Photoperiod-dependent changes in melatonin synthesis in the turkey pineal gland and retina.

The effect of photoperiod on melatonin content and the activity of the melatonin-synthesizing enzymes, namely, serotonin N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase, were investigated in the pineal gland and retina of turkeys. The birds were adapted to 3 different lighting conditions: 16L:8D (long photoperiod), 12L:12D (regular photoperiod), and 8L:16D (short photoperiod). Pineal, retinal, and plasma melatonin concentrations oscillated with a robust diurnal rhythm, with high values during darkness. The duration of elevated nocturnal melatonin levels in the turkey pineal gland, retina, and plasma changed markedly in response to the length of the dark phase, being longest during the short photoperiod with 16 h of darkness. These photoperiodic variations in melatonin synthesis appear to be driven by AANAT, because changes in the activity of this enzyme were closely correlated with changes in melatonin. By contrast, pineal and retinal hydroxyindole-O-methyltransferase activities failed to exhibit any significant 24-h variation in the different photoperiods. A marked effect of photoperiod on the level of melatonin production was also observed. Peak values of melatonin and AANAT activity in the pineal gland (but not in the retina) were highest during the long photoperiod. During the light phase, mean melatonin concentrations in the pineal gland and retina of turkeys kept under the long photoperiod were significantly higher compared with those from birds maintained under the regular and short photoperiods. In addition, mean circulating melatonin levels were lowest in the short photoperiod. Finally, the magnitude of the light-evoked suppression of nighttime pineal AANAT activity was also influenced by photoperiod, with suppression being smallest under the long photoperiod. These findings show that in the turkey, photoperiod plays an important role in regulating the melatonin signal.

Seasonal variations in the nycthemeral rhythm of plasma melatonin in the camel (Camelus dromedarius).

Seasonal changes in the pattern of plasma melatonin were investigated in two groups of camels (Camelus dromedarius): 11 adult and six young camels. Animals were subjected to the outdoor conditions of a desert environment. Blood samples were taken at regular intervals of about 3 hr (added to particular samples at 1 hr before then 30 min and 1 hr after sunset, and 1 hr before and 1 hr after sunrise) for 24 hr at both solstices and equinoxes of the year. The plasma melatonin levels steeply increased soon after sunset and remained elevated throughout all the night. Then, melatonin concentrations progressively declined shortly before sunrise and returned to daytime basal levels 1 hr later. There was no seasonal variation in the amplitude or in the offset of the melatonin peak or in the daytime basal levels. The onset of the nocturnal peak was delayed by 2 hr in June at the summer solstice (P < 0.05), which can be related to the changes in night length between the two solstices. A significant effect of age was observed in all seasons. Melatonin levels were higher in the young camel group (fall equinox: P < 0.001; spring equinox: P < 0.01; winter solstice: P < 0.01; summer solstice: P < 0.05). The pattern of melatonin secretion in the camel showed a significant seasonal variation parallel to the photoperiodic changes of the year. The observed decline of melatonin levels during an extra-light pulse in the middle of the night indicates the light control of melatonin synthesis. It is not yet known if, in this low latitude desert region, the seasonal breeding period of the camel is cued by the photoperiod. The data obtained, however, clearly demonstrate that the camel has the capacity to follow and to integrate photoperiodic changes through melatonin changes.

Melatonin and 5-methoxytryptophol (5-ML) in nervous and/or neurosensory structures of a gastropod mollusc (Helix aspersa maxima): synthesis and diurnal rhythms.

Daily patterns of melatonin and 5-methoxytryptophol (5-ML) concentrations and of aryl alkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities have been measured in the cerebroid ganglions, visceral ganglions, and ocular tentacles of the gastropod mollusc Helix aspersa maxima. Melatonin concentrations are very low in all the studied structures, except a small peak at the end of the night in the cerebroid ganglions. 5-ML, which is quite undetectable in the cerebroid and visceral ganglions, shows clear daily variations in the ocular tentacles with low values in the middle of the light period and high values during the night. These results are opposite to what is known on daily variations of 5-ML in vertebrates. AA-NAT activity was not detected, while the presence of an HIOMT-like activity supports the hypothesis that 5-ML is synthesized in the ocular tentacles. The temporal relationships existing between the 5-ML rhythm in the ocular tentacles and the hemolymph suggest that 5-ML could be released in the general circulation. These preliminary results suggest that 5-ML could be an informative molecule involved in adaptative processes in the snail and they reinforce the hypothesis that the different 5-methoxyindoles could be implicated in the integration of environmental information.

Effect of melatonin supplementation on the sexual development in European quail (Coturnix coturnix).

At the end of their wintering phase, male European quails were exposed to a stimulation photoperiod of light/dark 12:12 h for 10 days to induce sexual development. A daily oral melatonin supplementation was then given to one group of treated males (N=11) and the alcohol solvent was given to a control group of males (N=10). These solutions were provided during the final 3 h of the photophase for 28 days, then during the final 4 h for 18 days. There were no significant differences between the two groups with respect to fat levels. However, 3 weeks after the beginning of melatonin supplementation, the sexual development of the treated birds slowed down. The importance of this decline varied to a greater or lesser degree between individual birds. When melatonin supplementation stopped, sexual development resumed. Activity recordings revealed a decrease in feeding activity when melatonin supplementation was provided. However, this response showed important interindividual variability. The birds that produced the most marked responses to melatonin during the first 3 weeks of supplementation were those that also showed the most obvious decline in sexual development. It seems that, in European quail, a wild migratory species that always shows a natural biological annual rhythm, a melatonin signal could play a role in regulating reproduction.

Effects of cycloheximide and aminophylline on 5-methoxytryptophol and melatonin contents in the chick pineal gland.

The chick pineal gland rhythmically synthesizes two 5-methoxyindoles, melatonin and 5-methoxytryptophol. These rhythms are circadian in nature and have opposite phases. The aim of this study was to determine the effects of cycloheximide, a protein synthesis inhibitor, and aminophylline, an inhibitor of phosphodiesterase, on 5-methoxytryptophol content in the chick pineal gland and to compare this with the drugs' action on pineal melatonin production. Inhibition of melatonin biosynthesis by cycloheximide (1 mg/kg, i.p. ), revealed by a marked reduction in the nighttime activity of serotonin N-acetyltransferase (AA-NAT; a key regulatory enzyme in melatonin synthesis) and melatonin concentrations, was accompanied by a significant increase in 5-methoxytryptophol content. In contrast, administration of aminophylline (100 mg/kg, i.p.) to light-exposed chicks significantly increased pineal AA-NAT activity and melatonin levels and decreased 5-methoxytryptophol concentrations. It is concluded that in the chick the production of pineal 5-methoxytryptophol and melatonin is inversely correlated.

Long-term daily melatonin infusion induces a large increase in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland and eye of pinealectomized male Siberian hamsters (Phodopus sungorus).

The effects of long-term daily melatonin infusions on the melatonin synthetic pathway in the Harderian glands and eyes of male Siberian hamsters were studied. Hamsters were pinealectomized (PX) and infused daily for 8 hr with either melatonin (6 microg/hr) or vehicle for 7 days in short photoperiod (SP, 10L:14D), followed by 14 wk in either SP (SP group) or in constant darkness (DD group). After the infusion period (15 wk), the infusion was stopped and animals were transferred into SP for 3 wk. The hamsters were then killed at midday or midnight. Exogenous melatonin infusion caused an increase in the Harderian gland weight, which was still evident 3 wk after the end of the treatment. In addition, exogenous melatonin increased endogenous melatonin concentrations (4-fold) and hydroxyindole-O-methyltransferase (HIOMT) activity (2-fold). N-acetyltransferase (NAT) activity, however, was not increased, and no day/night difference in melatonin content and HIOMT activity was observed in the Harderian glands. In the eye, melatonin infusions significantly increased day and night-time melatonin levels (up to 3-fold) and both NAT and HIOMT activities (up to 3.5-fold). This effect of melatonin treatment was observed in both SP and DD groups. These observations demonstrate that exogenously-infused melatonin at relatively high doses activates the synthesis of endogenous melatonin in the Harderian gland and eye of the Siberian hamster. Circulating levels of melatonin were also markedly increased, indicating that in these conditions melatonin may be released from extra-pineal sites.

Phase-shifting effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin in the chick pineal gland.

In the chick pineal gland, 5-methoxytryptophol and melatonin concentrations fluctuate in a rhythmic manner. These rhythms are circadian in nature persisting in constant darkness and have opposite phases. Acute exposure of chicks to white light (30 lux for 5, 10, 20, and 30 min) at night increased the amount of pineal 5-methoxytryptophol and decreased pineal melatonin content. A 6 hr pulse of light (100 lux) applied early in the subjective night (CT12-CT18) caused a delay in the phase of the circadian rhythms of 5-methoxytryptophol and melatonin by 3.7 and 4.5 h, respectively, compared to untreated controls. When the 6 hr light pulse was given during the late subjective night (C18 CT24) it advanced the phase of the 5-methoxytryptophol and melatonin rhythms by 8.1 and 11.9 h, respectively. In the chick pineal the phase-advancing effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin were more pronounced than the phase-delaying effects. Our results provide the first evidence that light is capable of phase shifting the 5-methoxytryptophol rhythm in a manner similar to its action on the melatonin rhythm.

Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice.

In rodents, the nocturnal rise and fall of arylalkylamine N-acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)-inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin-deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin-deficient C57BL mouse and the melatonin-proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin-deficient and that this melatonin-deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.

Overexpression of neuropeptide Y induced by brain-derived neurotrophic factor in the rat hippocampus is long lasting.

Brain-derived neurotrophic factor (BDNF) plays an important role in hippocampal neuroplasticity. In particular, BDNF upregulation in the hippocampus by epileptic seizures suggests its involvement in the neuronal rearrangements accompanying epileptogenesis. We have shown previously that chronic infusion of BDNF in the hippocampus induces a long-term delay in hippocampal kindling progression. Although BDNF has been shown to enhance the excitability of this structure upon acute application, long-term transcriptional regulations leading to increased inhibition within the hippocampus may account for its suppressive effects on epileptogenesis. Therefore, the long-term consequences of a 7-day chronic intrahippocampal infusion of BDNF (12 microg/day) were investigated up to 2 weeks after the end of the infusion, on the expression of neurotransmitters contained in inhibitory hippocampal interneurons and which display anti-epileptic properties. Our results show that BDNF does not modify levels of immunostaining for glutamic acid decarboxylase, the rate-limiting enzyme for gamma-aminobutyric acid (GABA) synthesis, and somatostatin. Conversely, BDNF induces a long-lasting increase of neuropeptide Y (NPY) in the hippocampus, measured by immunohistochemistry and radioimmunoassay, outlasting the end of the infusion by at least 7 days. The distribution of BDNF-induced neuropeptide Y immunoreactivity is similar to the pattern observed in animals submitted to hippocampal kindling, with the exception of mossy fibres which only become immunoreactive following seizure activity. The enduring increase of neuropeptide Y expression induced by BDNF in the hippocampus suggests that this neurotrophin can trigger long-term genomic effects, which may contribute to the neuroplasticity of this structure, in particular during epileptogenesis.

Potentiation effect of vasopressin on melatonin secretion as determined by trans-pineal microdialysis in the Rat.

The mammalian pineal gland is known to receive a noradrenergic innervation originating from the superior cervical ganglion which corresponds to the primary regulatory input for melatonin synthesis. However, many peptidergic fibers containing peptides such as vasopressin and oxytocin have also been found in the rat pineal gland. The present study was performed to investigate the possible role of vasopressin and oxytocin on melatonin secretion in vivo. Therefore, both neuropeptides were delivered for 2 h through a trans-pineal microdialysis probe directly into the gland at different times during the nocturnal phase of the light:dark cycle. At the same time pineal dialysates were collected continuously. Melatonin concentrations were measured by radioimmunoassay. Melatonin synthesis potentiation was achieved when vasopressin was infused locally in the pineal, during the onset of nocturnal melatonin secretion. In order to assess the possible role of a physiological increase of endogenous circulating vasopressin on pineal metabolism, melatonin synthesis was recorded in the same animals before and after a prolonged dehydration period. Night time melatonin concentration was increased after the water deprivation vs control conditions. Contrary to that, oxytocin seems not to affect pineal metabolism in the rat since no significant change was observed on melatonin secretion in response to a local oxytocin infusion. These results show that vasopressin can modulate melatonin synthesis in the rat pineal whereas no effect was obtained with oxytocin, at least under the present experimental conditions.

Interindividual differences in the pattern of melatonin secretion of the Wistar rat.

In vivo trans-pineal microdialysis was performed in male Wistar rats maintained under a 12 hr light:12 hr dark (LD 12:12) cycle. Collected dialysates were assayed by radioimmunoassay for melatonin concentrations. A non-linear regression was fitted through the obtained datapoints to determine the time points at which a 50% increase (IT50) and decrease (DT50) of the nocturnal melatonin peak were reached. In a first experiment, the nocturnal melatonin profiles of four animals were determined throughout 5 consecutive days. In a second experiment, we analysed the melatonin profiles during the night in rats originating from three different breeding colonies (Dépré Harlan, and Iffa-Credo). A low intraindividual variability was found on the phase markers IT50 and DT50, as on peak duration of melatonin rhythms estimated over 5 subsequent days in the same animal. In contrast, animals showed a large interindividual variability in their profile phase markers and the values were dependent on the origin of the breeding colony. Each rat colony was characterized by early or late IT50 and DT50 as long or short peak length. It is concluded from experiment 1 that the melatonin rhythm is a very stable circadian marker. Nevertheless, great caution must be taken in the choice of animal groups while studying circadian rhythms due to the large interindividual variability observed in experiment 2. Therefore, as the technique allows the use of the animal as its own control, the present study demonstrated that the use of the microdialysis technique is of interest in studies on the circadian system.

Daily and light-at-night induced variations of circulating 5-methoxytryptophol (5-ML) in ewes with respectively high and low nocturnal melatonin secretion.

The aim of the present study was to determine whether the genetic differences previously reported in ewe plasma melatonin concentrations were correlated with differences in the synthesis and release of other 5-methoxyindoles. To determine if 5-methoxytryptophol (5-ML), which is known to be present in large amounts in the sheep pineal gland, is released, as is melatonin, into the general circulation, and if some temporal relationships between 5-ML and melatonin release could be observed, two groups of ewes were selected with respect to their endogenous melatonin secretion: in the first experiment, ten ewes from the low melatonin group (low group) and ten ewes from the high melatonin group (high group). 5-ML was measured every hour during a 24-hr period by radioimmunoassay. In all ewes, 5-ML was released during day-time, the rhythm of 5-ML concentrations being inversely related with the melatonin rhythm. Both day-time and night-time 5-ML concentrations were higher in the ewes from the high group than in the ewes from the low group (14.7 +/- 1.0 pg/mL plasma versus 6.4 +/- 0.3 pg/mL plasma during the day, 3.1 +/- 0.2 pg/mL plasma versus 1.9 +/- 0.2 pg/mL plasma during the night). The 5-ML/melatonin ratio appeared much higher during the day than during the night but was very similar in both groups (day-time: 1.03 in the high group versus 1.16 in the low group, night-time: 0.01 in both groups). In a second experiment, six low group and seven high group ewes were submitted to 1 hr of extra light at night. 5-ML increased and melatonin decreased during extra light. Our results clearly show for the first time a daily variation in circulating 5-ML, and that the strong genetic contribution in the variability in melatonin concentrations in sheep are clearly correlated with a similar variability in 5-ML concentrations. Whether 5-ML, like melatonin, plays a physiological role in the different adaptation processes to the environment remains to be determined.

Increased sympathetic activity during sleep and nocturnal hypertension in Type 2 diabetic patients with diabetic nephropathy.

AIMS: To elucidate the putative factors involved in the blunted nocturnal blood pressure reduction in hypertensive Type 2 diabetic patients with diabetic nephropathy. METHODS: Extracellular fluid volume and fluid shift from interstitial to plasma volume (haematocrit), sympathetic nervous activity (plasma noradrenaline and adrenaline) and the internal 'body clock' (serum melatonin) were investigated in 31 hypertensive Type 2 diabetes mellitus (DM) patients with diabetic nephropathy (24 males, age 60 (45-73) years). All variables, except extracellular volume, were measured repeatedly with the patients lying awake in bed from 21:30 to 23:00 h (baseline) and during sleep from 23:00 to 07:00 h. Using the median nocturnal blood pressure reduction (8.4%) as a guide, the patients were divided into groups; group 1 with the highest and group 2 with the lowest nocturnal blood pressure reduction. RESULTS: Haematocrit decreased from baseline to the sleep period in group 1 by a mean (95% confidence interval (CI)) of 1.7 (0.3-3.1)%, but it increased by 0.5 (-1.0-1.9)% in group 2, mean difference (95% CI), -2.1 (-4.0 to -0.2)% (P = 0.029). Noradrenaline decreased from baseline to the sleep period, mean (95% CI), by 13.3 (0.0-25.0)% in group 1 but rose by 7.7 (-9.7-28.4)% in group 2, mean difference (95% CI), -19.6 (-35-0.0)% (P = 0.049). The nocturnal blood pressure change correlated to the nocturnal change in both noradrenaline (r = 0.51, P = 0.004) and haematocrit (r = 0.42, P = 0.018). Adrenaline remained constant in both groups. Extracellular fluid volume and plasma melatonin levels were comparable in the two groups. CONCLUSION: Sustained adrenergic activity during sleep is associated with blunted nocturnal blood pressure reduction in hypertensive Type 2DM patients with diabetic nephropathy, probably mediated through a lack of peripheral vasodilatation whereas changes in extracellular fluid volume distribution and melatonin secretion have no impact.

Immunohistochemical evidence for the presence of tryptophan hydroxylase and serotonin in the rodent Harderian gland.

The Harderian gland is considered as being an extrapineal source of melatonin. In most rodents, the Harderian gland contains two epithelial cell types (I and II). The aim of this study has been to define which cell type is involved in indoleamine synthesis. The presence and localization of serotonin (melatonin precursor) and tryptophan hydroxylase (the rate-limiting enzyme for serotonin synthesis) have been investigated by immunohistochemistry in male Wistar rats, Syrian hamsters and Djungarian hamsters. The results of the present study show that immunoreactivity for tryptophan hydroxylase and serotonin is confined to the type I cell, suggesting that this cell type is involved in indoleamine synthesis in the rodent Harderian gland.

Interstrain differences in activity pattern, pineal function, and SCN melatonin receptor density of rats.

We investigated the possibility that strain-dependent differences in the diurnal pattern of wheel running activity rhythms are also reflected in the melatonin profiles. The inbred rat strains ACI/Ztm, BH/Ztm, and LEW/Ztm. LEW were examined for diurnal [12:12-h light-dark (LD)] wheel running activity, urinary 6-sulphatoxymelatonin (aMT6s) excretion, melatonin concentrations of plasma and pineal glands, and melatonin receptor density in the suprachiasmatic nuclei (SCN). ACI rats displayed unimodal activity patterns with a high level of activity, whereas BH and LEW rats showed multimodal activity patterns with ultradian components and reduced activity levels. In contrast, the individual daily profiles of aMT6s excretion and mean melatonin synthesis followed a unimodal time pattern in all three strains, suggesting that different output pathways of the SCN are responsible for the temporal organization of locomotor activity and pineal melatonin synthesis. In addition, melatonin synthesis at night and SCN melatonin receptor density at day were significantly higher in BH and LEW rats than in ACI rats. These results support the hypothesis of a long-term stimulating effect of melatonin on its own receptor density in the SCN.

Immunohistochemical characterisation of epithelial cells of rodent harderian glands in primary culture.

The aims of the current investigation were (1) to establish an efficient procedure for the isolation of rodent harderian gland cells and to define conditions for maintenance of viable differentiated cells; (2) to compare the in vitro growth pattern of cultured epithelial cells; and (3) to characterise the cultured epithelial cells from 3 rodent species: Wistar rats, Syrian hamsters and Djungarian hamsters. We have established primary culture conditions that permit the maintenance of viable and differentiated secretory cells from adult rodent harderian gland. This study demonstrates that the cell growth pattern is faster in hamsters than in rats and despite morphological changes, epithelial cells reestablish their distinctive (biochemical/metabolic) phenotype as indicated by lipid-containing vacuoles, porphyrin pigment and serotonin and tryptophan hydroxylase labelling.

Daily variations in pineal melatonin concentrations in inbred and outbred mice.

Melatonin was measured using a specific radioimmunoassay in 1 strain of outbred mice (OF1 Swiss) and 4 strains of inbred mice, 2 of them being known to synthesize melatonin (CBA and C3H) and the 2 others being controversial (BALB/c and C57BL/6). In this study, the 5 mouse strains were able to synthesize melatonin, but the basal levels as well as the diurnal variations were very different from one strain to another. CBA and C3H strains showed a clear-cut day-night rhythm of pineal melatonin concentration, with peak levels of 276 +/- 22 pg/pineal in CBA and 135 +/- 12 pg/pineal in C3H. In BALB/c, the authors confirmed the presence of a very short melatonin peak (15 min) in the middle of the dark period. In C57BL/6 and OF1 Swiss, a very small but significant peak was observed in the middle of the darkness. In the former, another small peak was also observed at light onset. Whether these very small peaks, which may be related to the deficience of N-acetyl transferase activity reported by others, have a physiological meaning remains to be determined.

Effect of NMDA receptor antagonist MK-801 on light-induced Fos expression in the suprachiasmatic nuclei and on melatonin production in the Syrian hamster.

In mammals, circadian rhythms generated by the suprachiasmatic nuclei (SCN) are daily synchronized by a light-dark cycle. Photic information is transmitted to the SCN mainly through the direct retinohypothalamic tract, the neurotransmitters involved being excitatory amino acids. It is also commonly accepted that photoperiodic information coming from the retina via the SCN is transduced by the pineal into a nocturnal signal, i.e. melatonin production. Light exposure at night induces (1) an inhibition of melatonin synthesis and (2) an expression of c-fos in numerous cells of SCN. To determine the role of the NMDA receptor in these effects, we treated Syrian hamsters with ip injections of MK-801, a noncompetitive NMDA receptor antagonist. Several subpopulations of light-sensitive cells in the SCN are affected by MK-801. According to previous studies, MK-801 inhibits light-induced Fos immunoreactivity mainly in the most ventral part of the SCN. However, we observed that numerous other cells are still activated by light. When light is applied in the middle of the night, MK-801 pretreatment does not reduce Fos-ir in the dorsal SCN. At the beginning of the night, labeled cells in this part of the nucleus appear even more numerous after MK-801. We also found that MK-801 fails to reduce the light-induced inhibition of melatonin synthesis. Moreover, in control animals, which received no light stimulation, ip injection of MK-801 induces by itself a dose-dependent inhibition of melatonin production.

Evidence for melatonin synthesis in rodent Harderian gland: a dynamic in vitro study.

Melatonin content and release from Harderian glands (HGs) has been measured by an in vitro perifusion technique in three rodent species: Wistar rat, Syrian hamster, and Siberian hamster. Melatonin immunoreactive concentrations in HGs of animals killed at 10.00 hr were 0.31 +/- 0.031 pg/mg gland in male Wistar rat, 0.54 +/- 0.026 pg/mg gland in male Siberian hamster, 0.17 +/- 0.070 and 0.20 +/- 0.059 pg/mg gland in male and female Syrian hamster, respectively. In all species examined, isolated HGs perifused for 9-15 hr released melatonin but did not stabilize their melatonin release rate. No sex-related difference could be noted in the HG melatonin release rate. The total amount of melatonin released over a 15 hr long perifusion was about 0.075 +/- 0.004 ng/15 h/mg gland and 0.063 +/- 0.010 ng/15 hr/mg gland in male and female Wistar rat, respectively; 0.155 +/- 0.019 ng/15 hr/mg gland and 0.141 +/- 0.006 ng/15 hr/mg gland in male and female Siberian hamster, respectively; 0.035 +/- 0.003 ng/15 hr/mg gland and 0.045 +/- 0.004 ng/15 hr/mg gland in male and female Syrian hamster, respectively. This amount, which is higher than the tissue levels, demonstrates the de novo melatonin synthesis. This is confirmed by the fact that infusion of the indoleamine precursor, tryptophan (TRP), stimulated melatonin secretion from HGs. The melatonin release is increased by 2.5-fold in male and female Wistar rat, 1.5-fold in male and female Siberian hamster, and 2.0- and 3.0-fold in male and female Syrian hamster, respectively. Treatment with a TRP hydroxylase inhibitor, para-chlorophenylalanine, reduced basal melatonin release and inhibited the TRP-induced melatonin stimulation. Kinetics and amounts of melatonin released were not affected by pinealectomy, ruling out a possible plasmatic origin of the HG melatonin. Isoproterenol, a beta-adrenergic agonist, and dibutyryl cyclic AMP, a cyclic AMP analogue, failed to stimulate HG melatonin secretion. In conclusion, these results confirm the presence of melatonin in the HGs and demonstrate that melatonin is synthesized in and released from isolated rodent HGs.