Vasoactive intestinal peptide stimulates N-acetyltransferase and hydroxyindole-O-methyltransferase activities and melatonin production in cultured rat but not in Syrian hamster pineal glands

The purpose of this study was to compare the responses of the Syrian hamster and rat pineal glands in organ culture to vasoactive intestinal peptide (VIP). The endpoints in these studies were the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as pineal and medium melatonin levels. When rat pineal glands were incubated with either VIP (1 microM) or isoproterenol (1 microM), a beta-adrenergic agonist, a significant increase in NAT and HIOMT activities and melatonin levels were observed within 3 hr. Conversely, during the day, VIP (1 microM) was ineffective in stimulating these parameters in hamster pineal gland after incubation times of either 2, 4, 6, or 8 hr. In another experiment, hamster pineal glands were collected from animals killed in the late dark period (after 30 min light exposure). In these glands, isoproterenol promoted NAT activity and melatonin production; however, VIP was ineffective in stimulating either NAT or HIOMT activities; likewise, VIP had no stimulatory effect on pineal melatonin levels at night. Finally, when hamster pineal glands at night were incubated with either 0, 10 nM, 100 nM, 10 microM, or 100 microM VIP, no changes in any parameter of melatonin synthesis were measured. The results indicate that the hamster pineal gland, unlike that of the rat, may not respond to VIP with an increased melatonin production.     

Effects of different doses and durations of melatonin infusions on plasma melatonin concentrations in pinealectomized Syrian hamsters: Consequences at the level of sexual activity

The effect of different doses and durations of melatonin infusions on plasma melatonin concentrations has been studied in pinealectomized Syrian hamsters maintained under short photoperiod at either 7 degrees C or 18 degrees C. The effects of the infusions on plasma melatonin concentrations and on gonadal activity were compared. The results show that the minimal effective quantity of infused melatonin that induced gonadal atrophy was 40 ng/h at 7 degrees C and 20 ng/h at 18 degrees C. An infusion of 8 hr duration per day is necessary to inhibit sexual activity, while an infusion of 6 hr duration was ineffective. This finding suggests that the critical duration of melatonin infusion is between 6 and 8 hr. Despite the various doses of melatonin infused, plasma melatonin concentrations measured in the middle of the infusion period did not differ significantly from concentrations measured in intact animals. This finding suggests that the metabolism of infused melatonin increases as the dose of melatonin increases. Moreover, the different physiological effects observed after the various melatonin infusions cannot be explained by variations in plasma melatonin concentrations.     

Physiological concentrations of melatonin inhibit the nitridergic pathway in the Syrian hamster retina

In the present work, the effect of melatonin on the hamster retinal nitridergic pathway was examined. When the retinas were incubated in the presence of low concentrations (1 pM-10 nM) of melatonin for 15 min, a significant decrease of nitric oxide synthase (NOS) activity was observed. However, when crude retinal homogenates were preincubated with melatonin for 15 min, no changes in NOS activity were detected, despite the fact that under the same conditions trifluoperazine, a calmodulin inhibitor, significantly decreased enzymatic activity. Kinetic analysis showed that melatonin decreased the V(max) of retinal NOS without changes in the K(m). On the other hand, low concentrations (100 pM) of melatonin significantly reduced retinal L-arginine influx. A decrease in the V(max) of L-arginine uptake was observed in the presence of melatonin, whereas the K(m) remained unchanged. Melatonin significantly inhibited the accumulation of cyclic guanosine monophosphate (cGMP) levels induced by both L-arginine and sodium nitroprusside (SNP). In summary, the present results indicate that melatonin could be a potent inhibitor of the retinal nitridergic pathway.     

Role of extracellular calcium on the regulation of melatonin synthesis in the Syrian hamster pineal gland

The role of extracellular calcium on the induction of cyclic AMP production, N-acetyltransferase (NAT) activity and melatonin synthesis, induced by forskolin, was investigated in Syrian hamster pineal glands. Pineals were removed from hamsters killed either in the first half of the normal dark period or late in the dark period. Forskolin immediately increased NAT activity and melatonin levels only when the glands were collected late in the dark period, while in the first half of the dark phase, hamster pineals responded to forskolin with an increase of NAT activity and melatonin production only after 6 hr of incubation. The absence of calcium prevented the induction of melatonin synthesis by forskolin only when glands were collected early in the dark phase and incubated for 6 hr. In the second half of the normal dark period removal of calcium markedly decreased NAT activity and melatonin levels in glands incubated with forskolin for either 4 or 6 hr. However, the absence of extracellular calcium had no significant effect on the induction of cyclic AMP production by forskolin in pineals collected either early in the dark period or late in the dark phase. These data indicate that at least part of the action of extracellular calcium is indirect and that it affects steps in the induction of melatonin synthesis beyond the accumulation of cAMP.     

Regulation of the androgen receptors in the Harderian gland of the male Syrian hamster: Influence of photoperiod, castration, and chronic melatonin treatment

Male Syrian hamsters that were exposed for 8 weeks to short photoperiod (LD 10:14) or treated with melatonin in the late afternoon under long photoperiod conditions (LD 14:10) had a significantly higher content of androgen receptors in the Lipidex-purified soluble fractions isolated from the Harderian glands as compared to the long photoperiod (LD 14:10) exposed controls. Simultaneous computer-assisted analyses of all series of saturation and competition experiments revealed that the numerical value of the apparent Kd, as determined by using the synthetic androgen R-1881 (methyltrienolone), was not different between the experimental groups, and ranged from 0.050 to 0.067 nM. Of the principal natural androgens, testosterone (T) was most potent in inhibiting methyltrienolone binding to the receptor (Ki values from 0.33 to 0.55 nM), and 5 alpha-dihydrotestosterone (DHT) and delta 4-androstenedione (AD) were less effective (Ki values between 1 and 1.9 nM). In the hypothalami and pituitaries of the same animals, used in parallel control assays, DHT was twice as potent as T. Short-term castration (24 hr post-orchidectomy) did not result in significant changes in the receptor binding characteristics. Following 8 weeks exposure to a long photoperiod (LD 14:10) the Bmax values demonstrated a four-fold increase in castrated animals (179 fmoles/mg protein vs. 47 fmoles/mg protein) over intact controls. The relative binding affinity of the major androgens under these conditions remained unchanged, with the exception of AD, where a five-fold increase in the numerical Ki values (decrease in the binding affinity) was recorded (Ki = 9.6 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexpression of MT1 and RORalpha1 melatonin receptors in the Syrian hamster Harderian gland

Melatonin acts through several specific receptors, including membrane receptors (MT(1) and MT(2)) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT(1) and RORalpha(1) in this gland was determined by nested RT-PCR followed by amplicon sequencing and Western-blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT(1) receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal-synthesized melatonin. On the contrary, although a RORalpha(1)-immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORalpha(1) on the role of this indoleamine against oxidative stress is discussed.     

Changes with age in daytime and nighttime contents of melatonin, indoleamines, and catecholamines in the pineal gland: A comparative study in rat and Syrian hamster

Abstract: Previous studies in rodents showed a severe deterioration of pineal physiology with aging. The present study investigated the age-related changes in the content of monoamines and metabolites in rat and Syrian hamster pineal gland. In addition to melatonin, the levels of 5-hydroxytryptophan (5HTP), serotonin (5HT), 5-hydroxyindoleacetic acid (5HIAA), N-acetylserotonin (N-Ac-5HT), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and noradrenaline (NA) were measured by HPLC. Pronounced reductions were found in 5HT and 5HIAA contents during daytime in rats of 24 months, which had not been observed in animals of 12 months. In addition, nighttime pineal 5HIAA, N-Ac-5HT, and melatonin contents were decreased in the old rats, although a significant day: night variation persisted. Also a diurnal fluctuation in NA, DA, and DOPAC contents was present in young and middle-aged rats but not for NA and DOPAC in the oldest rats due to a decrease in the nighttime levels. Pineal DA levels were also reduced in 24-month-old rats during the night, although a marked day: night change was still found. In the Syrian hamster pineal, significant reductions in daytime 5HT and 5HIAA were found respectively at 12 and 18 months, while nighttime levels of these compounds were decreased from 18 months. The nocturnal content of N-Ac-5HT dropped gradually from 12 months, and melatonin was reduced by 74% and 86% in hamsters of 18 and 24 months, respectively. In all these compounds, a significant day: night variation was observed irrespective of age. However, neither a day: night variation nor an effect of aging was found in terms of pineal NA content. In contrast, pineal DA and DOPAC levels displayed a diurnal variation in hamsters of 1.5 and 6 months, but not in animals of 12 and 18 months due a reduced nighttime content. These data suggest that the decline of pineal melatonin with age is a consequence of a deficit in the pathway of serotonin utilization. This probably is explained by a reduced N-acetyltransferase activity, which may be linked to impaired pineal catecholaminergic neurotransmission.     

Chronic administration of melatonin induces changes in porphyrins and in the histology of male and female hamster Harderian gland: Interrelation with the gonadal status

In this paper, we have investigated the influence of melatonin on the histology and porphyrin content of the Syrian hamster Harderian glands. Daily afternoon injections of 25 micrograms of melatonin to female hamsters for 12 weeks resulted in the discontinuity of estrous cyclicity, a marked decrease in the Harderian gland intraluminal area occupied by porphyrins, and in a significant rise in the number of Type II cells. A similar decrease in porphyrins was observed after 8 weeks of ovariectomy. However, if the melatonin injections were given for only 8 weeks (without inducing gonadal atrophy), no changes were observed in the area occupied by intraluminal porphyrins, suggesting that the effects of melatonin in female Syrian hamsters might be associated with the subsequent gonadal atrophy. Castration of male hamsters induced a significant increase in porphyrins and a clear drop in the number of Type II cells. These changes were totally prevented when melatonin was administered daily from the day of castration. Our results suggest that melatonin, at least in male Syrian hamsters, plays a role in Harderian metabolism, acting directly on the Harderian secretory cells or indirectly through pituitary hormones.     

Influence of melatonin on the proliferation of hepatoma cells in the Syrian hamster: In vivo and in vitro study

A study was carried out to determine the possible influence of melatonin on the proliferation of Kirkman-Robbins hepatoma cells in Syrian hamsters, both in vivo and in vitro. A highly significant inhibition of the proliferation of transplanted hepatoma cells was observed in vivo following melatonin administration (proliferation index 10.3 +/- 1.5; mean +/- SD) in comparison with controls (proliferation index 23.3 +/- 2.2), whereas in vitro melatonin (at concentrations of 10(-5)-10(-11) M) was ineffective. These results indicate that inhibitory effect of melatonin on transplantable hepatoma cells in vivo may be indirect.     

Afternoon injections of melatonin in the Djungarian hamster Phodopus sungorus: Long lasting sex-specific effects and influence of acute treatment on the endogenous pineal melatonin rhythm

Abstract: The effects of exogenous melatonin on gonadal function and on the endogenous melatonin profiles of male and female Djungarian hamsters were investigated. Daily afternoon subcutaneous injections of melatonin (50 μ.g) in hamsters kept in long photoperiods (LD 16: 8) led to gonadal inhibition in all the treated females (n=13) but in only 2 of 13 males within the treatment period of 8 weeks (uteri: 244±11 mg in controls vs. 79±4 mg in treated hamsters, P <0.001; ovaries: 13.6±0.6 mg in controls vs. 7.9±0.7 mg in treated hamsters, P <0.001; testes: 1,021±54 mg in controls vs. 732±100 mg in treated hamsters, P <0.05; and accessory glands: 641±38 mg in controls vs. 548±70 mg in treated hamsters, P >0.05). These results indicate that there are some circumstances under which the gonadal responses of the reproductive organs of male and female Djungarian hamsters differ. Interestingly, pineal concentrations of melatonin were found to be significantly higher in males ( P <0.01), possibly indicating a more robust endogenous supply with the hormone, whereas serum melatonin levels were not significantly different between males and females. However, the subcutaneous injection of melatonin (5, 25, and 125 μg) exerted no acute effect on the endogenous, circadian melatonin-profile, independent of dosage and sex.