THE PINEAL GLAND is a neuroendocrine structure that

Transcription

1 /03/$15.00/0 Endocrinology 144(6): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /en Transcription Factors May Frame Aa-nat Gene Expression and Melatonin Synthesis at Night in the Syrian Hamster Pineal Gland MARIE-LAURE GARIDOU, ELENA DIAZ, CHRISTIANE CALGARI, PAUL PÉVET, AND VALÉRIE SIMONNEAUX Laboratoire de Neurobiologie des Rythmes (M.-L.G., C.G., P.P., V.S.), Unité Mixte Recherche-Centre National de la Recherche Scientifique-Université Louis Pasteur 7518, F Strasbourg, France; and Departamento de Biología Funcional (E.D.), Area Fisiología, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain Pineal melatonin synthesis is stimulated at night following an increase in arylalkylamine-n-acetyltransferase (AA-NAT) activity. Depending on the species, two mechanisms of enzyme activation have been described: a camp/phospho-camp response element-binding protein-dependent stimulation of Aanat gene transcription in the rat, presumed to occur in all rodents, or a posttranslational regulation of AA-NAT protein in ongulates. The present data obtained in the Syrian hamster indicate another route of AA-NAT regulation. Elevated nocturnal levels of Aa-nat mrna were strongly suppressed following light exposure or adrenergic antagonist administration, demonstrating the involvement of norepinephrine in the stimulation of melatonin synthesis. However, administration of adrenergic agonists during the day did not increase Aa-nat THE PINEAL GLAND is a neuroendocrine structure that synthesizes and releases the hormone melatonin with both daily and seasonal rhythms (1). This peculiar pattern of melatonin release gives the hormone a pivotal function in the synchronization of numerous circadian and annual functions with the cyclic changes of environmental factors, especially light (2). The main afferent nervous pathway controlling the rhythmic synthesis of melatonin includes the retina, hypothalamic suprachiasmatic nuclei containing the endogenous biological clock, and superior cervical ganglia from which neurons project to the pineal gland and release norepinephrine (NE) only during the night (3). Melatonin is synthesized from serotonin following acetylation by arylalkylamine-nacetyltransferase (AA-NAT, EC ) and methylation by hydroxyindole-o-methyltransferase (EC ) (4). The hormone, immediately released after synthesis, conveys both daily and seasonal information because it is present in the blood with duration proportional to the duration of darkness (1, 5). In mammals, the cellular and molecular mechanisms involved in the regulation of melatonin synthesis have been studied mainly in the rat and bovine pineal gland. The main Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; CRE, camp response element; CREB, CRE-binding protein; CYCLO, cycloheximide; DA, dopamine; DD, constant dark conditions; ICER, inducible camp early repressor; IEG, immediate early gene; ISO, isoproterenol; LL, constant light; NE, norepinephrine; P-CREB, phospho-creb; PHE, phenylephrine; PRAZ, prazosin; PROP, propranolol; SSC, sodium saline citrate. mrna unless a protein synthesis inhibitor was given during the previous night. This indicates that an inhibitory protein, synthesized at night, prevents melatonin synthesis during the day. By contrast, a protein synthesis inhibitor given at the beginning of the night markedly reduced Aa-nat mrna, suggesting that a stimulatory protein (transcription factor?) is necessary for Aa-nat gene transcription at night. Noteworthy, hamsters raised in long photoperiod were responsive to adrenergic agonist injection only in the first hour after light onset, a response that may be important in this photoperiodic species in which the melatonin peak extends into the morning hours in a short photoperiod. (Endocrinology 144: , 2003) difference between both species depends on the regulatory mechanisms of AA-NAT activation, being mainly transcriptional in the rat and posttranslational in the bovine pineal (6 9). In the rat, currently considered to be representative of rodent species, synthesis of melatonin is triggered by the nighttime release of NE (10, 11). NE binding to -adrenergic receptors increases intracellular concentrations of camp, allowing a protein kinase A-dependent phosphorylation of the camp response element-binding protein (CREB) into phospho-creb (P-CREB; Ref. 12). NE also binds to -adrenergic receptors, leading to activation of a Ca 2 /protein kinase C pathway, which potentiates the -adrenergic-induced camp increase (13). P-CREB directly activates Aa-nat gene transcription leading to a 100- to 150-fold increase in Aa-nat mrna at night (6, 14). Translation of Aa-nat mrna results in production of AA-NAT protein (70-fold nocturnal increase) immediately activated by the fixation of proteins in a camp-dependent manner (15 18). The resulting AA-NAT activation drives a 10-fold increase in melatonin synthesis and release approximately 5 6 h after the beginning of the night. The decrease in melatonin synthesis at the end of the night depends on posttranslational mechanisms triggered by termination of NE release from ganglionic terminals (11). The large decrease in camp levels induces a very rapid degradation of AA-NAT protein (half-life of AA-NAT protein/activity is approximately 3 min) by proteasomal proteolysis and dephosphorylation by protein phosphatase (19 23). The decline in Aa-nat mrna, which appears later 2461

2 2462 Endocrinology, June 2003, 144(6): Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal and more slowly, is proposed to result from a decrease in P-CREB together with an increase in the inducible camp early repressor (ICER), a transcription factor inhibiting camp response element (CRE)-induced gene transcription late in the night (24, 25). The mechanisms involved in the decrease in melatonin production in the early morning can be reproduced by application of light or -adrenergic antagonists at night. The regulation of melatonin synthesis has also been studied in another rodent, the Syrian hamster, a species usually studied for the seasonal regulation of physiological functions, especially reproduction. Melatonin content displays a marked day/night rhythm with a nocturnal peak occurring late in the night (26). Although this question was initially debated, several experiments have now demonstrated that the nocturnal synthesis of melatonin in the Syrian hamster is stimulated by NE at night (27 29). Administration of a -adrenergic antagonist or exposure to light at night inhibits AA-NAT activity and melatonin synthesis (30 32). In addition, the synthesis and content of catecholamines, NE, and dopamine (DA) show daily variations with a peak occurring during the night (33, 34). In contrast to the rat, however, in which melatonin synthesis may be acutely stimulated by NE at any time in vivo or in vitro (35 38), all experiments performed so far in the Syrian hamster have failed to stimulate melatonin synthesis during the light phase. NE or / adrenergic agonists or forskolin (an activator of adenylate cyclase) administered acutely, chronically, or combined with other transmitters, like DA, have not induced AA-NAT activity or melatonin synthesis in in vivo (27, 32, 39 41) or in vitro (42 45) conditions. Strikingly, even though isoproterenol or forskolin failed to enhance melatonin synthesis during the day, they elicited camp accumulation during this period (43). These data suggest that the mechanisms regulating AA- NAT activation and melatonin synthesis in the Syrian hamster pineal gland are different from those described in the rat. We recently reported that Aa-nat mrna expression is strongly increased in the second half of the night in the Syrian hamster pineal (46), showing that AA-NAT activation and melatonin synthesis are regulated at a transcriptional level similar to other rodents: rat (6), Arvicanthis (47), and European hamster (48). In the present study, we further investigated the mechanisms involved in the NE stimulation of Aa-nat gene transcription with a special focus on whether newly synthesized regulatory protein-like factors are involved in the melatonin synthesis during the late part of the night. Animals Materials and Methods Female Syrian hamsters were bred in our animal facilities under constant conditions of ambient humidity and temperature. They were raised under a 14-h light (200 lux light intensity), 10-h dark (2 lux dim red light) lighting schedule (with lights on from h) with food and water ad libitum. For each experiment, 2- to 3-month-old Syrian hamsters (n 5 6 per experimental group) were killed by decapitation. For Aa-nat gene expression studies, the whole brain with the pineal gland attached was carefully removed, frozen at 30 C in isopentane and then stored at 80 C until sectioning for in situ hybridization. For the plasma melatonin assay, trunk blood was taken from each animal, centrifuged for 20 min at 2000 rpm, and approximately 1 ml of plasma was recovered and stored at 20 C until assay. All experiments were performed in accordance with the National Institutes of Health Principles of Laboratory Animal Care (National Institutes of Health publication 86-23, revised 1985) and French national laws. All efforts were made to minimize animal suffering and the number of animals used to produce reliable scientific data. Experimental protocols 1. Noradrenergic regulation of Aa-nat gene expression and melatonin synthesis at night: hypothesis of a stimulatory proteic factor Experiment a: effect of / -adrenergic antagonist administration and light exposure at night. To determine which adrenergic receptors were involved in the regulation of Aa-nat gene expression during the night, three groups of hamsters were injected ip at night (2200 h) with the -adrenergic antagonist, prazosin (PRAZ) (20 mg/kg, Sigma, St. Louis, MO) or the -adrenergic antagonist, propranolol (PROP) (20 mg/kg, Sigma) or vehicle, dimethylsulfoxide (25% in saline). Hamsters were killed 4 h after injection (0200 h). For comparison, untreated animals were killed during the day (1300 h) or night (0200 h). To study whether light inhibits Aa-nat mrna transcription and a -adrenergic agonist can reinitiate Aa-nat mrna expression (as observed for melatonin synthesis) (40), two groups of Syrian hamsters were exposed to continuous light starting 0730 h after the beginning of the dark phase and one group (untreated control) remained in darkness. Thirty minutes after light exposure, hamsters were injected ip with the -adrenergic agonist isoproterenol (ISO; 3 mg/kg, Sigma) or vehicle. Groups of untreated and treated hamsters were subsequently killed 1, 3, and 5 h later. Experiment b: effect of repeated injections of / -adrenergic agonists administered late in the day. Although melatonin synthesis is restricted to the second half of the dark phase, Gonzalez-Brito et al. (49) have shown that repeated injections of a -adrenergic agonist 4, 2, and 0 h before lights off advanced the nocturnal peak of melatonin with a delay between drug injection and melatonin onset similar to that observed in natural conditions. The authors hypothesized that this delay was required for Aa-nat gene transcription. To explore this hypothesis, three groups of hamsters were injected ip every 2 h from h with ISO (3 mg/kg) or ISO combined with the -adrenergic agonist, phenylephrine (PHE) (3 mg/kg of both, Sigma) or vehicle (saline). Animals were killed every 2 h from h (i.e. 2, 4, and 6 h after the last injection). Experiment c: effect of protein synthesis inhibition during the night. To test the hypothesis that Aa-nat gene transcription requires the synthesis of a stimulatory protein-like factor, two groups of hamsters were injected ip with the protein synthesis inhibitor, cycloheximide (CYCLO, 20 mg/ kg; Sigma) or vehicle (ethanol:saline, 25:100) early in the night (2100 h). Animals were killed either 4 h (0100 h) or 6 h (0200 h) after drug injection. 2. Absence of noradrenergic induction of Aa-nat gene expression and melatonin synthesis during the day: hypothesis of an inhibitory proteic factor Experiment a: effect of adrenergic and/or dopaminergic agonists administration during the day. The acute and chronic effects a -adrenergic agonist alone or together with DA were tested during the day on Aa-nat gene transcription and melatonin synthesis. For the acute study, four groups of hamsters were injected ip with ISO (3 mg/kg), DA (3 mg/kg, Sigma), ISO DA (3 mg/kg of each), or vehicle (saline) at 1100 h and killed 1 h (1200 h) or 3 h (1400 h) after injection. For the chronic study, four groups of hamsters were given the same drugs every 2 h from h and killed 1 h after the last injection (1700 h). The acute and chronic effects of - and/or -adrenergic agonists were tested during the day on Aa-nat gene transcription and melatonin synthesis. For the acute experiment, two groups of hamsters were injected ip at 1400 h with ISO PHE (3 mg/kg of each) or vehicle (saline) and killed 1 h (1500 h) or 3 h (1700 h) after injection. For the chronic study, four groups of hamsters were injected ip with ISO (3 mg/kg), PHE (3 mg/kg), ISO PHE (3 mg/kg of each), or vehicle (saline) every 2 h from h and killed 1 h after the last injection (1700 h). Experiment b: effect of a -adrenergic agonist given to animals kept in constant darkness or light. To determine whether light could be a daytime

3 Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal Endocrinology, June 2003, 144(6): inhibitor of the ISO-stimulated melatonin synthesis, two groups of hamsters were kept in constant dark conditions (DD) for 3 d. They were subsequently injected ip with ISO (3 mg/kg) or vehicle at 1100 h (subjective day) and killed 1 and 3 h after injection. In another experiment to test whether prolonged exposure to light may repress a daytime inhibitory factor, two groups of animals were maintained in constant light (LL) for 3 d and then injected ip with ISO (3 mg/kg) or vehicle (saline) at 1100 h and killed 1 and 3 h after injection. Experiment c: effect of a protein synthesis inhibitor given during the late night/early day. In this set of experiments, the presence of an inhibitory protein-like factor during the day was evaluated with the use of the protein synthesis inhibitor CYCLO. We first investigated whether a putative inhibitory transcription factor would be synthesized in the early morning. Two groups of hamsters were first injected ip 3 h (0800 h) after lights on with either CYCLO (20 mg/kg) or vehicle (ethanol:saline 25:100). Four hours later (1200 h), both groups were given ISO (3 mg/kg) and then killed 2 h later (1400 h). In a second experiment to test whether the putative inhibitory factor would be synthesized earlier (i.e. in the second half of the night) was investigated. Two groups of hamsters were given CYCLO or vehicle at 0200 h; then 4 h later (0600 h) both groups were given ISO (3 mg/kg). The animals were killed 1 h (0700 h) or 3 h (0900 h) after the last injection. One control group received vehicle at the time of each injection, and animals were killed 1 or 3 h after the last injection. In a third experiment, the effect of acute administration of -adrenergic agonist at different times of the early light phase was examined. Groups of hamsters were injected ip with a single dose of ISO (3 mg/kg) or vehicle (saline) at 0700 h, 0800 h, 0900 h, and 1100 h and killed 1 h after injection. 3. Analysis of the photoperiodic variation in Aa-nat mrna expression. To analyze whether the diurnal rhythm of Aa-nat mrna expression varies with photoperiod, Aa-nat mrna was measured in the pineal gland of Syrian hamsters maintained either in long photoperiod (14 h light/10 h dim red light; with lights on at 0500 h) or short photoperiod (10 h light/14 h dark with lights on at 0900 h). Animals were killed at the following time points under the two photoperiods: 1400, 1700, 1800, 2000, 2100, 2400, 0200, 0400, 0600, 0800, 1000, 1200, and 1400 h. In situ hybridization Coronal brain sections (20 m) were cut at 16 C in a cryostat and thaw mounted onto gelatin-coated slides. The slides were stored at 80 C until hybridization according to a protocol previously described (46) with some modifications. Briefly, radioactive antisense and sense probes were synthesized for 2 h at 37 C and hydrolyzed for 27 min at 60 C. Brain sections were exposed to different prehybridization treatments (fixation, acetylation, glycine treatment, and dehydration in graded alcohol baths). Sections were incubated overnight at 54 C in a medium containing 80 amol riboprobe/ l. Posthybridization treatments consisted of a 10-min wash in 2 sodium saline citrate (SSC); an incubation in X-A ribonuclease (0.02 kunitz units/ml, Sigma) during 30 min at 37 C; two 15-min washes in 2 SSC at room temperature; a 20-min wash in 2 SSC; then in 0.2 SSC at 53 C; and finally a dehydration in graded alcohol baths. Finally, the slides together with 35 S standards (lab made) were exposed to an autoradiographic film (Hyperfilm MP, Kodak, Orsay, France) for 3 d. Quantitative analysis of the autoradiographs was performed with the computerized analysis system Biocom-program RAG 200 (Biocom, Les Ulis, France). The specific labeling was determined as the difference between total (antisense) and nonspecific (sense) hybridization, both being run in parallel in each experiment. Plasma melatonin assay Melatonin was extracted from plasma samples using dichloromethane as previously described (50) and quantified by RIA using rabbit antiserum (R 19540, INRA, Nouzilly, France) and iodinated melatonin as described in Vakkuri et al. (51). Melatonin assay detected values as low as 5 pg/ml plasma and the intra- and interassay coefficients of variation were between 2.5% and 6.5% for 80 and 400 pg/ml. Data analysis Aa-nat mrna levels were measured using internal standards and expressed in disintegrations per minute. All data are given as the mean sem of five to six animals. Statistical analyses were performed using Student-Newman-Keuls multicomparison test following one-way ANOVA. The differences are considered significant for P 0.05 (*). Results 1. Noradrenergic regulation of Aa-nat gene expression and melatonin synthesis at night: hypothesis of a stimulatory proteic factor 1a. Administration of adrenergic antagonists or exposure to light in the second half of the night reduces Aa-nat mrna and melatonin content. To evaluate whether NE activates - and/or -adrenergic receptors to induce Aa-nat mrna expression, - (PRAZ) or - (PROP) adrenergic antagonists were administered during the night. In untreated animals, Aa-nat mrna content showed a significant day/night variation with nighttime values ( dpm) being approximately 35-fold higher than daytime values ( dpm). Both antagonists strongly reduced Aa-nat gene expression (Fig. 1A) and melatonin content (Fig. 1B), the -adrenergic antagonist being more effective than the -antagonist. PROP significantly reduced Aa-nat mrna levels by 93%, close to daytime values, whereas PRAZ decreased this level by only 69% (Fig. 1A). This result suggests that NE acts synergistically through both - and -adrenergic receptors to fully stimulate Aa-nat gene transcription and melatonin synthesis at night. It is known that light exposure at late night rapidly decreases AA-NAT activity and melatonin synthesis (31). Whether Aa-nat mrna levels could also be altered by light exposure during the night was examined. Because melatonin synthesis can be induced by ISO after light exposure (40), we also assessed whether Aa-nat mrna transcription could be involved in this ISO stimulatory mechanism. As expected, plasma melatonin was strongly suppressed 30 min after light exposure at night (0300 h) and again induced following adrenergic agonist injection (Fig. 1D). Aa-nat mrna levels were also markedly reduced after 30-min light exposure ( dpm), compared with the untreated control group maintained in darkness ( dpm). Aa-nat mrna levels continued to decrease in the hours following light exposure (Fig. 1C). ISO did not prevent the light-induced decrease of Aa-nat mrna 1 or 3 h after injection (Fig. 1C) although it reinduced melatonin synthesis (Fig. 1D), suggesting that the remaining level of Aa-nat mrna was still sufficient to allow AA-NAT activation and melatonin synthesis. 1b. Repeated injections of / adrenergic agonists in the late day/early night advance the nocturnal increase in Aa-nat mrna and melatonin content. To determine whether the rise in Aa-nat mrna level may be advanced by an early adrenergic stimulation, repeated injections of ISO, associated or not with PHE, were given 4, 2, and 0 h before lights off. Aa-nat mrna and plasma melatonin levels were measured 2, 4, and 6 h after the last injection. The nocturnal peaks of Aa-nat mrna (Fig. 2A) and plasma melatonin (Fig. 2B) were both significantly advanced by the repeated injections of the adrenergic

4 2464 Endocrinology, June 2003, 144(6): Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal FIG. 1. Nocturnal regulation of Aa-nat mrna and melatonin content in the pineal gland of Syrian hamsters. A and B, Effect of -adrenergic or -adrenergic antagonists on nocturnal Aa-nat mrna expression (A) and melatonin content (B). At 1300 h, a group of untreated Syrian hamsters was used as a daytime control (C, control). At 2200 h, four groups of Syrian hamsters were untreated (C, control) or injected ip with vehicle (V, saline/dimethylsulfoxide, 75/25 vol/vol), the -adrenergic antagonist PROP (20 mg/kg), or the -adrenergic antagonist PRAZ (20 mg/kg) and killed 4 h later. C and D, Effect of light and ISO on Aa-nat mrna expression (C) and melatonin content (D) in the second half of the night. From 0230 h, one group of Syrian hamsters was left in darkness (untreated) and two groups of animals were exposed to continuous light and received an ip injection of either ISO (3 mg/kg) or vehicle (saline) 30 min later. Animals were killed at the times indicated. Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h. The brain was taken out with the pineal attached and processed for in situ hybridization with the Aa-nat antisense or sense probe; trunk blood was taken and processed for plasma melatonin assay. Each point is the mean SEM of five to six animals. *, P 0.05, compared with night control animals;, P 0.05, compared with saline-injected animals. agonists with a stronger effect of ISO PHE, compared with ISO alone. 1c. Administration of a protein synthesis inhibitor at early night markedly reduces Aa-nat gene expression. To test the hypothesis that a stimulatory protein-like factor, synthesized de novo at the beginning of the night, is necessary for the induction of Aa-nat gene expression, the effect of a protein synthesis inhibitor was assessed (Fig. 3). CYCLO was injected at 2100 h, and 4 h after injection, Aa-nat mrna was markedly reduced ( dpm), compared with vehicle-injected animals ( dpm). The inhibition induced by CYCLO had a long-term effect because 6 h after injection, Aa-nat mrna levels were still reduced ( dpm), compared with vehicle-injected animals ( dpm). In parallel experiments performed in the rat, in contrast, CYCLO injection significantly increased Aa-nat mrna levels from dpm up to dpm (n 5) 6 h later.

5 Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal Endocrinology, June 2003, 144(6): FIG. 3. Effect of a protein synthesis inhibitor on nocturnal Aa-nat mrna in the pineal gland of Syrian hamsters. At 2100 h (2 h after lights off), two groups of hamsters received an ip injection of either CYCLO (20 mg/kg) or vehicle (25% ethanol in saline) and were killed 4 or 6 h later. Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h. Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe. Each point is the mean SEM of five to six animals. *, P 0.05, compared with vehicleinjected animals. FIG. 2. Noradrenergic regulation of Aa-nat mrna and melatonin content in the late day/early night in the pineal gland of Syrian hamsters. Three groups of Syrian hamsters received ip injection of either the -adrenergic agonist ISO (3 mg/kg) or both ISO (3 mg/kg) and the -adrenergic agonist PHE (3 mg/kg) or vehicle (saline) every 2 h from h (time of lights off). Animals were killed 2, 4, and 6 h after the last injection. Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h. Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe (A); trunk blood was taken and processed for plasma melatonin assay (B). Each point is the mean SEM of five to six animals. *, P 0.05, compared with vehicle-injected animals;, P 0.05, compared with ISO-injected animals. 2. Absence of noradrenergic induction of Aa-nat gene expression and melatonin synthesis during the day: hypothesis of an inhibitory proteic factor 2a. Daytime injection of adrenergic and/or dopaminergic agonists does not increase Aa-nat gene expression and melatonin synthesis. The experiments were conducted to determine whether the reported inability of adrenergic and dopaminergic agonists to increase daytime melatonin synthesis was related to their failure to stimulate Aa-nat gene transcription. A single injection of ISO during the day did not increase Aa-nat mrna (Fig. 4A) and melatonin content (data not shown), compared with untreated animals. Similarly, an acute injection of DA (Fig. 4A), ISO DA (Fig. 4A), or a mixture of / -adrenergic agonists (Fig. 5A) had no effect on daytime Aa-nat mrna expression and melatonin levels (data not shown). Repeated injections (every 2 h from h) of ISO (Fig. 4B), PHE (Fig. 5B), or DA (Fig. 4B) also had no effect on Aa-nat mrna levels and melatonin content (data not shown). Only when both / -adrenergic agonists were injected repeatedly, a small but significant increase in Aa-nat mrna was observed (Fig. 5B). However, no effect on pineal melatonin content was noted (data not shown). The results of these experiments showed that daytime activation of adrenergic or dopaminergic receptors had no effect on Aa-nat gene expression and suggested that during the day an inhibitory element was present. The following experiments were performed to test this hypothesis. 2b. Effect of an acute -adrenergic administration in constant darkness or light. To test whether light itself could act as an inhibitor thereby preventing daytime stimulation of melatonin synthesis, hamsters were maintained in DD for 3 d and then injected with a single dose of ISO during the subjective day. ISO failed to increase Aa-nat mrna levels 1 or 3 h after injection (Fig. 6A). To determine whether there could be an inhibitory mechanism triggered during the night but active during the day, hamsters were maintained in LL for 3 d and then injected with a single dose of ISO during the subjective day. In these conditions, ISO significantly increased Aa-nat mrna levels 3 h after injection (Fig. 6B) although to a level lower than the nighttime values (see Fig. 1A).

6 2466 Endocrinology, June 2003, 144(6): Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal FIG. 4. Effect of a -adrenergic agonist, alone or in combination with DA, on Aa-nat mrna expression in the pineal gland of Syrian hamsters. A, Acute effect of the -adrenergic agonist ISO and/or DA. At 1100 h, four groups of Syrian hamsters were injected ip with ISO (3 mg/kg), DA (3 mg/kg), ISO and DA (3 mg/kg of each), or vehicle (saline) and killed 1 or 3 h later. B, Effect of repeated injections of ISO and/or DA. From 1000 h, four groups of Syrian hamsters were injected ip with ISO (3 mg/kg), DA (3 mg/kg), ISO and DA (3 mg/kg each), or saline (vehicle) every 2 h until 1600 h. All groups were killed 1 h after the last injection (1700 h). Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h. Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe. Each point is the mean SEM of five to six animals. 2c. Administration of a protein synthesis inhibitor at late night allows daytime adrenergic stimulation of Aa-nat gene expression and melatonin synthesis. To find out whether the putative inhibitory mechanism suppressing daytime melatonin synthesis could involve synthesis of an inhibitory protein early in the day, hamsters were given CYCLO or vehicle at the beginning of the light phase and then a single dose of ISO 4 h FIG. 5. Effect of acute or repeated daytime injections of / -adrenergic agonists on Aa-nat mrna in the pineal gland of Syrian hamsters. A, Acute effect of the combination of - and -adrenergic agonists. At 1400 h, two groups of Syrian hamsters were injected ip with either both - and -adrenergic agonists (ISO/PHE, 3 mg/kg of both) or vehicle (saline) and killed 1 or 3 h later. B, Effect of repeated administration of the combination of - and -adrenergic agonists. From 1000 h, four groups of Syrian hamsters were injected ip with ISO (3 mg/kg), PHE (3 mg/kg), or ISO/PHE (3 mg/kg of each) or vehicle (saline) every 2 h until 1600 h. All groups were killed 1 h after the last injection (1700 h). Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h. Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe. Each point is the mean SEM of five to six animals. *, P 0.05, compared with vehicle-injected animals. later. No effect of ISO was observed when given alone or after CYCLO injection (Fig. 7A). The experiment was repeated but with CYCLO or vehicle given late during the night (0200 h) and ISO or vehicle injected 4 h later (0600 h). In CYCLO-treated hamsters, in contrast to vehicle-treated animals, acute injection of ISO

7 Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal Endocrinology, June 2003, 144(6): FIG. 6. Effect of a -adrenergic agonist on Aa-nat mrna transcription in the pineal gland of Syrian hamsters kept in constant darkness or light for 3 d. Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h and then exposed to either DD (A) or LL (B) for 3 d. On d 3 of exposure, Syrian hamsters were injected ip at 1100 h with the -adrenergic agonist ISO (3 mg/kg) and killed 1 or 3 h later. Data were compared with untreated animals killed at the time of injection (1100 h). Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe. Each point is the mean SEM of five to six animals. *, P 0.05, compared with to untreated animals. markedly increased Aa-nat mrna levels (Fig. 7B) to values similar to nighttime levels 1 and 3 h after ISO injection. Surprisingly, a single dose of ISO at 0600 h, in the absence of CYCLO pretreatment, also induced a marked increase in Aa-nat mrna 1 h after injection (Fig. 7B). To confirm this result and establish whether it was restricted to the early part of the light phase, a single dose of ISO was given at a number of time points early in the day, and hamsters were killed 1 h after ISO injection. Only when given at 0600 h could ISO induce a strong increase in FIG. 7. Effect of a protein synthesis inhibitor combined with a daytime injection of a -adrenergic agonist on Aa-nat mrna in the pineal gland of Syrian hamsters. A, Injection of the protein synthesis inhibitor early in the day. Two groups of hamsters were first injected ip at 800 h (3 h after lights on) with the protein synthesis inhibitor CYCLO (20 mg/kg) or vehicle (25% ethanol in saline), and then 4 h later, both groups were injected ip with the -adrenergic agonist ISO (3 mg/kg) and were killed 2 h after the last injection. B, Injection of the protein synthesis inhibitor during the late night. At 0200 h (3 h before lights on), one group of Syrian hamsters was injected ip with CYCLO (20 mg/kg), and two groups were injected ip with vehicle (25% ethanol in saline). Four hours later, the CYCLO group and one vehicle-injected group were injected ip with ISO (3 mg/kg). One vehicle group was reinjected with vehicle. Animals were killed 1 or 3 h after the last injection. Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h. Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe. Each point is the mean SEM of five to six animals. *, P 0.05, compared with vehicle-injected animals. Aa-nat mrna (Fig. 8A) and melatonin content (Fig. 8B), close to nighttime values. These results suggest that an inhibitory protein-like fac-

8 2468 Endocrinology, June 2003, 144(6): Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal or long (14 h light/10 h dark) photoperiods (Fig. 9, A and B). In both photoperiods, Aa-nat mrna levels increased significantly 5 h after lights off and reached a maximum 9 h after lights off. The decline in Aa-nat mrna levels occurred earlier in the long photoperiod (timed to light onset) than in the short photoperiod. This resulted in a larger nocturnal peak of Aa-nat mrna in short photoperiod, compared with long photoperiod. Noteworthy, the amplitude of the Aa-nat mrna peak was approximately 2-fold higher in the long photoperiod, compared with the short photoperiod. FIG. 8. Effect of a single injection of a -adrenergic agonist, given early in the day, on Aa-nat mrna and melatonin content in the pineal gland of Syrian hamsters. Acute ip injections of the -adrenergic agonist ISO (3 mg/kg) were given at 0600 h, 0700 h, 0800 h, and 1000 h in the morning. Animals were killed 1 h after injection. Hamsters were raised under 14 h light/10 h dark with lights on at 0500 h. Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe (A); trunk blood was taken and processed for plasma melatonin assay (B). Each point is the mean SEM of five to six animals. *, P 0.05, compared with vehicle-injected animals. tor is synthesized during the late night. This factor would be effective during the day, except during the first hour after lights on, in preventing stimulation of melatonin synthesis. 3. Analysis of photoperiodic regulation of Aa-nat gene transcription The daily profile of Aa-nat mrna expression was measured in hamsters raised under short (10 h light/14 h dark) FIG. 9. Photoperiodic variation in Aa-nat mrna levels in the pineal gland of Syrian hamsters raised under long or short photoperiod. Hamsters were raised under 14 h light/10 h dark (lights on at 0500 h; long photoperiod, LP; dark gray and black circles) or exposed to 10 h light/14 h dark (lights on at 0900 h; short photoperiod, SP, light gray and white circles) for 8 wk. Animals were killed at the indicated times. Data are expressed as raw values (A) or as percentage of the maximum nighttime value (B). Pineal tissue was processed for in situ hybridization with the Aa-nat antisense or sense probe. Each point is the mean SEM of five to six animals. *, P 0.05, compared with daytime (1400 h) value.

9 Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal Endocrinology, June 2003, 144(6): Discussion This study has investigated the mechanisms involved in the transcription of the gene coding for Aa-nat, the ratelimiting enzyme for melatonin synthesis, in the pineal gland of the Syrian hamster. The results show that NE, acting through both - and -adrenergic receptors, is the main neurotransmitter responsible for the enzyme coding-gene transcription, but, in contrast to the rat, the mechanisms involved may require stimulatory and inhibitory protein-like (transcription) factors. Similar to all rodents studied so far, nocturnal AA-NAT activity and melatonin production depend on a marked increase in Aa-nat gene transcription (7, 46, this study). Nocturnal stimulation of the melatonin synthesis pathway mainly requires -adrenergic receptor activation because the -adrenergic antagonist PROP significantly reduces melatonin content at night (30) but also -adrenoceptors because the specific antagonist PRAZ decreases melatonin production during the night (52). In the current study, we further show that inhibition by the antagonists occurs at the transcriptional level because both PROP and PRAZ reduce Aa-nat mrna levels, the effect of PROP being stronger. In the rat, adrenergic potentiation involves a Ca 2 -dependent protein kinase C activation, which augments intracellular camp levels and AA-NAT activity (53 58). In contrast to the Syrian hamster, however, the -adrenergic antagonist does not inhibit the nocturnal increase in Aa-nat mrna (6, 59). These results indicate that full nocturnal stimulation of melatonin synthesis in the Syrian hamster requires synergistic - and -adrenergic activation. Light exposure at night induces a rapid decrease in AA- NAT activity and melatonin content in the Syrian hamster (31, this study) as well as the rat (19, 21, 60). In the rat, light exposure at night causes termination of NE release from the sympathetic nerve endings (11) and therefore a marked decrease in camp content (20). This results, on the one hand, in rapid AA-NAT protein deactivation (16 18) and degradation by proteasomal proteolysis (23) and, on the other hand, in a slow decrease in Aa-nat mrna levels (6) (Simonneaux, V, unpublished data). Similar mechanisms probably occur in the Syrian hamster pineal gland. In contrast to the rat, however, we have observed that the decline in Aa-nat mrna levels is larger and faster, indicating that the mrna may be less stable. Administration of a -adrenergic agonist, 30 min after light exposure, reverses the inhibition of AA- NAT activity and melatonin content (40, 41, this study) but does not prevent the decrease in Aa-nat mrna levels. Noteworthy, melatonin production was capable of being stimulated, although the level of Aa-nat mrna was significantly reduced after light exposure. In earlier experiments, Reiter et al. (40) have shown that the induction of melatonin synthesis is restricted to the second half of the night. Another group (49), however, managed to advance the nocturnal peak of AA-NAT activity and melatonin content by giving chronic/repeated injections of NE at the end of the day (thus advancing the NEergic stimulation). The delay between the first NE injection and a significant effect on melatonin synthesis was 7 8 h, a delay similar to what is observed at night. The authors suggested that the long delay between endogenous or exogenous NE stimulation and onset of melatonin synthesis was required for Aa-nat gene transcription. Following the same protocol, we observed that repeated injections of adrenergic agonists 6 h before dark onset advanced the nocturnal increase in Aa-nat mrna levels, thus strengthening the hypothesis of Gonzalez-Brito et al. (49). In agreement with our previous experiments, the effect of both - and -adrenergic agonists was stronger than with the -adrenergic agonist alone. In the rat, the key event in turning Aa-nat expression on is the -adrenergic-controlled camp-dependent phosphorylation of the transcription factor CREB that binds to CREs located on the Aa-nat gene promotor (12, 61). Thus, NEinduced transcription of the Aa-nat gene at the beginning of the night is not blocked by the inhibitor of protein synthesis, CYCLO (6, 62, this study). By contrast, in the Syrian hamster, administration of a protein synthesis inhibitor in the early night markedly reduced Aa-nat mrna levels. This reduction cannot be the result of a general inhibitory effect of the protein synthesis inhibitor because in other experiments the same dose of CYCLO (given late at night) allowed the -adrenergic stimulation of Aa-nat mrna. This result strongly suggests that Aa-nat gene transcription, and therefore melatonin production, depends on a NE-induced synthesis of a protein-like (transcription) factor early in the night. This hypothesis agrees with the observation of a long delay between dark onset and the beginning of Aa-nat gene expression and melatonin synthesis. The products of the immediate early genes (IEGs) may be good candidates because they are rapidly expressed after stimulation of numerous organs including the pineal gland (63, 64). The IEG c-fos and its partner for DNA binding, JunB, are of particular interest because their transcription in the rat pineal gland is mediated by -adrenergic (c-fos, JunB) and -adrenergic (JunB) receptors (65). Strikingly, FOS immunoreactivity appears earlier in the pineal gland of the Syrian hamster (5 h after dark onset) than in the rat (7 h after dark onset) (66), although it is the opposite for melatonin synthesis. To further confirm this hypothesis, it will be necessary to establish the 24-h profiles of the various IEGs and P-CREB in the Syrian hamster pineal gland and look at whether the Syrian hamster Aa-nat gene promoter contains an activator protein-1 site (as in the rat promoter) (61). Earlier studies reported that AA-NAT activity and melatonin production in the Syrian hamster cannot be stimulated during the day by adrenergic agonists (27, 32, 39 41, 67). In agreement with the previous data, acute injection of a adrenergic agonist given alone or in combination with an -adrenergic agonist did not increase Aa-nat gene expression or melatonin content. Similarly, repeated administration of either an - or -adrenergic agonist had no effect on the Aa-nat mrna. Only a combination of both agonists was able to induce a small increase in Aa-nat mrna with no significant effect on melatonin content. In addition, the effect of DA, another pineal neurotransmitter, whose content displays a marked day/night variation with maximal values at night (34, 68 70), was tested. DA, however, given acutely or chronically during the day, alone or in combination with a -adrenergic agonist, could not induce Aa-nat gene transcription and melatonin synthesis.

10 2470 Endocrinology, June 2003, 144(6): Garidou et al. Aa-nat Gene Expression in Syrian Hamster Pineal Several hypotheses may explain the inability of melatonin synthesis to be stimulated during the day. It could be related to the absence or low sensitivity of adrenoceptors in the daytime. Several arguments, however, do not support this hypothesis, namely - and -adrenergic-binding sites display a day/night variation with maximal values during the day (71, 72); -adrenergic receptor affinity is constant throughout 24 h (72); a -adrenergic agonist elicits camp accumulation during the day (43); and camp or forskolin induces AA-NAT activity and melatonin synthesis in the pineal gland of Syrian hamsters killed during the night but not during the day (45, 67). These published results together with our observations on Aa-nat mrna regulation prompted us to investigate whether an inhibitory factor is present during the day preventing Aa-nat gene transcription and melatonin synthesis. We first explored whether light itself could be involved in this daytime inhibition. In hamsters kept in DD for 3 d, a -adrenergic agonist given in the subjective day had no effect on Aa-nat mrna and melatonin synthesis. By contrast, injection of a -adrenergic agonist in the subjective day to hamsters kept for 3 d in LL induced a significant increase in Aa-nat gene expression (although not up to nighttime values). This effect could be explained by a phenomenon of adrenergic receptor hypersensitivity caused by the 3-d-long absence of NE stimulation as already reported in the rat (73 75). Another hypothesis could be that absence of NE abolishes the synthesis of an inhibitory factor usually present during the day. To test this hypothesis that a daytime inhibitory factor inhibits Aa-nat gene transcription, the effect of a protein synthesis inhibitor was studied. CYCLO given in the early part of the day did not permit -adrenergic stimulation of Aa-nat expression, suggesting that the putative inhibitory protein-like factor is not synthesized early in the day. In a second experiment, when CYCLO was given during the second part of the night (i.e. when pineal metabolism is maximal), daytime injection of a -adrenergic agonist induced a marked increase in Aa-nat mrna levels similar to nighttime values. This result strongly suggests that an inhibitory protein-like (transcription) factor starts to be synthesized during the second half of the night (probably following NE stimulation) to further inhibit Aa-nat gene transcription and melatonin synthesis during the day. This would involve an inhibitory protein with a long half-life. The nature of this inhibitory transcription factor remains to be established. It could be ICER, an inhibitory transcription factor induced by camp-dependent mechanisms, which has been shown to repress Aa-nat transcription by competition with P-CREB binding on the CRE site of the Aa-nat promotor (24, 25, 76, 77). Preliminary experiments are indeed showing that Icer mrna is expressed in the Syrian hamster pineal gland with a NE-driven daily rhythm (78). It should be noticed that in hamsters kept for3dinll,the level of Aa-nat mrna reached within 3 h after a -adrenergic agonist injection is still lower that the nighttime level. This is fully consistent with our hypothesis that Aa-nat mrna expression is induced by a slow process involving the synthesis of a stimulatory transcription factor. Strikingly, 2 h after lights on, but no later, an acute injection of a -adrenergic agonist induced Aa-nat gene transcription and melatonin synthesis, even without pretreatment with CYCLO. It is possible that the inhibitory factor was not yet present or effective, whereas the stimulatory factor was still present during the few hours after lights on. This may suggest the existence of an early morning window of noradrenergic sensitivity in the Syrian hamster pineal gland. This observation might be of importance for the seasonal/photoperiodic regulation of melatonin synthesis because the nocturnal peak of Aa-nat mrna (this study) and melatonin content (34) displays an increase in duration in short photoperiod, compared with a long photoperiod with a lengthening of the nocturnal peak toward the late night/early day. Analysis of pineal metabolism in the Syrian hamster raised under long or short photoperiods also shows that the lengthening of Aa-nat gene transcription drives that of melatonin content, as already observed in the rat (79) and European hamster (48). Consistent with the pineal melatonin pattern (34, 80), Aa-nat mrna levels increased at the same time after darkness but decreased with light onset in the long photoperiod and before light onset in the short photoperiod. In addition to photoperiodic changes in peak duration, the amplitude of the peak of Aa-nat mrna levels displays photoperiodic modification with the amplitude being twice as high in the long, compared with the short, photoperiod. This has also been observed in the Siberian hamster (81) and, to a lesser extent, in the rat (79) and was hypothesized to be related to photoperiodic variation in the amount of ICER (82). In conclusion, this study shows that melatonin synthesis is regulated through adrenergic regulation of Aa-nat transcription as reported for several rodents (9). In contrast to the rat, however, the molecular mechanisms of Aa-nat gene transcription in the Syrian hamster appear to involve both inhibitory and stimulatory protein (transcription) factors that frame melatonin synthesis in the late part of the night. It is possible that this balance between repressive and permissive processes allows a strict control of the duration of nocturnal melatonin synthesis, a pivotal parameter for the transmission of photoperiodic information and therefore synchronization of Syrian hamster reproductive status with seasons. Interestingly, we recently observed that in the European hamster as well, melatonin synthesis is also restricted to the nighttime (48). Experiments are currently in progress to identify these putative transcription factors. Although it is now accepted that daily and photoperiodic information is integrated by the endogenous clock (83, 84) and transmitted to the pineal gland through NE release, our data indicate that the melatonin peak may also be regulated through pineal-specific mechanisms. Acknowledgments The authors are very grateful to Dr. Debra Skene for correcting the English language and Dr. Christophe Ribelayga for discussion. The work of Daniel Bonn for taking care of animals and helping during experiments was appreciated. Received January 15, Accepted March 3, Address all correspondence and requests for reprints to: Valérie Simonneaux, Laboratoire de Neurobiologie des Rythmes, Unité Mixte Recherche-Centre National de la Recherche Scientifique-Université Louis Pasteur 7518, 12, rue de l Université, F Strasbourg, France. simonneaux@neurochem.u-strasbg.fr.

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