Sexual dimorphism in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland of Syrian hamsters: changes following gonadectomy

The activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin content of the Harderian glands of intact and gonadectomized male and female Syrian hamsters were studied. NAT activity in intact male Harderian glands was twice that of the female. Prepubertal or adult castrated males exhibited a decrease in NAT activity to a level comparable to that seen in the female. Testosterone implants in the castrated males led to a recovery of the original male NAT levels. Intact male hamsters had very low levels of Harderian HIOMT activity and melatonin content in comparison with the glands of the females. Prepubertal gonadectomy but not castration of adult males raised the levels of HIOMT activity and the melatonin content to those of the females. Bilateral ovariectomy had no effect on melatonin content, NAT activity, or HIOMT activity in the female hamster Harderian gland.     

Effects of diazepam and its metabolites on nocturnal melatonin secretion in the rat pineal and Harderian glands. A comparative in vivo and in vitro study

We investigated the effects of diazepam (DZP) and its three metabolites: nordiazepam (NZP), oxazepam (OZP), and temazepam (TZP) on pineal gland nocturnal melatonin secretion. We looked at the effects of benzodiazepines on pineal gland melatonin secretion both in vitro (using organ perifusion) and in vivo in male Wistar rats sacrificed in the middle of the dark phase. We also examined the effects of these benzodiazepines on in vivo melatonin secretion in the Harderian glands. Neither DZP (10^-5-10^-6 M) nor its metabolites (10^-4-10^-5 M) affected melatonin secretion by perifused rat pineal glands in vitro. In contrast, a 10^-4 M suprapharmacological concentration of DZP increased melatonin secretion of perifused pineal glands by 70%. In vivo, a single acute subcutaneous administration of DZP (3 mg/kg body weight) significantly affected pineal melatonin synthesis and plasma melatonin levels, while administration of the metabolites under the same conditions did not. DZP reduced pineal melatonin content (-40%), N-acetyltransferase activity (-70%), and plasma melatonin levels (-40%), but had no affects on pineal hydroxyindole-O-methyltransferase activity. Neither DZP nor its metabolites affected Harderian gland melatonin content. Our results indicate that the in vivo inhibitory effect of DZP on melatonin synthesis is not due to the metabolism of DZP. The results also show that the control of melatonin production in the Harderian glands differs from that observed in the pineal gland.     

Bromocriptine prevents the castration-induced rise in porphyrin concentration in the harderian glands of the male Syrian hamster, Mesocricetus auratus

The Harderian glands in Syrian hamsters exhibit a striking sexual dimorphism. Male Harderian glands show two cell types and low levels of porphyrins and melatonin. Of the enzymes involved in the synthesis of melatonin, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) show high and low activity levels, respectively. Female Harderian glands show but one cell type and have high porphyrin and melatonin levels, low NAT activity, and high HIOMT activity. In castrated males, the Harderian glands exhibit a female pattern of morphology, porphyrin levels, and indoleamine metabolism. In an attempt to determine whether prolactin in involved in this sexually dimorphic response of the Harderian glands, intact and castrated male and intact female hamsters were injected daily with 500 micrograms of bromocriptine, a dopamine agonist. Bromocriptine led to reduced serum prolactin levels in all groups. It had no apparent effect on the Harderian glands of intact males. In contrast, in castrated males bromocriptine prevented the postcastrational rise in porphyrin levels but had no effect on NAT or HIOMT activities. In females, bromocriptine treatment had no effect on porphyrin concentrations or HIOMT activity; it led to a statistically significant increase in NAT activity. We propose that testosterone inhibits Harderian porphyrin synthesis while dopamine or prolactin stimulates it.     

N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin levels in the Harderian glands of the female Syrian hamster: changes during the light:dark cycle and the effect of 6-parachlorophenylalanine administration

The activities of NAT and HIOMT and the melatonin content of the Harderian glands of female Syrian hamsters were studied. When hamsters were kept under a light:dark cycle of 14:10 (lights on at 06.00 h), NAT activity exhibited a sharp, short term rise at one hour after lights on. Simultaneously, the activity of HIOMT, which forms melatonin, exhibited a rapid decline. Melatonin levels, like HIOMT activity, also showed a precipitous drop at one hour after light onset. After the respective changes, both NAT and HIOMT activity reverted back to night time levels. Melatonin levels remained depressed for several hours but by 1400 h (8 hours after lights on), nighttime melatonin values were re-established. Treatment of female hamsters with PCPA, a trytophan hydroxylase inhibitor, led to depressed levels of Harderian melatonin without affecting the activities of either NAT or HIOMT.     

Effects of prepubertal manipulation with androgens on the development of sexual differences in the harderian glands of Syrian hamsters.

The onset of sexual differences in the metabolism of porphyrins and melatonin in the Harderian glands of Syrian hamsters was studied. Three weeks after birth, the porphyrin concentrations were already higher in glands of females than in those of males. Castration of 22-day-old male hamsters led to an increase in Harderian porphyrin concentrations, although the levels of intact females were not reached. The administration of testosterone to 22-day-old female hamsters resulted in a marked decrease in porphyrin concentrations. Study of the development of sexual differences in the enzymes involved in melatonin synthesis, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) indicated that not all the sexual differences observed in these glands begin at the same time. Thus, while differences in NAT activity were detected after the age of 3 weeks, male-female differences in HIOMT activity were only observed after 7 weeks. Castration of prepubertal male hamsters lowered NAT but not HIOMT activities. The administration of testosterone to prepubertal female hamsters led to male activity levels in both enzymes. Although circulating androgens seem to have a crucial role in maintaining sexual differences, other hormones including those from the pituitary and thyroid glands are probably also important for generating these sexual differences.     

Simultaneous determination of N-acetyltransferase activity, hydroxyindole-O-methyl-transferase activity, and melatonin content in the pineal gland of the Syrian hamster

The activities of serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin content were measured in Syrian hamster pineal glands at 2-hr intervals over a period of 24 hr. NAT and HIOMT are the two enzymes which catalyze the formation of melatonin from serotonin. The use of micromethods for determination of the enzyme activities allowed concurrent measurement of NAT and melatonin or HIOMT and melatonin in the same gland. HIOMT activity showed no significant diurnal rhythm whereas NAT activity and melatonin content exhibited distinct peak values late in the dark phase as described previously. Despite an apparent parallelism between the NAT activity rhythm and melatonin content, no correlation exists between these parameters in single pineal glands.     

Gender differences and time course of castration-induced changes in porphyrins, indoles, and proteins in the Harderian glands of the Syrian hamster.

Sexual differences and the effects of orchidectomy were determined for porphyrin and melatonin concentrations and for the activities of the enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase, which synthesize melatonin from serotonin, in the Harderian glands of the Syrian hamster. Porphyrin concentrations in intact males were about 1/400th those of intact females. Castration for 1 week increased male Harderian porphyrin concentrations 10-fold; by 3 weeks, castrated male porphyrin levels were 140 times those of control values. N-Acetyltransferase activity in intact male Harderian glands was about 4 times that of females. Castration led to a drop in N-acetyltransferase activity to female levels within 2 weeks. Hydroxyindole-O-methyltransferase activity was 7 times higher in females than in males and castration had no effect on male Harderian hydroxyindole-O-methyltransferase activity. Neither gender nor castration influenced Harderian melatonin concentrations. Soluble proteins in Harderian glands from male and female hamsters and from male hamsters castrated for 1 and 4 weeks were examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The gel profiles revealed several differences among the protein distribution in male and female gland lysates. Orchidectomy led to a female protein pattern within 4 weeks.     

Melatonin and porphyrin in the harderian glands of the Syrian hamster: circadian patterns and response to autumnal conditions

1. Adult male Syrian hamsters were killed at nine intervals during a 24 hr period in the autumn, after 2 months either indoors in controlled conditions or in natural outdoor conditions. 2. Harderian glands were taken for determination of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities and melatonin and porphyrin concentrations. 3. Mean 24 hr Harderian NAT and melatonin values were lower outside than inside. 4. Twenty-four hour melatonin rhythms were detected with similar daytime (afternoon) acrophases in both environmental conditions. 5. An NAT rhythm was seen only in animals kept inside, with a circadian maximum in the late dark phase. 6. Mean 24 hr HIOMT activity was slightly higher outdoors than indoors, and 24 hr rhythms were not detected in either condition. 7. Mean porphyrin concentrations were higher outdoors, with 24 hr rhythms detected in both conditions and a significantly earlier nocturnal circadian maximum outdoors.     

Melatonin in the lacrimal gland: first demonstration and experimental manipulation

NAT, HIOMT and melatonin are described in the extra-orbital lacrimal glands. The extra-orbital lacrimal glands of female Syrian hamsters contain higher NAT activity and melatonin levels than those in male glands, while male glands have higher HIOMT activity. Castration did not change melatonin in the lacrimal glands, although NAT and HIOMT activities were altered. The exposure of female hamsters to light in the morning (0600h) was associated with a reduction in both NAT activity and melatonin levels. Porphyrins were not detected in the lacrimal glands of either male or female hamsters.     

N-acetyltransferase activity and indole contents of the male Syrian hamster Harderian gland: changes during the light:dark cycle

The activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the indole contents of the Harderian glands of male Syrian hamsters were studied throughout a 24-h period. NAT activity exhibited a sharp rise 1 h after lights on, decreasing to basal levels 1 h later. Neither a HIOMT activity nor a melatonin concentration rhythm was detected throughout the 24 h. The 5-hydroxytryptamine (serotonin) concentration was highest during the dark phase reaching a peak at 0300 h; with light onset serotonin levels exhibited a rapid short-term drop. The 5-hydroxytryptophol concentration was highest during the mid- to late photophase; the lowest values to this constituent were measured late in the dark phase and at 1 h after lights on. The 5-hydroxyindole acetic acid concentration of the Harderian glands was rather stable throughout the 24-h period but levels did show a short-lived drop 1 h after light onset. Only a few animals contained detectable amounts of N-acetyl-5-hydroxytryptamine (N-acetylserotonin) in their Harderian glands. In agreement with previous work on the Harderian glands of female Syrian hamsters, the present results in males suggest that light onset is associated with marked changes in Harderian indoleamine metabolism.     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.     

Lack of effect of ghrelin treatment on melatonin production in rat pineal and Harderian glands

The effects of ghrelin, a peptide hormone secreted from the stomach, on melatonin remain unknown. The aim of the study was to investigate possible ghrelin-melatonin interactions by studying the effect of ghrelin treatment on melatonin production in rat pineal and Harderian glands. Young (9 weeks) and old (20 months) male Wistar rats, maintained under a light:dark cycle regimen of 12:12, were assigned randomly to either a single subcutaneous (s.c.) injection of saline or ghrelin (1 microg/rat or 15 microg/rat) 1 h before sacrifice in the middle of the dark phase, or repeated s.c. saline or ghrelin injections (15 microg/rat), 3, 2 and 1 h before sacrificed in the middle of the dark phase. Neither ghrelin doses (1 microg/rat or 15 microg/rat) nor type of treatment (acute or repeated) influenced melatonin levels or the melatonin synthesizing enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase activities, either in pineal gland or in Harderian glands. At the concentrations used, ghrelin does not influence melatonin production in rat pineal and Harderian glands, and therefore is not involved in the regulation of melatonin secretion, at least under our experimental conditions.     

The influence of different light irradiances on pineal N-acetyltransferase activity and melatonin levels in the cotton rat, Sigmodon hispidus

Wild-captured cotton rats (Sigmodon hispidus) trapped and tested in September and October exhibited a rapid reduction in pineal N-acetyltransferase (NAT) activity and melatonin levels after exposure to a light irradiance of 300 ωW/cm² during the dark period. The half-time for the depression of both NAT and melatonin was on the order of 2 min. The exposure of cotton rats during darkness to much lower irradiances of light, i.e., 5.0, 0.04, 0.03 or 0.01 W/cm², for 32 min also greatly diminished pineal NAT activity and radioimmunoassayable melatonin levels; however, a light irradiance of 0.005 ωW/cm² failed to significantly depress either the acetylating enzyme or the melatonin content of the pineal gland. The results show that the pineal gland of the wild-captured cotton rat, as judged by NAT activity and melatonin levels, is inhibited even by very low irradiances of light.     

Swimming-induced suppression of rat pineal melatonin is prevented by pretreatment with calcium channel blockers

Young adult male rats were treated with isoproterenol during the day to induce high levels of pineal N-acetyltransferase (NAT) activity and melatonin. Roughly 2 hr later when pineal NAT activity and melatonin levels were elevated, animals were given either an injection of a calcium channel blocker, i.e., either nifedipine or verapamil, or diluent. The rats were then forced to swim for 10 min in room temperature (22 degrees C) water. Fifteen minutes after swimming onset, pineal glands were collected for measurement of NAT activity and melatonin. Swimming caused a dramatic reduction in pineal melatonin content without influencing NAT activity. Nifedipine substantially and verapamil completely blocked the drop in pineal melatonin levels due to swimming without influencing NAT activity. The results suggest that calcium may be somehow directly or indirectly involved in melatonin release from the rat pineal gland.     

Harderian glands of golden hamsters: morphological and biochemical responses to thyroid hormones

Summary Manipulation of circulating levels of thyroid hormones modifies Harderian gland structure and porphyrin concentrations in male and female golden hamsters. Specifically, thyroxine (T₄) and triiodothyronine (T₃) induce the morphological conversion of the Harderian glands of females to approximate those of the male. Further, porphyrin concentrations are markedly decreased by this treatment. This effect occurs in ovariectomized animals as well, indicating that the gonads are not involved. Suppression of thyroid function by potassium perchlorate (KClO₄) drastically reduces Harderian gland weight in both males and females. However, KClO₄ decreases porphyrin levels in the Harderian glands of females and increases it in the male. Concurrently, KClO₄ also induces a morphological conversion of the Harderian glands of males to the female type. This effect is evident in photoperiods of either 14:10 (h) or 8:16 (h).     

Cold prevents the light induced inactivation of pineal N-acetyltransferase in the Djungarian hamster,Phodopus sungorus

Summary In the Djungarian hamster seasonal acclimatization is primarily controlled by photoperiod, but exposure to low ambient temperature amplifies the intensity and duration of short day-induced winter adaptations. The aim of this study was to test, whether the pineal gland is involved in integrating both environmental cues. Exposure of hamsters to cold (0 °C) reduces the sensitivity of the pineal gland to light at night and prevents inactivation of N-acetyltransferase (NAT). The parallel time course of NAT activity and plasma norepinephrine content suggests that circulating catecholamines may stimulate melatonin synthesis under cold load.Abbreviations NAT N-acetyltransferase - NE norepinephrine - T _a ambient temperature     

Testosterone increases N-acetyltransferase activity in both male and female Syrian hamster harderian glands

N-acetyltransferase (NAT) activity was studied in the Harderian glands of intact and castrated (with or without subcutaneous testosterone implants) male and female Syrian hamsters. Castration in male hamsters produced a significant drop in the NAT activity. Castrated males with testosterone implants had NAT activity levels comparable to those in intact males. Ovariectomy did not modify NAT activity. Ovariectomized hamsters with testosterone implants exhibited a significant increase in the Harderian NAT activity reaching the same values as those in the glands of the male hamsters.     

A comparative study of melatonin production in the retina,pineal gland and Harderian gland of Bufo viridis and Rana esculenta

1. The circadian patterns of melatonin and of its synthesizing enzyme N-acetyltransferase (NAT) were investigated in the serum, retina, pineal gland and Harderian gland (HG) of two amphibian species, Bufo viridis and Rana esculenta.2. Serum melatonin levels showed no diurnal fluctuations in Bufo viridis, whereas, in Rana esculenta, they exhibited a circadian rhythm, with the highest values occurring during the night. Retina melatonin exhibited characteristic circadian patterns in both species, with the highest values occurring during the day, in Bufo, and the highest concentrations occurring at night in Rana.3. In the retina, NAT activity peaked at night in both amphibians, but in Bufo the levels were up to 30 times higher than in Rana. In the HG and in the pineal gland, NAT activity showed different patterns in the two species with no diurnal variations in Bufo, and characteristic circadian rhythms in Rana.4. In the HG and pineal gland of both species, melatonin was only occasionally detectable over the 24-hr period.5. This is the first report exploring melatonin production in Bufo viridis and Rana esculenta. In our experimental conditions, marked differences emerged between the two species.     

Androgenic control of N-acetyltransferase activity in the harderian glands of the Syrian hamster is mediated by 5 alpha-dihydrotestosterone

N-acetyltransferase (NAT) activity in the Harderian glands of intact and gonadectomized male and female Syrian hamsters was evaluated. The exogenous administration of 5 alpha-dihydrotestosterone (DHT) to castrated males and intact females produced an increase in NAT values, which reached the values present in the glands of intact males. The administration of a 5 alpha-reductase inhibitor to intact males led to a decrease in NAT activity, suggesting that testosterone is converted in DHT within the glands. It is concluded that NAT activity in the Syrian hamster Harderian glands is under androgenic control, the active steroid being DHT.     

Evidence that interferon-gamma alters pineal metabolism both indirectly via sympathetic nerves and directly on the pinealocytes.

1. Interferon-gamma (IFN-gamma) has been shown to suppress N-acetyltransferase (NAT) activity in cultured rat pineal glands when stimulated with isoproterenol (ISO). 2. Conversely, IFN-gamma has also been shown to increase the melatonin content of the rat pineal gland in organ culture. 3. Circumstantial evidence leads to a hypothesis that the NAT suppressive effect may be due to the action of IFN-gamma on the sympathetic nerve terminals. 4. To test this hypothesis, pineal glands from intact (INT) and superior cervical ganglionectomized (SCGX) rats, which had been operated 5 days earlier, were cultured with either ISO or ISO + IFN-gamma. 5. The concentration of ISO was 10(-8) M and that of IFN-gamma was 300 antiviral units/ml. 6. The pineals were incubated for a total period of 5.5 hr, after which the activities of NAT and hydroxyindole-O-methyltransferase (HIOMT) and the levels of melatonin and cAMP were estimated. 7. Suppression of NAT by IFN-gamma was observed in the pineals from INT rats, but not in those from SCGX animals. 8. IFN-gamma significantly enhanced melatonin levels over those in ISO-stimulated pineals and culture media from the SCGX animals, but not from the INT animals. 9. IFN-gamma treatment had no effect on either the HIOMT activity or cAMP levels. 10. The results indicate that the IFN-gamma-induced NAT suppression requires the integrity of the sympathetic nerve terminals and the IFN-gamma-induced enhancement of melatonin production is accomplished through its direct action on pinealocytes.