane to adenylate cyclase on the inner membrane. This leads to in- creases in the formation of cyclic AMP and synthesis of N-acetyltransferase when the g-adrenergic receptor interacts with catecholamines. The reverse occurs when the §-adrenergic receptor is exposed to excessive amounts of catecholamines; ATP, adenosine triphosphate. of its agonist. It is also possible that receptor sites become more available when exposed to few agonist molecules or less available when exposed to an excess of molecules. Changes in sub- and supersensitivity can occur relative- ly rapidly, which makes changes in conformation or availability of the B- adrenergic receptor a likely possibility and a parsimonious adaptive mech- anism for responsive cells. Supersensitivity and tolerance to physiologic agents and drugs is not un- common. Repeated administration of narcotic drugs results in rapid develop- ment of tolerance. On the basis of enzyme studies, it has been proposed that tolerance to opiates is caused by reduced availablity of receptor sites resulting from a continuous interaction of receptor with the narcotic drugs (58). In denervated muscle, an in- crease in the number of acetylcholine binding sites appears on the postsynap- tic membrane (59), and the reverse oc- curs with exposure to large amounts of cholinergic agents (60). The number of insulin receptors decreases in fat cells of obese rats with high plasma insulin concentrations (6/). Adenylate cyclase in the adrenal cortex is more responsive to adrenocorticotrophic hormone in rats in which the hormone has been previously depleted by hypo- physectomy (62). In view of our find- ings with the pineal, changes in many physiologic and pharmacologic _ re- sponses might be due to alterations in conformation or availability of receptor sites of responsive cells imposed by absence or excess of physiologic agents and drugs. Summary There are circadian rhythms in sero- tonin, serotonin N-acetyltransferase, N- acetlserotonin, and melatonin in the pineal which persist in continuous dark- ness and are abruptly abolished by exposure to light. These rhythms are generated by diurnal changes in the release of the neurotransmitter nor- adrenaline from sympathetic nerve ter- minals innervating the pineal. An in- creased discharge of noradrenaline at night stimulates the @-adrenergic re- ceptor, which causes increased syn- thesis of serotonin N-acetyltransferase molecules inside the cell by mediation of an adenylate cyclase system. 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