INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MELATONIN (Mel), which production from the pineal gland follows daily and seasonal rhythms, plays an important role in the timing of many physiological functions. This hormonal message is mainly regulated by the daily and seasonal changes in the environmental light cycle. The synthesis and release of Mel display a marked increase during nighttime, and the duration of the nocturnal peak is positively related to the duration of the night (review in Refs. 31, 43).

The molecular mechanisms involved in the regulation of Mel synthesis have been extensively investigated in the rat. The synthesis of Mel requires two enzymatic steps from serotonin (review in Refs. 15, 16). Serotonin is first N-acetylated by the arylalkylamine-N-acetyltransferase (AA-NAT; EC 2.3.1.37) into N-acetylserotonin (NAS), then O-methylated by hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4) into Mel. The photic information is conveyed from the retina to the pineal gland through a complex nervous pathway, including the suprachiasmatic nuclei of the hypothalamus (SCN), that contains the circadian pacemaker and the superior cervical ganglia, of which noradrenergic fibers innervate the pineal gland (review in Ref. 25). In addition, the pineal gland is innervated by various peptidergic fibers of different origins (review in Ref. 24). Some of these peptides have been reported to regulate or modulate Mel synthesis (review in Refs. 36, 37). The physiological role of these peptides remains to be determined, but they are strongly suspected to take part in the integration of environmental information by the pineal gland (review in Ref. 29). In the rat, the nocturnal stimulation of Mel synthesis is mainly driven by the nocturnal release of norepinephrine (5). Activation of both postsynaptic ₁- and ₁-adrenergic receptors leads to a 100-fold increase in AA-NAT activity (review in Ref. 17). A less-conspicuous nocturnal increase of HIOMT activity (by 1.5-fold) has been reported but does not result from adrenergic stimulation of the pineal gland, but rather neuropeptide Y (NPY) (33, 35). Because changes of AA-NAT activity parallel those of Mel production, this enzyme is considered as the rate-limiting enzyme for Mel synthesis (review in Ref. 17).

In all species, the profile of the daily rhythm of Mel displays seasonal variations especially dependent on the photoperiod (review in Ref. 30). It is clearly established that these variations constitute a temporal signal used to synchronize certain physiological functions such as reproduction, hibernation, or variations of the body mass with seasons (review in Ref. 31). To date, however, it is not clearly established how the photoperiodic regulation of Mel synthesis occurs, how the photoperiodic modifications of the Mel peak relay seasonal informations to the organism, and how the tissular targets integrate the message. Recently, it has been suggested that the SCN integrate photoperiodic variations of the environmental light cycle (review in Refs. 28, 42). The photoperiodic message elaborated in the endogenous clock is then transmitted to the pineal gland, probably through photoperiodic modifications of the duration of norepinephrine release. In addition, certain peptides present in the pineal gland may also be involved in this process, because some of them display seasonal variations of their contents in the gland, as established for NPY (23), vasopressin, and oxytocin (19, 26).

In most of the species, changes in the duration of the nocturnal peak of Mel appear to be sufficient to transmit the photoperiodic information to the organism (review in Refs. 2, 27). In the rat, it has been shown that the variations in the duration of the nocturnal peak of Mel directly depend on similar variations of mRNA expression and activity of AA-NAT (13, 14, 34). In addition, in the rat, there is also a photoperiodic regulation of HIOMT activity resulting from transcriptional and translational mechanisms (1, 32, 33, 39, 40, 47). The activity of HIOMT is higher on a short photoperiod (SP) than on long photoperiod (LP). These photoperiodic variations of HIOMT activity, however, do not appear to modify Mel synthesis in this nonphotoperiodic species (11, 16, 32).

In most of the photoperiodic species, photoperiodic variations in the amplitude of the nocturnal peak of Mel have also been reported in addition to changes in its duration (review in Refs. 30, 45). In the Siberian hamster (Phodopus sungorus) for example, the Mel peak amplitude is two times higher under SP than under LP (10, 12, 18, 22), although the amplitude of the peak of AA-NAT activity is equal (11) or even lower under SP than under LP (9, 41, 44). Consequently, photoperiodic changes in the amplitude of the nocturnal peak of Mel do not appear driven by AA-NAT activity, at least in the Siberian hamster.

The previous observations have led us to hypothesize that the photoperiodic variations in the amplitude of the Mel peak might be driven by HIOMT rather than AA-NAT. To test this hypothesis, we have studied the photoperiodic modifications of HIOMT, AA-NAT, and Mel in the pineal gland of the Siberian hamster.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Male and female Siberian hamsters (Phodopus sungorus) used in the different experiments were from our own breeding colony. They were kept from birth under LP (16:8-h light-dark cycle, with lights on at 0400). Adult animals (3-6 mo old) were transferred to SP (10:14-h light-dark cycle, with lights on at 0800) for at least 11 wk before experiments or kept under LP. A dim red light (<2 lx) was constantly present. Food and water were provided ad libitum, and temperature was set at 22 ± 1°C. The effect of SP was checked on fur color and testis weight in males.

Animals were killed by decapitation. Thereafter, the pineal gland was rapidly dissected out and frozen in liquid nitrogen until enzymatic assays. Enzymatic activities were determined within the 24 h after death. Animal experiments were performed in agreement with Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205] and French national laws.

Enzymatic assays. Pineal HIOMT and AA-NAT activities were assayed as previously described (4, 34). Individual pineal glands were sonicated in 100 µl of 0.05 M sodium phosphate buffer (pH 7.9 for HIOMT and 6.8 for AA-NAT assay).

For HIOMT activity assay, 50 µl of the tissue homogenate were incubated for 30 min at 37°C with 1 mM NAS (Sigma, Saint Quentin Fallavier, France) and 43.8 µM S-adenosyl-L-[¹⁴C]methionine (SAM; 59.3 mCi/mmol, NEN-Dupont, Le Blanc Mesnil, France) in a final volume of 100 µl. After incubation, the reaction was stopped by the addition of 200 µl of sodium borate buffer (12.5 mM, pH 10). For AA-NAT activity assay, 40 µl of homogenate were incubated for 20 min at 37°C with 192 µM ¹⁴C-labeled acetyl CoA (ACoA; NEN-Dupont) and 10 mM tryptamine (Sigma) in a final volume of 80 µl. Newly synthesized Mel or N-acetyltryptamine were measured after extraction in 1 ml of water-saturated chloroform and counting of the radioactivity after evaporation of the organic solvent. HIOMT and AA-NAT activities are expressed as picomoles per gland per hour.

Determination of optimal pH for HIOMT activity was conducted from a subset of 27 females raised under LP and killed at 1400. Individual pineals were sonicated in 0.05 M phosphate buffer at a range of pH's from 6.8 to 9.1. Fifty microliters of each homogenate were incubated with 43.8 µM SAM and 1 mM NAS in a final volume of 100 µl to assay HIOMT activity. Thirty microliters of the tissue homogenate were used to determine the amount of protein, following the protocol of Lowry et al. (20), with bovine serum albumin as standard. In this experiment, HIOMT activity was expressed in nanograms per milligram protein per hour.

The biochemical characteristics of HIOMT and AA-NAT activity were compared under LP and SP. Two groups of 10-13 females, one kept under LP, the other one under SP, were killed at the same time, between 0200 and 0300. Pineal homogenates from a same group were pooled. Enzymatic activities of both groups were determined concomitantly.

HIOMT activity was assayed in two conditions. First, 25 µl of the pineal homogenate containing 5 mM NAS were incubated with increasing concentrations of SAM (from 0 to 150 µM) in a final volume of 50 µl. Second, 25 µl of the pineal homogenate containing 100 µM SAM were incubated with increasing concentrations of NAS (from 0 to 5 mM).

AA-NAT activity was assayed in two conditions. First, 25 µl of the pineal homogenate containing 192 µM ACoA were incubated with increasing concentrations of tryptamine (from 0 to 10 mM) in a final volume of 50 µl. Second, 25 µl of the pineal homogenate containing 10 mM tryptamine were incubated with increasing concentrations of ACoA (from 0 to 300 µM).

Mel assay. Pineal Mel was measured directly in 20 µl of pineal homogenate by radioimmunoassay using rabbit antiserum (R 19540, Institut National de la Recherche Agronomique, Nouzilly, France) and iodinated Mel (21). This assay was validated in the Siberian hamster by Miguez et al. (22). Mel is expressed in picograms per gland.

Statistical analysis. For determination of kinetic characteristics, data were fitted to the Michaelis-Menten equation using the computer-assisted nonlinear regression system GraphPad 1.03 (Microsoft). With the use of this program, we estimated the Michaelis-Menten constant value (K_m) and the maximal reaction velocity (V_max), which are both expressed with their standard deviation estimates.

The daily profiles of Mel were characterized by performing nonlinear regression with SigmaPlot 4.0 (Jandel Scientific). Each profile was fitted with the following equation (logistic peak): y = y₀ + y_max/([1+exp(₁-x)] × {1 + exp[2(x-₂)]}), where y is the nth data point, x is the time of the nth data point, y₀ is the mean basal level, y_max is the maximum of the nocturnal peak (amplitude), ₁ is the inflection point at the onset of the peak, and ₂ is the inflection point at the decline of the peak. The duration of the peak was estimated as the difference between ₁ and ₂. The regression coefficients (y₀, y_max, ₁, and ₂) are given with their respective asymptotic SD estimates (32).

All the other data are expressed as the means ± SE. Statistical analyses were performed using Student-Newman-Keuls multicomparison test following one-way ANOVA (to compare >2 groups) or Student's t-test (to compare 2 groups).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All the Siberian hamsters kept for >11 wk under SP show the characteristic white pelage. In males, the testis weight was lower under SP than under LP [59 ± 7 (n = 12) vs. 617 ± 57 mg (n = 9); P < 0.001]. The body mass of the animals was lower under SP than under LP [33.56 ± 1.22 (n = 27) vs. 41.00 ± 0.83 g (n = 31); P < 0.001], whereas the total amount of protein per pineal was higher under SP than under LP [45.95 ± 2.18 (n = 39) vs. 38.41 ± 1.83 µg (n = 42); P < 0.001]. No statistical differences were observed between males and females for these two variables.

Pineal Mel content was determined in pineal homogenates of animals kept under LP or SP (Fig. 1). The daytime levels in Mel content were around the detection limit under both photoperiods. On the contrary, marked differences in the duration and the amplitude of the nocturnal peak of Mel were observed between the two photoperiods. Nonlinear regression performed from the individual values revealed that from LP to SP, the duration extended by 4 h (from 5 h 52 ± 36 min to 9 h 46 ± 46 min; P < 0.001), and the amplitude increased by 1.6-fold (from 714.35 ± 40.29 to 1,116.56 ± 81.18 pg/gland; P < 0.001).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Daily patterns of pineal Mel content in Siberian hamsters kept under short (SP; 10:14-h light-dark cycle) or long photoperiod (LP; 16:8-h light-dark cycle). Data are expressed as picograms per gland. Each point represents mean ± SE of 5 or 6 animals. Black bars indicate dark periods.

The characteristics of AA-NAT activity were determined in both photoperiods. In agreement with previous studies, the daytime values of AA-NAT activity were below the detection limit of the enzymatic assay, whereas the nocturnal values were elevated (data not shown). There was no difference in the daily pattern of AA-NAT activity between males and females (data not shown). Because AA-NAT activity was the highest between 0200 and 0300 under both photoperiods, we chose this time to collect pineals and perform enzymatic analyses. Determination of kinetic parameters of AA-NAT revealed that V_max was ~2-3 times higher under LP than under SP (Figs. 2 and 3 and Table 1). On the opposite, the affinity (K_m) of the enzyme for ACoA and tryptamine did not vary between the two photoperiods (Fig. 3 and Table 1).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Pineal arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities in Siberian hamsters kept under SP or LP. Animals were killed between 0200 and 0300 under dim red light conditions. AA-NAT activity was measured in presence of 192 µM 14C-labeled acetyl CoA (ACoA) and 10 mM tryptamine. HIOMT activity was measured in presence of 43.8 µM S-adenosyl-L-[14C]methionine (SAM) and 1 mM N-acetylserotonin (NAS). Data are expressed as picomoles per gland per hour. Each value represents mean ± SE of 5 or 6 pineals. * P < 0.001 when compared with respective LP value.

View larger version (1211K): [in this window] [in a new window]   Fig. 3.   Reaction velocity of pineal AA-NAT in Siberian hamsters kept under SP or LP. Animals were killed between 0200 and 0300 under dim red light conditions. A: AA-NAT activity was measured in presence of 192 µM ACoA and increasing concentrations of tryptamine. B: AA-NAT activity was measured in presence of 10 mM tryptamine and increasing concentrations of ACoA. Data are expressed as picomoles per gland per hour. Each value was determined in triplicate and is expressed as mean ± SE.

The previous observations demonstrate that photoperiodic changes in the amplitude of the nocturnal peak of Mel are not related to AA-NAT activity and led us to investigate the role of HIOMT in such a regulation. Determination of optimum pH revealed that HIOMT activity in the Siberian hamster pineal peaks at pH 7.9 (Fig. 4). The enzymatic characteristics of HIOMT were also determined under SP and LP in pineal homogenates from animals killed between 0200 and 0300. The estimated V_max was about two times higher under SP compared with LP in presence of saturable concentrations of substrates (Fig. 2 and Table 2; see also Fig. 6). Assay of HIOMT activity during 24 h in both photoperiods revealed that this difference was observed all along the daily cycle (Fig. 5). The mean daily level was higher under SP than under LP [440.19 ± 29.95 (n = 44) vs. 223.58 ± 13.18 pmol · gland¹ · h¹ (n = 40); P < 0.001]. These photoperiodic changes in HIOMT activity were observed in both males and females (data not shown). In contrast to the V_max, no difference was observed in HIOMT affinity for SAM or NAS between the two photoperiods (Fig. 6 and Table 2).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   pH dependence of HIOMT activity in pineal gland of Siberian hamster. Data are expressed as nanomoles per milligram protein per hour. Each point represents mean ± SE of 3 pineals.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Daily patterns of pineal HIOMT activity in Siberian hamsters kept under SP or LP. Data are expressed as picomoles per gland per hour. Each point represents mean ± SE of 5 or 6 animals. Black bars indicate dark periods.

View larger version (1212K): [in this window] [in a new window]   Fig. 6.   Reaction velocity of pineal HIOMT in Siberian hamsters kept under SP or LP. Animals were killed between 0200 and 0300 under dim red light conditions. A: HIOMT activity was measured in presence of 5 mM NAS and increasing concentrations of SAM. B: HIOMT activity was measured in presence of 43.8 µM SAM and increasing concentrations of NAS. Data are expressed as picomoles per gland per hour. Each value was determined in triplicate and is expressed as mean ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our observations made in the Siberian hamster confirm that the nocturnal peak of Mel is longer and higher under SP than under LP and strongly suggest that HIOMT, but not AA-NAT, drives the photoperiodic changes in the amplitude of the Mel peak.

In the rat, where the mechanisms regulating the rhythm of Mel synthesis have been extensively studied, the stimulation of Mel synthesis is mainly, although not exclusively, controlled by the sympathetic input to the pineal gland. The nocturnal SCN-driven release of norepinephrine induces a marked nocturnal increase of AA-NAT activity resulting from transcriptional and translational mechanisms (8, 15, 16, 18). This nocturnal increase in AA-NAT activity drives the nocturnal synthesis and release of Mel. In addition, the lengthening of the dark phase induces, probably through a longer noradrenergic stimulation of the gland, an extension of the period of elevated AA-NAT mRNA expression (32) and activity (11, 13, 14) and consequently increases the duration of the nocturnal peak of Mel. The synthesis of Mel in the pineal gland of the Siberian hamster is partly controlled by similar mechanisms. Indeed, AA-NAT mRNA and activity are almost undetectable during the photophase and dramatically increase during the scotophase (3, 9, 12, 44, this study). In addition, the duration of the nocturnal peak of AA-NAT activity, and thus of the Mel level, increases with the duration of the night (9, 12, 44). Thus it is very probable that in the Siberian hamster, like in the rat, AA-NAT drives the nocturnal increase in Mel synthesis but also the photoperiodic changes in the duration of the nocturnal peak of this pineal hormone.

In the Siberian hamster, in contrast to the rat, the amplitude of the nocturnal peak of Mel displays marked photoperiodic variations. Indeed, the maximal peak amplitude is about two times higher under SP than under LP (10, 12, 18, 22, this study). We report here that these photoperiodic variations do not imply AA-NAT activity because its nighttime value is about two times lower under SP than under LP. In addition, we show herein that this variation in AA-NAT activity is not accompanied with a change in the substrate affinity of the enzyme. Because in rodents, the level of AA-NAT activity tightly depends on the level of AA-NAT mRNA (17), it is most probable that the photoperiodic decrease in the nocturnal AA-NAT activity results from a photoperiodic decrease in the AA-NAT mRNA amplitude. We recently reported such a photoperiodic variation in the rat pineal gland with an amplitude of the nocturnal AA-NAT mRNA peak being significantly lower under SP than under LP (32). We speculate that this decrease in AA-NAT transcription results in a photoperiodic inhibition by the inducible cAMP early repressor (ICER). Indeed, ICER, whose expression is driven by norepinephrine (38), represses the cAMP-dependent (nocturnal) AA-NAT transcription (6). Because the rat pineal ICER displays photoperiodic variations with a higher level in SP (7), it may be responsible for the lower nocturnal AA-NAT transcription observed in the pineal gland of rodents raised under SP. In addition, we cannot exclude that some neuropeptides, whose pineal content display seasonal variations (19, 23, 26, 35), may be involved in the photoperiodic regulation of AA-NAT mRNA expression. Our observations clearly indicate that photoperiodic changes in the amplitude of the nocturnal peak of Mel synthesis are independent of AA-NAT activity, therefore suggesting a possible role for HIOMT in such a regulation.

Investigations on the characteristics of the Siberian hamster pineal HIOMT revealed that the optimal pH is ~7.9, as currently observed in other species (review in Ref. 15). In both photoperiods tested, the affinity of HIOMT for each of its substrates is identical and the estimated K_m values are similar to those measured in the rat (review in Ref. 15). However, the V_max was about two times higher under SP than LP, indicating that the pineal amount of HIOMT is probably higher under SP than LP. A similar photoperiodic regulation of HIOMT with an increase in the activity in response to an increase in night length has been described in the rat (1, 32, 33, 39, 40) and the European hamster (35). In the rat, the physiological impact of this regulation remains uncertain, because no change in the amplitude of the nocturnal peak of Mel occurs in response to photoperiod changes (11, 16, 32). One explanation would be that rat pineal HIOMT is not saturated by its substrates in situ (review in Ref. 16) or else that other photoperiodic mechanisms regulate Mel synthesis (7). In the European hamster (Cricetus cricetus), on the contrary, we have observed that marked seasonal changes in the synthesis of pineal 5-ML and Mel occur concomitantly with parallel variations in HIOMT activity (35, 46). In the Siberian hamster as well, we are reporting herein that photoperiodic variations of Mel as well as 5-ML (22) synthesis are positively related to HIOMT activity changes. Because changes in V_max induce changes in the reaction velocity, it seems probable that in these photoperiodic species, HIOMT is saturated by its substrates in in situ conditions. This interpretation implies an important role for HIOMT in the photoperiodic modulation of Mel synthesis.

Neurotransmitters and molecular mechanisms involved in the photoperiodic regulation of HIOMT activity have been investigated in the rat. We recently demonstrated that the nocturnal release of norepinephrine by the sympathetic fibers induces stimulation of the HIOMT gene expression and that this is required to uphold a constant level of enzyme activity over a few days (33). In the rat, we have shown that an increase in the duration of the night induces a similar increase in the duration of the nocturnal peak of the HIOMT gene expression and an increase in the daily level of HIOMT activity (32). All together, these data strongly suggest that the duration of the nocturnal adrenergic stimulation of the HIOMT gene expression drives the amount of the protein and thus of the enzyme activity (32, 47). Additionally, NPY, another sympathetic neurotransmitter of the rodent pineal gland, may be involved in the long-term regulation of HIOMT activity. Indeed, we recently reported that the seasonal variations of HIOMT activity are positively related with similar changes in the immunoreactivity of NPY in the pineal gland of the European hamster (23, 35). Moreover, we have observed a direct acute and chronic stimulatory effect of NPY on HIOMT activity in cultured rat pinealocytes (34). Consequently, even if the role of norepinephrine in the long-term regulation of HIOMT activity has been demonstrated in the rat, it is very probable that other neurotransmitters, especially NPY, can modulate HIOMT activity on a long-term range (34, 35). Thereby, it will be interesting to investigate whether the content of NPY displays photoperiodic changes in the Siberian hamster pineal gland.

Perspectives