Conditions for the use of specific antibodies for immunohistochemical visualization of serotonin and melatonin in the pineal gland of sheep

Having prepared antisera to serotonin and to melatonin, the authors were able to show that, in the pineal gland, the behaviour of these two antisera in immunohistochemical studies differs. The antiserum raised against 5HT, actually bound to the molecule formed by condensation of formaldehyde on 5HT and therefore could be used to reveal 5HT in tissue fixed with formaldehyde. On the other hand, the anti-melatonin antiserum bound to melatonin, and could therefore be used to reveal its presence in fresh tissue.     

Radioimmunoassay for melatonin and its application to fowl pineal research

A radioimmunoassay for melatonin has been developed after raising anti-melatonin antibodies in rabbit. Melatonin was extracted from serum or pineal gland of chickens (Gallus domesticus). The radioimmunoassay was performed by using 3H-melatonin as tracer. The standard curve covered the range 0.022-0.345 pmol/vial and the KD value for melatonin was estimated at 1.37 x 10(10) l/mol. The antiserum specificity has been analysed, none of the common melatonin analogues influencing this method of melatonin measurement. The intra-assay variability was 7.2% for serum samples and 8.6% for pineal extract. The inter-assay variability for this biological sample was 15.3% and 6.4% respectively.     

Melatonin Synthesis in the Bovine Pineal Gland Is Regulated by Type II Cyclic AMP-Dependent Protein Kinase

Abstract: We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RIα/RIIβ and high levels of Cα and Cβ mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both Cα and Cβ catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.     

Decreases in pineal melatonin content during the hibernation bout in the golden-mantled group squirrel, Spermophilus lateralis

The role of the pineal gland in modulating the rhythmic bouts of hibernation in the golden-mantled ground squirrel (S. lateralis) was explored by comparing pineal melatonin content in hibernating animals with that of euthermic animals at the same time of year. Significant decreases in pineal melatonin content were found in hibernating versus euthermic animals. In addition, significantly lower values for pineal melatonin were observed in hibernating animals that were sacrificed in the late bout period, just prior to expected spontaneous arousal, as compared to hibernating animals that were sacrificed on the first day of their respective bouts. A strong correlation was evident between pineal melatonin content and the duration of the individual hibernation bout. These data suggest that pineal melatonin may be important in determining the duration of individual bouts of hibernation in this species.     

The effect of different wavelengths of light during incubation on the development of rhythmic pineal melatonin biosynthesis in chick embryos

Rhythmic pineal melatonin biosynthesis develops in chick embryos incubated under a light (L)-dark (D) cycle of polychromatic white light. The spectral sensitivity of the embryonic pineal gland is not known and was investigated in this study. Broiler breeder eggs (Ross 308, n=450) were incubated under white, red, green or blue light under the 12L : 12D cycle. Melatonin was measured in extracts of pineal glands by radioimmunoassay. The daily rhythm of pineal melatonin levels in 20-day-old chick embryos was confirmed during the final stages of embryonic life under all four wavelengths of light with expected higher concentrations during dark- than light-times. The highest pineal melatonin levels were determined in chick embryos incubated under red and white light and lower levels under green light. The incubation under blue light resulted in the lowest melatonin biosynthesis. Pineal melatonin concentrations increased substantially on post-hatching day two compared with pre-hatching levels and we did not find differences between birds incubated and kept in either white or green light. Our results demonstrate a selective sensitivity of the chick embryo pineal gland to different wavelengths of light. Rhythmic melatonin production is suggested as a possible mechanism, which transfers information about the quality of ambient light to the developing avian embryo.     

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.     

The profile of melatonin production in tumour-bearing rats

The pineal gland is involved in the regulation of tumour growth through the anticancer activity of melatonin, which presents immunomodulatory, anti-proliferative and anti-oxidant effects. In this study we measured melatonin content directly in the pineal gland, in an attempt to clarify the modulation of pineal melatonin secretory activity during tumour growth. Different groups of Walker 256 carcinosarcoma bearing rats were sacrificed at 12 different time points during 24h (12h:12h light/dark cycle) on different days during the tumour development (on the first, seventh and fourteenth day after tumour inoculation). Melatonin content in the pineal gland was determined by high-performance liquid chromatography with electrochemical detection. During tumour development the amount of melatonin secreted increased from 310.9 ng/mg of protein per day from control animals, to 918.1 ng/mg of protein per day 14 days after tumour implantation, and there were changes in the pineal production profile of melatonin. Cultured pineal glands obtained from tumour-bearing rats turned out to be less responsive to noradrenaline, suggesting the existence, in vivo, of putative factor(s) modulating pineal melatonin production. The results demonstrated that during tumour development there is a modification of pineal melatonin production daily profile, possibly contributing to cachexia, associated to changes in pineal gland response to noradrenaline stimulation.     

Inconsistent suppression of nocturnal pineal melatonin synthesis and serum melatonin levels in rats exposed to pulsed DC magnetic fields

The purpose of these experiments was to determine whether the exposure of rats at night to pulsed DC magnetic fields (MF) would influence the nocturnal production and secretion of melatonin, as indicated by pineal N-acetyltransferase (NAT) activity (the rate limiting enzyme in melatonin production) and pineal and serum melatonin levels. By using a computer-driven exposure system, 15 experiments were conducted. MF exposure onset was always during the night, with the duration of exposure varying from 15 to 120 min. A variety of field strengths, ranging from 50 to 500 μT (0.5 to 5.0 G) were used with the bulk of the studies being conducted using a 100 μT (1.0 G) field. During the interval of DC MF exposure, the field was turned on and off at 1-s intervals with a rise/fall time constant of 5 ms. Because the studies were performed during the night, all procedures were carried out under weak red light (intensity of <5 μW/cm²). At the conclusion of each study, a blood sample and the pineal gland were collected for analysis of serum melatonin titers and pineal NAT and melatonin levels. The outcome of individual studies varied. Of the 23 cases in which pineal NAT activity, pineal melatonin, and serum melatonin levels were measured, the following results were obtained; in 5 cases (21.7%) pineal NAT activity was depressed, in 2 cases (8.7%) studies pineal melatonin levels were lowered, and in 10 cases (43.5%) serum melatonin concentrations were reduced. Never was there a measured rise in any of the end points that were considered in this study. The magnitudes of the reductions were not correlated with field strength (i.e., no dose-response relationships were apparent), and likewise the reductions could not be correlated with the season of the year (experiments conducted at 12-month intervals under identical exposure conditions yielded different results). Duration of exposure also seemed not to be a factor in the degree of melatonin suppression. The inconsistency of the results does not permit the conclusion that pineal melatonin production or release are routinely influenced by pulsed DC MF exposure. In the current series of studies, a suppression of serum melatonin sometimes occurred in the absence of any apparent change in the synthesis of this indoleamine within the pineal gland (no alteration in either pineal NAT activity or pineal melatonin levels). Because melatonin is a direct free radical scavenger, the drop in serum melatonin could theoretically be explained by an increased uptake of melatonin by tissues that were experiencing augmented levels of free radicals as a consequence of MF exposure. This hypothetical possibly requires additional experimental documentation. Bioelectromagnetics 19:318–329, 1998. © 1998 Wiley-Liss, Inc.     

Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.     

Differential effects of isoproterenol injections on the levels of pineal N-acetyltransferase, serum N-acetylserotonin and melatonin

Rats housed under diurnal lighting conditions were either injected with isoproterenol (ISO), 0.5 mg/kg subcutaneous (SC) and sacrificed at different times up to 180 minutes afterwards, or injected with different doses of ISO (0.2 mg/kg to 5.0 mg/kg intraperitoneally (IP] and sacrificed 120 minutes later. Pineal N-acetyltransferase (NATase), serum N-acetylserotonin (NAS) and serum melatonin (MT) levels were determined. It was found that both pineal NATase and serum MT responded to the injection with peak increase at 120 minutes after the injection. This increase in pineal NATase and serum MT levels were also found to be dose-dependent. It was also observed that at 30 minutes after ISO injection, the serum MT level already demonstrated a significant increase which preceeded any increase in the pineal NATase activity. The underlying mechanism for this observation remains undetermined. Unlike serum MT and pineal NATase, there were no changes in serum NAS levels after injections of ISO at all the doses tested or up to 180 minutes after injection of the drug at 0.5 mg/kg dose SC. This suggests that serum NAS level is neither regulated by pineal NATase activity nor is the pineal gland the major source of NAS in circulation. This also indicates that serum NAS level is not influenced by beta-adrenergic stimulation.     

Detection of specific protein bands with melatonin-like immunoreactivity in different cell lines and human brain regions

The pineal hormone melatonin regulates various neural and endocrine processes involved in mammalian circadian rhythms. To understand how melatonin mediates these functions, we investigated melatonin-like immunoreactivity (MLI) in cell extracts and human brain. In Western immunoblots, we detected high-molecular-mass protein bands (85-135 kDa) that specifically reacted with the anti-melatonin antibody. The specific protein bands were present in cell extracts from the human brain and cell lines of different origins. The immunoreactive signal of the 135-kDa protein band was highest in a neuroendocrine PC12 cell line, which was 10-fold higher than the signal observed in any cell extracts used. The commercial antibody employed in the Western blots was further purified against serum proteins and thyroglobulins. We have previously reported that the antibody against melatonin recognizes MLI as detected by a sensitive RIA. In the present report we have detected the putative melatonin-specific binding proteins, which could contribute to the MLI. Our results suggest that melatonin binds with specific proteins in different cellular and brain extracts, the protein(s) being maximally synthesized in PC12 cells. These results may indicate a group of yet unknown proteins sharing a melatonin-like epitope or the presence of melatonin-binding protein(s) that regulate availability of free melatonin, or both.     

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.     

Melatonin radioimmunoanalysis: evaluation of the pineal function in hyperprolactinemic male rats and controls

A sensitive and specific radioimmunoassay for melatonin quantification in rat pineal and biological fluids is described. The assay utilizes a specific antibody and H3-melatonin as tracer. Bound and free fraction were separated by a saturated sulphate ammonium solution. The sensitivity of the method is 9 pg/ml. The intra and interassay variation coefficient were 10.4 and 13.6% respectively. By means of this RIA the content of melatonin in the pineal gland in male rats made hyperprolactinemic on day 30 of life and their respective sham-operated controls has been evaluated. The results showed that the melatonin content measured at 2 a.m. was reduced in the transplanted animals when compared to control group, not only shortly (48 hours) after the transplant operation, but also in the chronic situation; though suggesting that further investigations are necessary to deepen and understand the interrelationships between prolactin and pineal gland and their effect on the hypothalamic-pituitary-gonadal axis.     

Immunohistochemical evidence for the presence of melatonin in the pineal gland,the retina and the Harderian gland

Summary The presence of melatonin is demonstrated in the pineal gland, the retina and the Harderian gland in some mammalian and non-mammalian vertebrates, using a specific fluorescence labelled antibody technique. Four different potent antibodies against melatonin have been used and compared. In the pineal gland of hamsters, mice, rats and snakes, specific fluorescence, mostly restricted to the cytoplasm of the cells, is detected in pinealocytes. Fluorescence is also detected in the pineal organ of fishes, tortoises and lizards, but it has not been possible, from cryostat sections of fresh tissue, to assert which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells). In the retina, fluorescence is almost exclusively restricted to the outer nuclear layer. In the Harderian gland of mammals and reptiles, fluorescence is localized in the secretory cells of the alveoli and mostly restricted to the cytoplasm surrounding the nucleus. These results are discussed in relation to the concept of melatonin synthesis at extrapineal sites independent of pineal production.Parts of this work have been presented in the Xth Conference of Comparative Endocrinologists, Sorrento, May 20–25, 1979 (Vivien-Roels and Dubois 1980) and the VIth International Congress of Endocrinology, Melbourne, February 10–16, 1980 (Vivien-Roels et al. 1980)The author wishes to thank Professor Lutz Vollrath who has accepted her in his laboratory for a short period, Doctor George M. Bubenik for his suggestions and critical remarks, Dr. L.J. Grota for producing the melatonin diazobenzoic acid-BSA and Dr. Castro for preparing one of the melatonin derivates     

Effect of melatonin and pineal extracts on human ovarian and mammary tumor cells in a chemosensitivity assay

Pinealectomy enhances tumor growth and metastatic spread in experimental animals. This effect is only in part due to melatonin since melatonin-free pineal extracts containing yet unidentified pineal substances have also shown tumor inhibiting activity. Despite numerous reports suggesting melatonin as a potential anti-cancer agent there have not been sufficient clinical trials to define the actual therapeutic potential of melatonin for the treatment of human cancers. To help fill this gap, we used a chemosensitivity assay designed to test the sensitivity of tumors from individual patients towards chemotherapeutic drugs for assessing the effect of melatonin and pineal extracts on primary human tumor cells. Primary cell cultures from seven ovarian and six mammary tumors were incubated with melatonin, the pineal extract YC05R (containing substances between 500 and 1000 daltons) and chemotherapeutic drugs. The pineal extract YC05R inhibited growth of all tumors in a dose-dependent manner. Physiological concentrations of melatonin (10(-8)-10(-10) M) inhibited the growth of one out of six mammary carcinomas in a dose-dependent manner. Primary cell cultures from three ovarian tumors were affected by melatonin in different ways, i.e., two were inhibited and one was slightly stimulated. There was no correlation between sensitivity towards melatonin and sex steroid receptor status, stage or grade of the tumor. It is concluded that, 1), melatonin may be an inhibitor of human mammary and ovarian carcinoma in individual cases and, 2), the pineal gland contains very active anti-tumor substances inhibiting both, the mammary and ovarian tumors, tested. These substances require chemical and biological identification.     

Thoughts on the causes of seasonality of menarche

The pineal gland plays an important role in the production of melatonin and in the synchronization of the reproduction process in seasonal breeding animals. Changes in the duration of day length are the most important stimulatory factors. In humans the pineal gland may not only have a physiological role in the seasonality of maturation, but also concerning the simultaneously observable changes of serum hormone and serum melatonin levels as well as in regard of changes in hair colour in prepuberal children. Therefore the pineal gland seems to play an important physiological role in the timing of human maturation.     

Melatonin rhythms in the pineal and non-pineal tissues and their physiological implications in subtropical fish

Abstract

Melatonin (N-acetyl-5-methoxy tryptamine), following discovery from the extracts of bovine pineal gland, has been detected in the pineal as well as several extra-pineal tissues/organs of different vertebrates including fish. The unique feature of melatonin in the pineal gland is its rhythmic biosynthesis and release in blood in synchronization with the environmental light-–dark cycle. Accordingly, melatonin produced in the pineal of an animal living in a changing environment is implicated to the regulation of seasonal reproduction by acting as a hormone at one or more levels of hypothalamo-hypophyseal-gonadal axis. Additionally, melatonin is known to act as a potent free-radical scavenger or antioxidant to influence maturation of oocytes. However, possible relationship between extra-pineal melatonin and seasonality of reproduction in any animal remains enigmatic. Perhaps, carp is the only known animal in which temporal patterns of melatonin levels in the serum as well as in the extracts of pineal, retina, ovary, gut, and liver have been studied in relation to the reproductive events in an annual cycle. The purpose of current review is to bring those fascinating, and arguably most important data together to underline their significance in the control of seasonal reproduction in subtropical fish in general and in carp in particular.     

Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Spermophilus richardsonii)

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.