Pineal Dependence of the Syrian Hamster''s Nocturnal Serum Melatonin Surge

The usual nocturnal surge of pineal melatonin content was blocked by bilateral superior cervical ganglionectomy in male Syrian hamsters. Ganglionectomy and pinealectomy each prevented the nocturnal rise of serum melatonin concentration seen in control animals. The normal nocturnal surge of circulating melatonin in this species appears to depend on the pineal gland and its sympathetic innervation.     

Pharmacological Studies on the Regulation of N-Acetyltransferase Activity and Melatonin Content of the Pineal Gland of the Syrian Hamster

Thus far, all attempts to stimulate melatonin synthesis by beta-adrenergic receptor agonists in the Syrian hamster pineal gland have failed. Neither a wide range of dosages of isoproterenol (0.5 mg/kg to 24 mg/kg), nor prolonged treatment with norepinephrine, the natural neurotransmitter, increased N-acetyltransferase (NAT) activity or melatonin production. In the present study, the administration of isoproterenol at night was likewise ineffective in advancing or enhancing the normal nightly melatonin peak. Also, we did not find a delayed effect 7 or 8 h after the administration of the drug. Furthermore, we tested the idea of coneurotransmitters such as octopamine or dopamine being possibly necessary for stimulation, but could not find any effect of these substances on melatonin synthesis. In addition, a parasympatholytic agent, atropine, did not increase the responsiveness to sympathomimetic agents. Administration of a phosphodiesterase inhibitor was also ineffective in stimulating NAT activity. On the other hand, isoproterenol did retard the drop in NAT and melatonin after lights-on at night, indicating that beta-receptors are involved in maintaining elevated melatonin levels.     

Failure of Prazosin to Mimic the Effects of Melatonin Under In Vivo and In Vitro Conditions

The gonads of male hamsters exposed to short photoperiod (LD, 10:14) or treated with melatonin in the late afternoon under long photoperiod (LD, 14:10) had undergone complete regression by the end of the treatments (8 weeks). Animals treated for the same period of time with prazosin (a putative melatonin analogue) under the same conditions failed to show a difference in their gonadal status as compared to the long photoperiod controls. Prazosin was unable to prevent melatonin-induced gonadal atrophy when injected either in the morning or 30 min before melatonin. Moreover, prazosin was without any effect on (and unable to prevent the melatonin-stimulated) progesterone production by rat adrenals under in vitro conditions. These data demonstrate that prazosin, which reportedly inhibits 2-[125I]iodomelatonin binding in the hamster brain in an affinity-related manner, does not possess properties of a biological melatonin analogue under conditions of two different model systems in two species.     

In Vitro Uptake of Benzodiazepines by Rat Pineal Gland

As a part of a study aiming to characterize the physiological and pharmacological significance of the high affinity pineal benzodiazepine (BZP) binding sites reported previously, we examined the uptake of the BZP derivative 3H-flunitrazepam (FNZP) by rat pineal glands in vitro. At 37 degrees C, 3H-radioactivity was taken up by tissue up to a pineal/medium concentration of about 12, while at 0 degrees C the uptake amounted to only one-third that at 37 degrees C. Reciprocal of uptake analyzed by Lineweaver-Burk plots indicated apparent Km's of 1.74 and 1.45 microM, and Vmax's of 1.32 and 1.04 pmol per min per mg tissue, for control and superior cervical ganglionectomized rats, respectively, suggesting that the neural compartment does not participate significantly in 3H-FNZP uptake. Cerebral cortex explants of similar size and weight as the pineal ones took up 3H-FNZP to a maximum tissue/medium concentration of about 2. Neither pineal nor cerebral cortex 3H-radioactivity uptake exhibited significant changes as a function of time of day. A number of agents, including several BZP analogues, cocaine, desipramine, melatonin, fluoxetine, nomifensine, and dipiridamol, as well as changes in the ionic environment or metabolic inhibitors, did not affect 3H-FNZP uptake significantly. Other tissues, such as liver, muscle, kidney, adrenal gland, or anterior pituitary, took up 3H-radioactivity to tissue concentrations slightly lower than those of the cerebral cortex, suggesting that drug liposolubility accounted only to a limited extent for the high in vitro uptake detected in incubated pineals.     

Immunohistochemical Assessment of Melatonin Binding in the Pineal Gland

Melatonin binding in the pineal gland of albino rats is estimated using an immunohistochemical procedure. Binding is saturable, has relatively high affinity (Apparent KD = 2.7 nM), and competition studies indicate binding of indoleamines possessing an N-acetyl group on the terminus of the side chain (N-acetylserotonin and melatonin). These data are consistent with the interpretation that immunohistochemically determined melatonin in unfixed pineal tissue is assessing binding of N-acetylated indolealkylamines to pineal cell components. In albino rats maintained on 12-hour light: 12-hour dark cycles, melatonin binding exhibits a diurnal rhythm with low levels of saturation (30%) early in the light and saturation by endogenous melatonin near the onset of darkness. An annual rhythm of melatonin binding was observed in albino rats with low levels during the summer and high levels during the winter. Other rats were maintained on 12-hour light:dark cycles and fed for 2 hours either early in the light period or early in the dark period. For both morning- and evening-fed animals, melatonin binding was high prior to feeding and dropped immediately after feeding. Changes in melatonin binding that occur in response to alterations of feeding and time of year suggest the possibility that this binding reflects a functional site for melatonin.     

Role of Postsynaptic α-Adrenergic Receptors in the β -Adrenergic Stimulation of Melatonin Production in the Syrian Hamster Pineal Gland in Organ Culture

The role played by postsynaptic alpha-adrenergic receptors in the stimulation of pineal melatonin production was investigated in the Syrian hamster. The studies were conducted using organ cultured pineal glands collected from both anatomically intact and superior cervical ganglionectomized hamsters. Results obtained indicate that phenylephrine, an alpha-adrenergic agonist, by itself has no effect in promoting melatonin production; however, it potentiates the stimulatory effects of isoproterenol, a beta-adrenergic agonist, on pineal melatonin production in nonoperated hamsters. Similar observations were obtained with pineal glands whose presynaptic terminals were removed by prior superior cervical ganglionectomy. However, a longer incubation time was required (4-6 hours vs. 2 hours) with pineal glands taken from ganglionectomized animals. Apparently, beta-adrenergic activation is an absolute requirement to stimulate pineal melatonin production, and an alpha-adrenergic receptor mechanism potentiates beta-adrenergic activation. In addition, the findings obtained with denervated pineal glands suggest that the regulation of pineal melatonin production by both alpha- and beta-adrenergic mechanisms is through receptors located on postsynaptic structures.     

Secretory Patterns of Pineal Melatonin in the Rat

The release patterns of pineal melatonin were studied by continuously monitoring melatonin levels in the confluens sinuum plasma in sighted and bilaterally enucleated rats in the light and dark periods. Plasma melatonin was determined by radioimmunoassay, and the data were analyzed by a computerized algorithm developed in our laboratory. Pulsations of melatonin levels were found in the confluens sinuum plasma in all the animals studied, suggesting episodic secretion of pineal melatonin in rats. Because the minimum melatonin levels in the confluens sinuum were over three times the melatonin levels in the general circulation, it is postulated that 1) there is an episodic release pattern of pineal melatonin superimposed on a basal release pattern and 2) there are two pools of melatonin in the pineal gland, a readily releasible pool responsible for the basal release and a bound pool responsible for the pulsatile release. In the sighted rats, there was no diurnal difference in mean melatonin concentration, mean pulse amplitude, mean pulse rate, mean minimum melatonin level, and mean maximum melatonin level in the confluens sinuum. In the bilaterally enucleated rats, with the exception of the mean pulse amplitude, diurnal rhythms were demonstrated in all the other parameters studied with, higher values in the dark period. This experimental model should be employed in future investigations on the regulation of secretory patterns of pineal melatonin. Results of these studies may provide important insight into the regulation of pulsatile release of neuroendocrine secretions in general.     

Alloxan-Induced Diabetes and the Pineal Gland: Differential Effects on the Levels of Pineal N-Acetylserotonin, Pineal Melatonin, and Serum Melatonin

Effects of alloxan treatment on the levels of pineal melatonin, pineal N-acetylserotonin, and serum melatonin were investigated. Male rats were housed under a photoperiod of 12 h light: 12 h darkness and a temperature of 23 +/- 3 degrees C. Three weeks after alloxan (170 mg/kg) or carrier injection (s.c.), the animals were killed at mid-light (1200 h) and mid-dark (2400 h). Pineal and serum indoles were extracted and quantified by radioimmunoassays. It was found that pineal levels of N-acetylserotonin in the diabetic rats were significantly higher (P less than 0.05) than those of the controls. Conversely, pineal and serum levels of melatonin in the control rats were significantly higher (P less than 0.05) than those of the alloxan-induced diabetics. Our results suggest that alloxan-induced diabetes may decrease pineal melatonin synthesis in rats by reducing the activity of hydroxyindole-O-methyltransferase, resulting in a decrease in pineal melatonin secretion.     

Melatonin Content of the Pineal Gland in Different Mouse Strains

Pineal melatonin content at several times during the day and night was measured in 36 inbred strains of mice (Mus musculus) kept under LD 12:12 cycles. The results have indicated that only five inbred strains have pineal melatonin content, with higher levels during the night and lower levels during the day; the other 31 strains do not contain detectable melatonin in their pineal gland at any of times examined. The former group includes two commonly used strains (C3H/He and CBA/Ms) and three wild-derived strains (Mol-A, Mol-Nis, MOM). C3H and CBA mice showed a similar pattern of pineal melatonin rhythm with a peak at 2 hours before lights on. The peak levels were about 150 pg/gland in both strains. The rhythmic patterns of melatonin content in Mol-A, Mol-Nis, and MOM were slightly different from those in CBA and C3H. In the wild-derived strains, the peak of melatonin content did not occur at 2 hours before lights on but tended to occur at midnight. The peak levels were 67-91 pg/gland at the highest point in these strains.     

Production of Methoxyindoles In Vitro From Methoxytryptophan by Rat Pineal Gland

Pineal glands were incubated in the presence of [3H] methoxytryptophan with and without methoxamine, epinephrine, and norepinephrine. The beta-adrenoceptor-stimulated pineal glands were capable of converting methoxytryptophan to methoxytryptamine, melatonin, methoxyindole acetic acid, and methoxytryptophol, albeit in small quantities. Only methoxyindole acetic acid was detectable after incubation of unstimulated and alpha-adrenergic-agonist-treated pineal glands. These results support the proposal that melatonin can be formed from methoxytryptophan although this is a minor synthetic pathway, and the classic pathway from serotonin via N-acetylserotonin should be considered to be responsible for the majority of pineal melatonin production.     

Effect of Selective Monoamine Oxidase Inhibitors on Rat Pineal Melatonin Synthesis In Vitro

Freshly cultured pineal glands respond to the monoamine oxidase A (MAO A) inhibitor clorgyline and to high concentrations of the MAO B inhibitor, deprenyl, with an increase in serotonin N-acetyltransferase activity and N-acetylated indoles and a fall in 5-hydroxylated serotonin degradation products. Glands cultured for 48 hours before challenge respond less. Response is absent in glands cultured for 72 hours and in glands from ganglionectomized animals cultured for 48 hours before challenge. These data are consistent with the hypothesis that these MAO inhibitors stimulate melatonin synthesis by protecting norepinephrine from degradation.     

Antigonadal Activity of the Melatonin Analogs 2-Iodomelatonin and 2-Chloromelatonin in the Juvenile Djungarian Hamster, Phodopus sungorus campbetti

The antigonadal effects of daily (20 micrograms, s.c.) injection of melatonin and two analogs, 2-iodomelatonin and 2-chloromelatonin, were compared in juvenile Djungarian hamsters housed under long photoperiod (L:D 16:8). Melatonin, 2-iodomelatonin, and 2-chloromelatonin injected 3 h before lights off for 16 days (17-34 days of age) significantly inhibited testis growth compared to vehicle-injected hamsters. In addition, melatonin and both analogs significantly reduced body weight gain. These 2-substituted analogs appear to be melatonin agonists with a potency in vivo similar to the parent compound, melatonin.     

The Influence of Various Irradiances of Artificial Light, Twilight, and Moonlight on the Suppression of Pineal Melatonin Content in the Syrian Hamster

The purpose of the present studies using artificial light was to determine how the timing and duration of exposure influence the light-induced suppression of pineal melatonin levels in hamsters. An 8-min exposure to 0.186 microW/cm2 of cool white fluorescent light caused a continued depression of pineal melatonin even when animals were returned to darkness. In addition, the pineal gland does not appear to change its sensitivity to light throughout the night. A 20-min exposure to 0.019 microW/cm2 of cool white fluorescent light did not significantly suppress pineal melatonin during any time of the melatonin peak, whereas a 20-min exposure to 0.186 microW/cm2 was capable of always suppressing melatonin. Furthermore, increasing the duration of 0.019-microW/cm2 exposure to 30, 60, 120, or 180 min does not increase the capacity of this irradiance to depress melatonin. Similar to artifical light, natural light has a variable capacity for suppressing nocturnal levels of pineal melatonin. Twilight irradiances of 0.138 microW/cm2 or less did not suppress nocturnal melatonin whereas twilight irradiances of 3.0 microW/cm2 or greater did suppress pineal melatonin. A few animals did have lower melatonin after a 40-min exposure to full moonlight during July (0.045 microW/cm2) or January (0.240 microW/cm2). However, pineal melatonin levels remained high in the majority of animals exposed to full moonlight.     

Alpha-1 Adrenoceptor Involvement in the Control of Melatonin Secretion in the Golden Hamster

The nocturnal peak in pineal and serum melatonin content was reduced following administration of the selective alpha-1 adrenoceptor antagonist prazosin in a dose of 0.25 mg/kg. The effect was pronounced one hour post treatment during the late dark phase of the daily photocycle. These data confirm the reported findings that the hamster pineal sensitivity to adrenergic challenge is confined to the second part of the dark phase and indicate that postsynaptically located alpha-1 adrenoceptors are also involved in the physiological control of melatonin synthesis and/or release in that species.     

Temporal Effects of Norepinephrine on Pineal Serotonin In Vitro

The purpose of this study was to examine the temporal effects of norepinephrine (NE) stimulation on pineal serotonin (5HT) in vitro. Rat pineal glands were individually incubated in nutrient media for 6 h. After a 2-h preincubation, the glands were exposed to tryptophan (TRYP) or TRYP + norepinephrine (10(-4)M each) for 4 additional h. Media and pineals were then analyzed for 5HT and 5HIAA content. Initial exposure to TRYP increased 5HT levels in the media. Norepinephrine increased media 5HT concentrations further and also caused 5HIAA levels to rise significantly. These findings suggest that 5HT secretion and/or oxidation is an early response to stimulation of the pineal gland by NE. Following prolonged NE exposure, media 5HT declined to control values while 5HIAA remained high. Similarly, intrapineal levels of 5HT fell after prolonged stimulation with NE. Thus, pineal responses to initial and continued stimulation with NE are differentiable as early and late effects upon 5HT. Early effects may include secretion and/or oxidation of 5HT followed later by utilization of 5HT in N-acetylation pathways such as that leading to melatonin synthesis. The finding that 5HT is secreted in response to NE suggests that the indoleamine may be a hormone of the pineal gland. Based upon its known influence on physiologic rhythms, daily secretion of 5HT from the pineal may be an important part of the gland's time-keeping function.     

"Synaptic" Ribbons and Spherules Lacking in the Pineal Gland of the European Hamster Appear After Ganglionectomy

"Synaptic" ribbons and spherules are typical organelles of the mammalian pinealocytes, that present quantitative day-night variations parallel to pineal melatonin levels. The European hamster is a wild photoperiodic mammal whose pineal melatonin concentrations do not vary during the 24 hr light-dark cycle. The aim of this study was thus to see if a rhythmic variation in synaptic structures was nevertheless present in this species. It was found that, in contrast to all of the photoperiodic species studied to date, the European hamster pinealocyte contains no ribbons, and virtually no spherules. Moreover, constant administration of melatonin does not result in the occurrence of these organelles; nevertheless, the pinealocytes have the potentiality to produce spherules and, to a lesser extent, ribbons, as they are observed in ganglionectomized animals and in the deep pineal of some hamsters after ablation of the superficial part of the gland. The formation of pineal synaptic structures, normally lacking in the European hamster, thus seems directly related to the noradrenergic innervation of the gland.     

Rhythmic Incorporation of Thymidine by Chick Pineal Glands In Vitro

Cultured chick pineal glands showed a cycle in the cumulative incorporation of thymidine into DNA. In undisturbed cultures the rate of thymidine incorporation, amount of thymidine incorporated per 1-day cycle, and persistence of the incorporation process were all markedly affected by the concentration of exogenous precursor. However, more than two full cycles of incorporation were found when culture medium of low thymidine content was renewed daily, or when medium of intermediate concentration was replaced on the 3rd day of culture. At a high thymidine concentration the second cycle of incorporation sometimes appeared to be impaired. At all concentrations tested, less than 2% of the available thymidine was incorporated.     

Melatonin Inhibits Mitotic Activity of Adrenocortical Cells In Vivo and in Organ Culture

The goal of this study was to examine the effects of melatonin, as well as those of melatonin and corticotropin (1-24 adrenocorticotrophic hormone (ACTH); Synacthen Depot) administered together, on the mitotic activity of adrenocortical cells in male and female mice. Melatonin was given subcutaneously once daily, in late-afternoon injections (between 16:00 and 18:00) in doses of 1 microgram, 10 micrograms, and 100 micrograms, and ACTH in a dose of 0.1 mg (10 U) daily for 10 days. Additionally, the highest dose of melatonin (100 micrograms daily) was administered together with ACTH. The metaphase-arrest technique using colchicine as a stathmokinetic agent was employed in the study. Melatonin, in all the examined doses, significantly decreased mean mitotic activity rate (MMAR) of the adrenal cortex in both male and female mice. Moreover, in a dose of 100 micrograms, melatonin suppressed the mitogenic effect of ACTH on the adrenal cortex. Furthermore, the present study investigated the effects of melatonin (5 x 10(-7)M), N-acetylserotonin (NAc-5HT) (5 x 10(-7)M), and ACTH (250 mU/ml or 1,000 mU/ml) alone as well as the effect of ACTH (250 mU/ml) applied jointly with melatonin on the mitotic activity of adrenocortical cells in rat adrenal explants incubated in vitro. Both pineal indoleamines (melatonin and NAc-5HT) significantly decreased the MMARs of adrenocortical cells. Corticotropin, as well as ACTH and melatonin applied together, also reduced the MMAR of adrenocortical cells. The present data suggest that melatonin may be directly involved in the inhibitory control of adrenocortical cell proliferation.     

Daily variation in the content of indoleamines, catecholamines and related compounds in the pineal gland of Syrian hamsters kept under long and short photoperiods

Abstract: This study examined the diurnal changes in the content of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), N-acetylserotonin (NAS), melatonin, 5-methoxytryptophol (5-ML), noradrenaline (NA), dopamine (DA), and 3,4-dihydroxyphenylacetic acid (DOPAC) in the pineal gland of Syrian hamsters kept under long (14L: 10D) and short (10L: 14D) photoperiods. The nocturnal increase in NAS and melatonin levels was dependent upon the prevailing photoperiod, with a prolonged duration when the night lengthened. In both photoperiods, NAS and melatonin contents increased several hours after the onset of darkness, and, in animals kept in short photoperiod, the levels of both compounds began to decrease before light onset. On the contrary, decreases were noted in 5-HT, 5-HIAA, and 5-ML contents during the night, which was directly proportional to the dark phase. 5-HTP levels did not show a rhythmic variation. Correlations between the mean values of 5-HT-related compounds showing daily rhythms were very high when group means were compared, but they decreased when values from individual animals were considered. In addition, when correlations were calculated on per-animal basis during the night phase, a weak negative correlation was found for 5-HT vs NAS and 5-HT vs melatonin, although the correlation of 5-HT with positively 5-HT-correlated compounds (5-HIAA and 5-ML) continued to be high. These results indicate that the nocturnal increase in the N-acetyl transferase activity is the major factor generating the rhythm of pineal 5-HT content, but that other photoperiod-dependent mechanisms (i.e., 5-HT synthesis or release) seem to be also implicated. On the other hand, this study shows that NA content in the Syrian hamster pineal gland does not exhibit daily variations, although marked nocturnal increases in the levels of DA and DOPAC were evident. These results suggest the existence of parallel daily alterations in pineal catecholamine synthesis and release, and suggest a role for DA in the pineal activation at night.     

Photoperiodic Control of Testicular Size in the Golden Hamster: Lack of a Role for the Deep Pineal Gland

Removal of the superficial pineal gland prevents testicular regression from occurring in golden hamsters exposed to a short-day photoperiod, but to date no studies have examined the role of the deep pineal gland in the photoperiodic control of gonadal function. We investigated the role of the deep pineal gland in this process by monitoring testicular size in hamsters with an electrolytic lesion of the deep pineal gland. Sexually mature hamsters that had been sham-lesioned or that had received a lesion aimed at the deep pineal gland were transferred from 14L:10D to 6L:18D or were maintained in 14L:10D for 73 d. Testicular widths were determined at 2-3-wk intervals and after 73 d the testes were removed and weighed. The mean testis widths of the seven animals with histologically confirmed deep pineal lesions and exposed to 14L:10D remained large, similar to those of the sham-operated control animals exposed to the same photoperiod. The testes regressed in the five animals with confirmed deep pineal lesions and exposed to 6L:18D in a similar manner to those of the sham-operated control animals exposed to the same photoperiod. No significant difference (P greater than .05) was found between the testicular weights of lesioned and unlesioned animals exposed to either photoperiod. The lack of a significant effect of a deep pineal lesion on testicular size demonstrates that this portion of the pineal complex is not required for normal regulation of testicular function by photoperiod. These results also suggest that nerve fibres projecting from the brain to the superficial pineal gland via the deep pineal gland are not involved in photoperiodic regulation of testicular function.