Methyltransferase (HIOMT) - CiteSeerX

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society

Vol. 140, No. 3 Printed in U.S.A.

Photoneural Regulation of Rat Pineal Hydroxyindole-OMethyltransferase (HIOMT) Messenger Ribonucleic Acid Expression: An Analysis of Its Complex Relationship with HIOMT Activity* CHRISTOPHE RIBELAYGA, FRANC ¸ OIS GAUER, CHRISTIANE CALGARI, PAUL PEVET, AND VALERIE SIMONNEAUX Neurobiologie des Fonctions Rythmiques et Saisonnie`res, Unite´ Mixte de Recherche-Centre National de la Recherche Scientifique 7518, Universite´ Louis Pasteur, F-67000 Strasbourg, France ABSTRACT In the pineal gland, synthesis of melatonin requires O-methylation catalyzed by hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4). We investigated in vivo the molecular mechanisms involved in the regulation of rat pineal HIOMT messenger RNA (mRNA) expression and activity using in situ hybridization and radioenzymatic assay. HIOMT mRNA levels and activity are both detectable during the daytime and display nocturnal increases of 100% and 30%, respectively. These variations are controlled by the endogenous clock, as they persist in constant darkness. The nocturnal increase in HIOMT mRNA mainly results from a b1-adrenergic stimulation of HIOMT gene expression without requiring de novo synthesis of a transcription factor. In contrast, the nocturnal increase in HIOMT

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NE OF THE most potent environmental factors regulating metabolism of the mammalian pineal gland is the light/dark cycle (1). The production of the pineal hormone melatonin (MEL) is restricted to the night, with a duration directly proportional to night length, thus being a time-giver hormonal message (2– 6). MEL is synthesized from the amino acid tryptophan (7) which is first converted into 5-hydroxytryptophan by tryptophan hydroxylase (EC 1.14.16.4) before being decarboxylated into serotonin (8). This latter compound is then acetylated by arylalkylamineN-acetyltransferase (AA-NAT; EC 2.3.1.87) (9, 10) and finally O-methylated by hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4) into MEL (11). In contrast to lower vertebrates, the mammalian pineal gland is not able to respond directly to light, but its metabolism is indirectly controlled by light via a photoneural system whose major components are the retina, the suprachiasmatic nuclei of the hypothalamus (SCN), and the superior cervical ganglia (1, 12, 13). In this neural network, the SCN play a central role, as they contain the endogenous clock controlling most circadian rhythms, including the rhythmic

Received June 1, 1998. Address all correspondence and requests for reprints to: Dr. Vale´rie Simonneaux, Neurobiologie des Fonctions Rythmiques et Saisonnie`res, Unite´ Mixte de Recherche-Centre National de la Recherche Scientifique 7518, Universite´ Louis Pasteur, 12 rue de l’Universite´, F-67000 Strasbourg, France. E-mail: [email protected]. * This work was supported by grants from La Fondation pour la Recherche Me´dicale et La Fondation Simone et Cino del Duca.

activity appears independent of b1/a1-adrenergic stimulation. A light pulse at night abolishes the nighttime increase in HIOMT mRNA, but not HIOMT activity. Constant light application for up to 11 days does not depress HIOMT mRNA levels lower than the daytime levels, but decreases enzyme activity down to 50% of the daytime level. This finding indicates that the nocturnal stimulation of HIOMT gene expression is required for sustaining a basal level of activity over a few days. Our data suggest 1) that HIOMT gene expression is partly regulated by b1-stimulation; and 2) that HIOMT activity is regulated over the short term by a nonnoradrenergic stimulus and over the long term by noradrenergic stimulation. (Endocrinology 140: 1375–1384, 1999)

synthesis of MEL (14). Even if the SCN oscillate autonomously on a 24-h basis, their activity is synchronized to exactly 24 h by light/dark information being conveyed from the retina by the retino-hypothalamic tract (12, 15, 16). Consequently, light also entrains the MEL rhythm to exactly 24 h. Additionally, when applied during the night, light depresses the SCN-driven nocturnal stimulation of MEL synthesis at least partly through AA-NAT activity breakdown (17–21). Neural outputs of the SCN relay to different central and peripheral structures (16, 22, 23), one being the superior cervical ganglia from which noradrenergic fibers terminate in the pineal gland (12, 24 –26). The noradrenergic input is considered to be the major input controlling metabolic activity of the pineal gland. Indeed, MEL synthesis is primarily controlled by the rhythmic release of noradrenaline (24, 25, 27–29). In the rat, nocturnal stimulation of the pineal gland by noradrenergic activation of both a1- and b1-adrenergic postsynaptic receptors leads to a 100-fold rise in the intracellular cAMP accumulation and consequently to a series of cascade events resulting in activation of MEL synthesis and release (24, 25, 29 –31). Elevation of cAMP levels leads to a slight increase in tryptophan hydroxylase messenger RNA (mRNA) expression (120%) (32) and activity (2-fold) (33). Concomitantly, a dramatic elevation in AA-NAT mRNA expression (150-fold) (19, 20, 34) causes a 70- to 100-fold increase in AA-NAT activity (35). Thus, nocturnal activation of MEL synthesis is primarily regulated by cAMP-mediated noradrenergic stimulation of AA-NAT gene expression and

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PHOTONEURAL REGULATION OF RAT PINEAL HIOMT

translation of its messenger into active molecules (19 –21). In addition, nocturnal stimulation of the pineal gland leads to an increase in HIOMT mRNA levels (2-fold) (36) and HIOMT activity (130 to 50%) (37– 40). It is suggested that the slight rhythm of HIOMT activity is not involved in the occurrence of the daily rhythm of MEL synthesis (24). The involvement of noradrenaline in HIOMT stimulation is quite complex. We have shown that elevation of the pineal HIOMT mRNA content can be produced by the b1-adrenergic agonist isoproterenol (ISO) (36). In addition, the crucial role of noradrenergic stimulation in the long term regulation of HIOMT activity has been well documented (40 – 44). In contrast, attempts to stimulate HIOMT activity acutely with noradrenergic agonists or cAMP analogs failed both ex vivo (45) and in vitro (40). These previous observations suggest a possible multiregulation of HIOMT activity over the short (day/night) and long (several days) term, but the relative importance of transcriptional, translational, and posttranslational regulation remains unclear. To investigate further the mechanisms regulating HIOMT and to assess the link between messenger and enzyme activity, we have studied in vivo HIOMT mRNA expression, HIOMT activity, and MEL levels concomitantly after various experimental procedures. The results of this and previous studies (36, 40, 42– 44) have led us to propose a model for the complex regulation of HIOMT in the rat pineal gland. Materials and Methods Animals All of the experiments were performed on adult male Wistar rats, weighing 150 –220 g, from Centre de Neurochimie (Strasbourg, France) under constant conditions of ambient humidity and temperature. Before experimentation, animals were adapted to our laboratory conditions for at least 1 week under a 12-h light/12-h dark (12L/12D) cycle (with lights on at 0700 h). During the day, light intensity was approximately 200 lux at the level of the cages. During the period of darkness, animals were exposed to constant dim red light (,2 lux). They were given food and water ad libitum. Animal experimentation was performed in agreement with the Principles of Laboratory Animal Care (NIH) and French national laws. HIOMT mRNA expression and HIOMT activity were followed in the pineal gland of rats in various experimental protocols. 1) Day/night variations in pineal HIOMT and circulating MEL were investigated in animals kept in 12L/12D cycle and killed at midday (1300 h) or at midnight (0100 h). 2) To determine whether day/night variations persisted in constant conditions, rats were housed in different lighting conditions [12L/12D, constant light (200 lux; L/L), or constant darkness (2 lux dim red light; D/D)] for 5 days. On the fifth day, they were killed either in the middle of the day (1200 h; or in the middle of the subjective day) or in the middle of the night (0100 h; or in the middle of the subjective night). 3) The chronic effect of light was investigated in rats kept in L/L for 11 days and compared with that in rats kept in standard 12L/12D conditions for 11 days. All animals were killed at 1300 h. 4) The effect of a light pulse (200 lux) was investigated. Firstly, rats housed in 12L/12D were submitted to a light pulse of various durations (from 5– 60 min) during the night. Secondly, rats were housed in D/D (starting at 1900 h) for 3 days, then subjected to a 1-h light pulse at different times: 1300, 2100, 0030, and 0500 h. Animals were killed at the end of light exposure together with control animals kept in darkness. 5) The effects of specific a1-adrenergic [phenylephrine (PHE)] and b1-adrenergic (ISO) agonists were investigated in rats kept under 12L/ 12D. Drugs were dissolved in 9% NaCl (wt/vol) and were injected ip at midday (1300 h). Initially, to determine a time-response curve for b1adrenergic stimulation of HIOMT, the effect of 5 mg/kg ISO was followed up to 6 h after injection. Thereafter, the following doses were

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tested: ISO, 0.1, 1, and 5 mg/kg; PHE, 1 and 5 mg/kg; and ISO and PHE, 1 and 1 mg/kg. Animals were killed 1 h after injection, and MEL, HIOMT activity, and mRNA content were measured in the pineal gland. For each experiment, control animals received an ip injection of vehicle solution (9% NaCl). 6) The effect of 1 mg/kg ISO on HIOMT mRNA was further investigated in the presence of cycloheximide (20 mg/kg; Sigma Chemical Co., Saint Quentin Fallavier, France) or actinomycin D (5 mg/kg; Sigma Chemical Co.). These drugs were dissolved in ethanol-saline (vol/vol, 1:1). Cycloheximide or actinomycin D was injected ip 20 min before ISO or 9% NaCl injection at midday (1300 h), and comparisons were made with control animals injected first with ethanol-saline (vol/vol, 1:1) and 20 min later with ISO or 9% NaCl. In all experiments, animals were killed by decapitation. For HIOMT activity and MEL analysis, the pineal gland was dissected out and rapidly frozen in liquid nitrogen, then kept at 280 C until assay. For HIOMT mRNA analysis, the whole brain with the pineal attached was carefully removed, frozen in 230 C isopentane, and then stored at 280 C until analysis. For each animal, trunk blood was sampled for plasma MEL assay.

In situ hybridization In situ hybridization was performed as previously described (46). Twenty-micron thick coronal sections of frozen brains were thawmounted onto gelatin-coated slides. All of the prehybridization steps were carried out at room temperature. Sections were incubated in 4% paraformaldehyde-1 3 PBS (10 3PBS is 1.37 m NaCl, 27 mm KCl, 81 mm Na2HPO4, and 15 mm KH2PO4, pH 7.4) for 15 min. They were washed successively into 1 3 PBS and 2 3 SSC (sodium saline citrate; 20 3 is 3 m NaCl and 0.3 m sodium citrate, pH 7.0) for 2 min each time. Sections were then acetylated with 0.5% acetic anhydrate-0.1 m triethanolamine (pH 7.4) for 10 min and rinsed in 2 3 SSC and 1 3 PBS for 2 min each. They were then incubated for 30 min in 0.1 m glycine-0.1 m Tris (pH 7.0) and rinsed in 2 3 SSC and 1 3 PBS before being dehydrated in graded ethanols (70%, 90%, 95%, and 100%, 1 min each) and dried at room temperature. The pBluescript plasmid containing the complementary DNA encoding HIOMT (1542 b) (36) was linearized with either BamHI or HindIII (Life Technologies, Cergy Pontoise, France). An antisense or a sense riboprobe was transcribed with T3 or T7 RNA polymerase, respectively (MAXIscript transcription kit, Ambion, Inc., Montrouge, France; [a-35S]UTP, 1250 Ci/mmol; New England Nuclear-DuPont, Le Blanc Mesnil, France). Both probes were hydrolyzed by alkaline treatment (0.1 m carbonate buffer, pH 10.2) for 42 min at 60 C to generate 200-bp long fragments. The size of the riboprobes were checked by electrophoresis on a polyacrylamide gel. For hybridization, dehydrated brain sections were incubated overnight at 54 C in a moist chamber with 50 attomoles (amol)/ml (corresponding to 10,000 cpm/ml on the reference day) antisense or sense probe in a hybridization solution containing 2 3 SSC, 20% dextran sulfate, 50% deionized formamide, 10 mm dithiothreitol, 1 3 Denhardt’s solution (from a 50-fold stock solution; 1 g/liter Ficoll, 1 g/liter polyvinylpirrolidone, and 1 g/liter BSA), 1 mg/ml salmon sperm DNA, and 200 mg/ml yeast RNA. The hybridization solution was laid down on sections (120 ml/slide) before being gently recovered by a siliconized coverslip. Posthybridization treatment consisted of washing the sections for 10 min at room temperature in 2 3 SSC before incubation for 30 min at 37 C with 0.14 Kunitz unit/ml ribonuclease type X-A (from bovine pancreas; Sigma Chemical Co.) in 0.5 m NaCl, 10 mm Tris (pH 7.4), and 10 mm EDTA. The sections were then washed three times in 2 3 SSC at room temperature before dehydration in graded ethanols (70%, 90%, 95%, and 100%, 1 min each) and air-dried. The slides were exposed to autoradiographic films (Hyperfilm MP, Amersham, Les Ulis, France) for 48 h at room temperature. Quantitative analysis of the autoradiograms was performed using the computerized analysis (Biocon, Les Ulis, France) program RAG 200. Specific hybridization was determined as the difference between total (antisense) and nonspecific (sense) hybridization.

PHOTONEURAL REGULATION OF RAT PINEAL HIOMT Northern blot analysis

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Pineal HIOMT activity was assayed as previously described (40). Briefly, single pineal glands were sonicated in 100 ml sodium phosphate buffer (0.05 m; pH 7.9). Fifty microliters of the tissue homogenate were incubated for 30 min at 37 C with 1 mm N-acetylserotonin and 43.8 mM S-adenosyl-l-[14C-methionine] (59.3 mCi/mmol; New England Nuclear-DuPont, Le Blanc Mesnil, France) in a final volume of 100 ml, then the reaction was stopped by the addition of 200 ml sodium borate buffer (12.5 mm; pH 10). Newly synthesized MEL was measured after extraction in 1 ml water-saturated chloroform and counting of the radioactivity after evaporation of the organic solvent. Protein content was measured in 30 ml tissue homogenate following the protocol of Lowry with BSA as standard (49).

band of approximately 1.7 kb was observed at both time points; Fig. 1). Plasma MEL concentrations showed the characteristic 10-fold nocturnal increase (from 8.5 6 1.6 pg/ml at midday to 104.0 6 6.5 pg/ml at midnight). Day/night variations in HIOMT mRNA, HIOMT activity, and plasma MEL were compared in parallel in rats maintained for 5 days under three different lighting conditions (L/L, 12L/12D or D/D) and killed either in the middle of the day (or subjective day) or in the middle of the night (or subjective night). The results (Fig. 2, A and B, C) show that day/night variations in both HIOMT mRNA and activity are totally abolished in L/L, but persist in D/D, indicating that they are controlled by the endogenous clock. As predicted, day/night variations in plasma MEL persist in D/D and are abolished in L/L. After 5 days, midday levels of both HIOMT mRNA and activity were not significantly different between L/D and L/L conditions (Fig. 2, A and B). To determine whether this could evolve with time, this experiment was extended to 11 days. Figure 3 shows that after 11 days in L/L, midday HIOMT activity was significantly decreased by 241% compared with the control value in 12L/12D, in agreement with previous studies (40, 42– 44). Surprisingly, no significant difference in pineal HIOMT mRNA content was observed between the two groups. This experiment was repeated once and gave the same result. Thus, even if HIOMT activity is altered by chronic light exposure, the basal level of HIOMT mRNA is not, at least for the first 11 days in L/L.

Circulating and pineal MEL assays

Acute effects of light on pineal HIOMT

Circulating MEL was extracted from plasma samples using dichloromethane as previously described (50). Pineal MEL was measured directly in 20 ml pineal homogenate. MEL was quantified by RIA using rabbit antiserum (R 19540, INRA Nouzilly, France) and iodinated MEL (51).

The effect of a light pulse applied at nighttime was investigated in different lighting conditions. Under standard 12L/ 12D conditions, a 1-h light pulse applied at midnight significantly decreased mRNA content to daytime values, but

Total RNA was extracted from rat pineal glands collected either at midnight (0100 h) or at midday (1300 h; n 5 10 in each group) using RNAzol (Tel-Test, Inc., Friendswood, TX) according to the protocol of Chomczynski and Sacchi (47). Ten micrograms of both day and night total RNA and a RNA ladder (Life Technologies, Gaithersburg, MD) were electrophoresed in a 1.5% formaldehyde-agarose gel with 1 3 MOPS buffer (10 3 is 0.2 m 3-[N-morpholino]-propanesulfonic acid, 0.05 m sodium acetate, and 0.01 m EDTA, pH 7.0). The gel was stained with ethidium bromide and examined under UV light. Total RNA was electrophoretically transferred overnight with a 0.5-A current onto a ZetaProbe membrane (Bio-Rad Laboratories, Inc., Hercules, CA) in 40 mm Tris-acetate and 2 mm EDTA (pH 8.5) buffer at 4 C, then baked at 80 C for 60 min. The blot was prehybridized and hybridized as previously described (48). Afterward, the blot was washed twice for 30 min each time at room temperature (2 3 SSC-0.5% SDS), twice for 30 min at 37 C (0.5 3 SSC-0.5% SDS), and finally for 20 min at 62 C (0.1% SSC-0.1% SDS). The blot was exposed to x-ray film for 4 days at 280 C.

Pineal HIOMT activity assay

Data analysis Specific in situ hybridization labeling is expressed as the relative optic density, HIOMT activity is expressed as nanomoles per mg protein/h, circulating MEL is expressed as picograms per ml plasma, and pineal MEL is expressed as picograms per mg protein coupled. All data are expressed as the mean 6 sem of n values. Statistical analyses were performed using Student-Newman-Keuls multicomparison test (after one-way ANOVA).

Results Day/night variations in pineal HIOMT activity and mRNA expression

A significant nocturnal increase in HIOMT mRNA content and activity was observed in the pineal of rats raised in 12L/12D in several independent experiments. HIOMT mRNA content increased by 95 6 19% from the midday value of 3.18 6 0.30 amol/section to the midnight value of 6.20 6 0.59 amol/section (P , 0.001; n 5 24 animals in 4 independent experiments) and HIOMT activity increased by 30 6 4% from the midday value of 0.97 6 0.03 nmol/mg proteinzh to the midnight value of 1.26 6 0.05 nmol/mg proteinzh (P , 0.001; n 5 42 animals in 8 independent experiments). Northern blot analysis of total RNA isolated from pineal glands collected either at midday or midnight showed that no variation in the size of the HIOMT transcript occurred (a single

FIG. 1. Northern blot analysis of HIOMT mRNA isolated from rat pineal glands collected either at midday (Day) or at midnight (Night). Total RNA from day or night rat pineal glands were extracted (n 5 10/group). Ten micrograms of total RNA were loaded onto a 1.5% agarose formaldehyde denaturing gel. After electroblotting onto a Zeta-Probe membrane, blots were probed with radiolabeled rat pineal HIOMT complementary DNA. The HIOMT mRNA-specific 1.7-kb band is indicated.

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FIG. 3. Chronic exposure to light depresses daytime HIOMT activity but not HIOMT mRNA levels. Animals were raised either in 12L/12D or in L/L for 11 days. Rats from both groups were killed on the 11th day at midday, and HIOMT activity and mRNA were determined as described in Materials and Methods. Results are given as the mean 6 SEM of six animals. ***, P , 0.001 compared with 12L/12D.

did not alter the nocturnal level of HIOMT activity (Fig. 4). As expected, nocturnal plasma MEL levels were strongly depressed after light application (from 45.1 6 6.7 to 12.2 6 2.0 pg/ml; P , 0.001; n 5 6 in each group). Because the inhibitory effect of light on HIOMT mRNA appeared maximal after 1 h of light exposure, we studied the effect of shorter duration light pulses (from 5– 60 min). The HIOMT mRNA content decreased rapidly after light exposure, with basal levels reached within 30 min (Fig. 5A). Circulating MEL levels also decreased with a similar pattern after various durations of light exposure (Fig. 5B). To verify whether there was a circadian period of sensitivity for the light effect on HIOMT mRNA, light pulses were applied for 1 h at different times (1300, 2100, 0030, and 0500 h) to rats kept in D/D for 3 days. The HIOMT mRNA level was significantly higher at 0030 and 0500 h than at 1300 h, confirming the circadian nature of this rhythm (Fig. 6A). Light had no effect on the basal level of mRNA at both 1300 and 2100 h, but inhibited the elevated nocturnal level as observed at 0030 and 0500 h. Interestingly, the mRNA level never decreased to below the basal 1300 h level. Determination of circulating MEL concentrations confirmed the inhibitory effect of light on pineal metabolism (Fig. 6B). Effect of adrenergic agonists on pineal HIOMT

FIG. 2. Day/night variations in HIOMT mRNA (A) and HIOMT activity (B) are driven by the endogenous clock. Rats were housed for 5 days in different conditions: 12L/12D (with lights on at 0700 h), D/D, or L/L. They were killed either at midday (1300 h) or at midnight (0100 h). Pineal HIOMT mRNA (A), HIOMT activity (B), and plasma MEL (C) were measured as described in Materials and Methods. Results are given as the mean 6 SEM of six animals. *, P , 0.05; **, P , 0.01; ***, P , 0.001 (compared with the respective midday value).

As our data indicated that nocturnal stimulation of both HIOMT mRNA and activity were under the control of the endogenous clock, we investigated whether this control occurs through the adrenergic input to the pineal gland. To assess the kinetics of the HIOMT response to b1-adrenergic stimulation, HIOMT mRNA, HIOMT activity, and plasma MEL were measured up to 6 h after a single ip injection of the b1-adrenergic agonist ISO (5 mg/kg) given at midday. HIOMT mRNA levels rapidly increased by 166 6 25% (n 5 6; P , 0.001) within 1 h after injection, but this increase was transient, as it returned to basal values 2 h after


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FIG. 4. A 1-h light pulse applied during the night inhibits the nocturnal increase in HIOMT mRNA, but not HIOMT activity, in the rat pineal gland. Animals were divided into three groups: one group of rats was killed at midday; one group received a 1 h-light pulse (200 lux) from 2330 – 0030 h and was killed afterward; and one group was kept in darkness and killed at 0030 h. Pineal HIOMT mRNA and activity were measured as described in Materials and Methods. Results are expressed as the mean 6 SEM of six animals. *, P , 0.5; ***, P , 0.001 (compared with the respective midday value).

ISO injection (Fig. 7A). Saline injections under similar conditions had no effect on HIOMT mRNA (data not shown). Interestingly, ISO had no effect on HIOMT activity measured 1 or 4 h after injection (Fig. 7A). The effect of ISO on pineal MEL was as expected, with maximum levels reached within 1 h followed by a fall to basal daytime values 6 h after drug injection (Fig. 7B). After this preliminary experiment, 1 h was considered to be the optimal time to study the effect of a single midday ip injection of various drugs on pineal HIOMT. Various concentrations of ISO (0.5, 1, or 5 mg/kg) induced a similar maximal increase of the messenger level (Fig. 8A). The magnitude of the ISO-induced increase in HIOMT mRNA was consistent with the amplitude of the endogenous nocturnal increase (see Figs. 2A, 4, and 6A). In contrast, 1 or 5 mg/kg of the a1-adrenergic receptor agonist, PHE, did not increase HIOMT mRNA levels. Interestingly, the ISO-induced 2-fold increase in HIOMT mRNA was not augmented by PHE injected at the same time. None of the above treatments increased HIOMT activity measured 1 h after injection (Fig. 8B), strengthening the hypothesis that the nocturnal increase in HIOMT activity may not be mediated by noradrenaline. As previously reported, the b1-adrenergic, but not the a1adrenergic, agonist stimulated MEL synthesis (Fig. 8C). b1-Adrenergic-induced increase in HIOMT mRNA requires gene transcription but not protein synthesis

To determine whether the b1-adrenergic-induced increase in HIOMT mRNA required gene transcription activation and/or protein synthesis, we tested the effect of actinomycin D (5 mg/kg) or cycloheximide (20 mg/kg) injected 20 min

FIG. 5. Light exposure during the night rapidly decreases HIOMT mRNA (A) and plasma MEL (B). Rats were kept in 12L/12D (with lights on at 0700 h) and were exposed to a 200-lux light pulse of different durations (5, 15, 30, and 60 min) starting at 0300 h. The animals were killed at the end of light exposure. Control animals, kept in darkness, were killed at 0300 and 0400 h (black bars). Pineal HIOMT mRNA analysis (A) and plasma MEL assay (B) were performed as described in Materials and Methods. Results are given as the mean 6 SEM of six animals. *, P , 0.05; **, P , 0.01; ***, P , 0.001 (compared with no light).

before the ip injection of ISO (1 mg/kg) at midday. The ISO-induced stimulation of HIOMT mRNA was totally inhibited by actinomycin D, but not by cycloheximide (Fig. 9A), whereas pineal MEL synthesis was inhibited by both drugs (Fig. 9B). Actinomycin D and cycloheximide had no effect per se on basal HIOMT mRNA or MEL levels (Fig. 9, A and B). These results demonstrate that the b1-adrenergic-induced

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FIG. 6. Light inhibition of pineal HIOMT mRNA (A) and plasma MEL (B) is restricted to the subjective night. Rats were housed under conditions of constant darkness for 3 days and exposed to a 1-h light pulse (200 lux) at different times of the third subjective day (1300 h) or night (2100, 0030, and 0500 h) and killed at the end of the light exposure. Control animals, kept in constant darkness, were killed at the same time as light-exposed animals. HIOMT mRNA content (A) was quantified as described in Materials and Methods. Plasma MEL (B) was assayed by RIA. Results are given as the mean 6 SEM of six animals. ***, P , 0.001 compared with the 1300 h value.

HIOMT gene expression does not require new transcription factor synthesis. Discussion

The results reported in the present study clearly establish that pineal HIOMT activity and HIOMT mRNA content dis-

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FIG. 7. A single injection of ISO during the day induces a rapid and transient increase in pineal HIOMT mRNA, but not HIOMT activity. A, Effect of a single injection of ISO during the day on HIOMT mRNA and HIOMT activity. Animals were housed under 12L/12D conditions (with lights on at 0700 h) and were injected ip at midday with the b1-adrenergic receptor agonist ISO (5 mg/kg). Animals were killed at the time of injection (0) or 1, 2, 4, or 6 h after injection. Pineal HIOMT mRNA and activity were measured as described in Materials and Methods. B, A single ip injection of ISO stimulates MEL production. MEL was measured by RIA in pineal homogenates. Results are given as the mean 6 SEM of six animals. *, P , 0.05; ***, P , 0.001 compared with no treatment.

play day/night variations controlled by the endogenous clock, because they persist under constant darkness. As clock input to the pineal is primarily mediated by noradrenergic fibers, it was tempting to hypothesize that both HIOMT


FIG. 8. Activation of b1-adrenergic receptors specifically stimulates HIOMT mRNA, but not HIOMT activity. The effects of different doses of b1- and a1-adrenergic receptor agonists on pineal HIOMT mRNA content (A), HIOMT activity (B), and plasma MEL levels (C) are shown. Animals were injected ip at midday with different doses of the b1-adrenergic agonist ISO and/or the a1-adrenergic agonist PHE. Control animals received vehicle (9% NaCl) only. Animals were killed 1 h after drug injection. Levels of pineal HIOMT mRNA, HIOMT activity, and MEL were measured as described in Materials and Methods. Results are given as the mean 6 SEM of six animals. *, P , 0.05; **, P , 0.01; ***, P , 0.001 (compared with control).

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FIG. 9. Activation of b1-adrenergic receptors stimulates HIOMT gene transcription de novo, but not protein synthesis. The effects of a specific inhibitor of transcription (actinomycin D) or protein synthesis (cycloheximide) on the ISO-induced increase in HIOMT mRNA (A) and plasma MEL (B) are shown. Animals were housed in 12L/12D, with lights on at 0700 h. They were injected ip at midday with actinomycin D (AD; 5 mg/kg), cycloheximide (cyclo; 20 mg/kg), or vehicle (ethanol/saline) (vol/vol, 1:1), then with ISO (1 mg/kg) or vehicle (9% NaCl) 20 min later. Animals were killed 1 h after the last injection. HIOMT mRNA and pineal MEL were measured as described in Materials and Methods. Results are given as the mean 6 SEM of six animals. *, P , 0.05; ***, P , 0.001 (compared with control).

mRNA level and HIOMT activity were stimulated by the nighttime release of noradrenaline. Our observations, however, suggest that although the nocturnal increase in HIOMT mRNA content is induced by b1-adrenergic stimulation, the nocturnal increase in enzyme activity does not appear to be a short (few hours) response of translational events and is probably controlled by nonnoradrenergic transmitters.

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This study confirms that HIOMT mRNA displays a day/ night variation, with a 2-fold increase in nighttime levels (36). It also shows that this day/night variation persists under constant darkness and is abolished by light applied at night, indicating that HIOMT gene expression is controlled by the endogenous clock. This is also confirmed by our observation that the b1-adrenergic agonist ISO is able to stimulate daytime HIOMT mRNA levels to the same extent as observed during the night. In our study, the a1-adrenergic agonist PHE did not stimulate or potentiate the increase in HIOMT mRNA, suggesting that noradrenaline stimulates HIOMT mRNA primarily through b1-adrenergic receptor activation. The nocturnal activation of b1-adrenergic receptors leads to the accumulation of cAMP and a subsequent protein kinase A-dependent phosphorylation of the constitutive cAMP responsive element (CRE)-binding protein (CREB) (52–54). Nocturnal AA-NAT gene expression is triggered by phosphorylated CREB (P-CREB) (20, 55). As the transcription inhibitor, actinomycin D, but not the protein synthesis inhibitor, cycloheximide, inhibited the ISO-induced increase in HIOMT mRNA levels, it is possible that P-CREB also directly mediates noradrenergic-induced stimulation of HIOMT gene expression. The involvement of P-CREB remains hypothetical, but a putative CRE has been characterized in one of the two human HIOMT gene promoters (56). In addition, Northern blot analysis revealed that no difference in the size of the transcript between day and night existed. Therefore, the nocturnal increase in HIOMT mRNA involved an accumulation of the same transcript. Thus, the most probable scenario is that noradrenaline, released at night, activates b1-adrenergic receptors, which trigger a cAMP-dependent phosphorylation of CREB and consequently P-CREBinduced stimulation of HIOMT gene expression, leading to a doubling of the pineal HIOMT mRNA content. Acute light application rapidly depressed the nighttime values of HIOMT mRNA, with the basal level being reached within 30 min. Interestingly, the HIOMT mRNA half-time (t1/2 5 ;15 min) appears much shorter than that of AA-NAT mRNA (t1/2 5 ;2.5 h) (19). Considering the relatively short half-life of HIOMT mRNA and the very transient effect of ISO on HIOMT mRNA, the nocturnal elevation in HIOMT mRNA probably reflects a sustained adrenergic activation of its gene expression. Although light totally inhibited the nocturnal increase in HIOMT gene expression, it did not ever reduce HIOMT mRNA levels lower than the basal daytime value. Even after 11 days of constant light exposure, the level of HIOMT mRNA was similar to the daytime value observed in 12L/12D animals. This result is surprising, because several days of constant light has been reported to decrease HIOMT activity to 50% (but never less) of its basal daytime value (40, 42– 44), reflecting a decrease in the protein content (57). This discrepancy could be explained if HIOMT gene expression was stimulated by at least two pathways: one involving the noradrenaline/b1-adrenergic/cAMP/P-CREB pathway working every night and another one, as yet to be defined, working during the day and night, independently of the endogenous clock. The latter pathway would be responsible for the remaining 50% of HIOMT activity observed after several days in L/L. Both pathways together would be involved in the basal diurnal HIOMT activity observed in

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12L/12D. A similar multiple regulation of HIOMT gene expression has been also suggested in human retinoblastoma Y79 cells (58). The present in vivo results confirm our hypothesis on the short term independent regulation of HIOMT expression and HIOMT activity (40). Various concentrations of ISO (up to 5 mg/kg) alone or together with PHE, although stimulating HIOMT gene expression within 1 h, displayed no effect on HIOMT activity up to 4 h after drug injection. These observations are in agreement with those of previous studies reporting no acute effect of adrenergic agonists or cAMP analogs on HIOMT activity either ex vivo (45) or in vitro (40). Theoretically, considering the low turnover of the protein (.24 h in the chicken pineal gland) (59) and its large distribution among pineal proteins (.4%) (60), a nocturnal increase of 50% in its intracellular amount would inevitably lead to a gradual daily accumulation of the protein within the cell, which we have never observed (Ribelayga, C., personal observation). Thus, we postulate that daily fluctuations of HIOMT activity occur independently of HIOMT gene expression and probably reflect posttranslational events. A short light pulse at night, which inhibits noradrenaline release (61), abolished the rapid nocturnal increase in HIOMT mRNA and MEL, but did not alter HIOMT activity. This result strengthens our hypothesis of an independent regulation of HIOMT gene expression and activity. HIOMT activity is not altered by a 1-h light pulse, whereas it is lower during the day than at night. Possibly, the light application was too short to disclose an effect on HIOMT activity. Alternatively, the mechanisms involved in both conditions may be different. Daily variations in HIOMT activity persist after 5 days in constant darkness and are abolished after 5 days in constant light, indicating that the daily regulation of HIOMT activity is under control of the endogenous clock, but probably through a nonnoradrenergic mechanism. Neuropeptide Y (NPY) appears to be a good candidate for the daily control of HIOMT activity in the rat pineal gland. It originates mainly from the sympathetic nerve fibers where it is colocalized with noradrenaline (62, 63) and displays daily and circadian variations of its content in the rat pineal gland (64). Moreover, we have shown that in vitro NPY stimulates HIOMT activity up to 50% within 6 h (40, 65). These observations lead us to consider NPY to be a putative regulator of circadian HIOMT activity. In conclusion, in contrast to AA-NAT activity, which directly reflects the AA-NAT mRNA level, HIOMT activity regulation appears more complex. At the transcriptional level, HIOMT gene expression appears to be regulated by at least two pathways. One involves the b1-adrenergic/cAMP/ P-CREB pathway, which induces a 2-fold nighttime increase in HIOMT mRNA. The other pathway, as yet to be defined, is responsible for the high daytime level of HIOMT mRNA observed in animals kept for up to 11 days in constant light. This dual stimulation of HIOMT gene expression would be involved in the long term regulation of HIOMT activity. In addition, our data strongly indicate that daily variations in HIOMT activity reflect posttranslational events triggered by a nonnoradrenergic transmitter, possibly NPY.

PHOTONEURAL REGULATION OF RAT PINEAL HIOMT Acknowledgments The authors are grateful to Mrs. Aurore Senser and Mr. Daniel Bonn for taking care of the animals, to Dr. Andre´ Malan for his many helpful discussions, and to Dr. Debra Skene for linguistic review of the manuscript.

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Erratum In the article “Glicentin and oxyntomodulin modulate both the phosphoinositide and cyclic adenosine monophosphate signaling pathways in gastric myocytes” by Jarrousse and co-workers (Endocrinology 140: 22–28), 1999, please note the following correction. The first author’s name is listed incorrectly. Her correct name is Genevie`ve Rodier. The authors regret this error.