Showing papers in "Journal of Pineal Research in 1994"

High melatonin solubility in aqueous medium.

Abstract: The pineal hormone melatonin (5-methoxy-N-acetyl-tryptamine) has been reported to participate in important physiological processes. Although some of its biological actions seem to depend on a protein receptor at the membrane surface, melatonin is known to interact with a large variety of tissues and cells, suggesting that the molecule may not necessarily interact through a specific membrane receptor at a specific cell. Most discussions of melatonin activity have assumed that the molecule is highly hydrophobic. Contrary to belief, the present work shows that melatonin is soluble in a purely aqueous medium up to 5 x 10(-3) M and describes a new method of melatonin preparation which shows the high hydrophilicity of the molecule. The results presented will affect the current biological hypothesis on the need of a melatonin carrier in the blood stream or the mechanisms which allow the hormone to cross the cell membrane and interact at the level of the nucleus.

Characterization of high‐affinity melatonin binding sites in purified cell nuclei of rat liver

Abstract: Acuna-Costroviejo D, Reiter RJ, Menendez-Pelaez A, Pablos MI, Burgos A. Characterization of high-affinity melatonin binding sites in purified cell nuclei of rat liver. J. Pineal Res. 1994: 16: 100–112. Abstract High-affinity 2-125I-iodomelatonin binding sites in homogenates of purified cell nuclei from rat liver were localized and characterized using biochemical binding techniques. Binding at these sites was found to be rapid, reversible, saturable, and to demonstrate pharmacological selectivity. At 0°C, binding reached equilibrium in about 10 min. Scatchard analysis of the data at equilibrium revealed a single class of binding sites with a dissociation constant of KD= 190 ± 47 pM, Bmax= 9. 8 ± 0. 6 fmol/mg protein, and a Hill coefficient of nH= 1. 02 ± 0. 034. Kinetic analysis of the association and dissociation curves indicated a kinetic KD= 148 ± 41 pM, which is in good agreement with the value obtained at equilibrium. The specific binding of 2-125I-iodomelatonin (45 pM) (0. 51 ± 0. 04 fmol/mg protein) was significantly improved (0. 79 ± 0. 04 fmol/mg protein) when the homogenates of purified liver cell nuclei were reinsulated with DNase (2 μg/ml at 37°C for 20 min) before being used in binding experiments. After the addition of either proteinase K or trichloroacetic acid to DNase-treated purified cell nuclear homogenates, the specific binding disappeared. This suggests that the specific binding of 2-125I-iodomelatonin in liver cell nuclei is associated with nuclear protein. Competition experiments show that N-acetyl-serotonin (Ki= 81. 3 nM) was more potent than 5-hydroxytryptamine (Ki > 1 μM) and 5-methoxytryptamine (Ki≫ 10 μM) in inhibiting 2-125I-iodomelatonin binding (Ki melatonin = 146 pM). These data indicate that specific 2-125I-iodomelatonin binding sites exist in the cell nuclei of rat liver, and that they may comprise a locus for the intracellular action of melatonin. The correlation between the KD and Bmax values with melatonin concentrations in nuclei suggest that these binding sites may be a physiological melatonin receptor, which could explain the described genomic effects of the pineal hormone.

Inhibitory effect of melatonin on cataract formation in newborn rats: Evidence for an antioxidative role for melatonin

Abstract: We evaluated the inhibitory effect of melatonin, a recently discovered scavenger of free radicals, on cataract formation in the newborn rat. The glutathione synthesis inhibitor, buthionine sulfoximine (BSO) (3 mmol/kg), was intraperitoneally injected into newborn rats for 3 consecutive days starting on day 2 after birth. These glutathione depleted rats develop cataracts. Melatonin (4 mg/kg) was injected intraperitoneally into half of the rats once a day beginning at day 2 after birth; the other half of the animals received solvent daily. The incidence of cataract was observed on day 16, after the eyes of the newborn animals had opened. Both reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were measured. Cataracts were observed in all animals (18/18) treated with BSO plus solvent. The incidence of the cataract in the animals cotreated with melatonin was only 6.2% (1/15). Total lenticular glutathione (GSH + GSSG) levels in BSO only treated rats were reduced by 97%. The total glutathione in the lens of the BSO plus melatonin group was significantly higher (by 3%) than that of the BSO only group. The percentage of the total glutathione as GSSG for the BSO plus solvent group was higher than the control value. Cotreatment of BSO injected rats with melatonin (4 mg/kg/day) clearly reduced cataract formation proving that it is directly or indirectly protective against oxidative stress which accompanies glutathione deficiency. The inhibitory effects of melatonin on cataract formation in this study could be due to melatonin's free radical scavenging activity or due to its stimulatory effect on glutathione production.

Effects of melatonin on microtubule assembly depend on hormone concentration: Role of melatonin as a calmodulin antagonist

Abstract: Huerto-Delgadillo L, Anton-Tay F, Benitez-King G. Effects of melatonin on microtubule assembly depend on hormone concentration: rate of melatonin as a calmodulin antagonist. J. Pineal Res. 1994: 17: 55–62. ©Munksgaard, 1994 Abstract Melatonin may play a key role in cytoskeletal rearrangements through its calmodulin antagonism. In the present work, we tested this hypothesis by studying melatonin effects on both microtubule polymerization in vitro and cytoskeletons in situ. Microtubule assembly is a dynamic process inhibited by Ca2+/calmodulin. Calmodulin antagonists prevent the inhibition by binding to Ca2+-activated calmodulin, thus causing microtubule enlargement. In the presence of calmodulin (5 μM) and CaCl2 (1 mM), polymerization at equilibrium was inhibited by 40%. Complete reversal of the Ca2+/calmodulin effect on microtubules was observed with 10-9 M melatonin or with 10-5 M trifluoperazine or 1 μg/ml of compound 48/80. In the absence of Ca2+/calmodulin, melatonin at 10-5 M inhibited tubulin polymerization like 10-4 M trifluoperazine does. Melatonin effects on microtubule assembly at both nanomolar and micromolar ranges were corroborated in cytoskeletons in situ. Therefore, it is suggested that at a low concentration (10-9 M), cytoskeletal melatonin effects are mediated by its antagonism to Ca2+/calmodulin. At a higher concentration (10-5 M), non-specific binding of melatonin to tubulin occurs, thus overcoming the melatonin antgonism to Ca2+/calmodulin. The results support the hypothesis that under physiological conditions, melatonin synchronizes different body rhythms through cytoskeletal rearrangements mediated by its calmodulin antagonism.

Acute 60 Hz magnetic field exposure effects on the melatonin rhythm in the pineal gland and circulation of the adult Djungarian hamster.

Abstract: Yellon SM. Acute 60Hz magnetic field exposure effects on the nucleation rhythm in the pineal gland and circulation of the adult Djungarian hamster. J. Pineal Res. 1994; 16: 136–144. Abstract Adult male and female hamsters in long days (16 hr of light) were exposed to a 1 gauss 60 Hz magnetic field for 15 min starting 2 hr before lights off. Sham-exposed controls were placed in an adjacent exposure system but current was not applied. Hamsters were decapitated at 0.5–2 hr intervals from 1 hr before lights off to 1 hr after lights on (n = 4–6/clocktime/group); sera were harvested and pineal glands obtained for melatonin radioimmunoassay. In controls, pineal melatonin significantly increased from an average daytime baseline of less than 0.3 ng/gland to 3 ng/gland by 3 hr after lights off (P < 0.05, ANOVA). This increase was sustained for the duration of the night and returned to baseline within 1 hr after lights on. A similar melatonin rhythm was found in serum; concentrations ranged from 30 to 50 pg/ml at night and returned to a baseline of 12 pg/ml or less by 1 hr before lights on. The single magnetic field exposure reduced the duration and blunted the rise in the nocturnal melatonin rhythm. The study was then repeated in its entirety 6 months later. The same magnetic field treatment significantly suppressed pineal melatonin content at 5 hr after lights off and reduced serum melatonin concentrations at 3 and 5 hr after dark onset compared to sham-exposed controls. Thus, the acute magnetic field exposure was again found to blunt the increase and suppress the duration of the nighttime melatonin rise. Point-by-point comparisons with the first study, however, did not replicate the magnetic field-associated reduction in pineal melatonin content at 3 hr into the night, as well as in pineal and serum melatonin at 7. 5 hr after lights off. Concern about this divergence led us to repeat the experiment for a second time 6 months later. In both sham- and magnetic field-exposed groups, melatonin increased within 3 hr after lights off and this rise was sustained until 0. 5 hr before lights on; nighttime melatonin content in the pineal gland was approximately 2 ng while in circulation melatonin concentrations averaged 60 pg/ml or less. No statistical differences were evident between the control and magnetic field exposed hamsters at any clocktime (P < 0. 05, ANOVA). Thus the absence of an effect of magnetic field exposure on the melatonin rhythm in either the pineal gland or circulation in this second replicate study contrasts with the clear suppression of the nocturnal melatonin rhythm in two previous experiments. Further work is needed to define the parameters of magnetic field exposure that consistently affect the pineal gland and its circadian melatonin rhythm. The time of the year for experimentation, animal age, or the endogenous response to exposure may be variables that might be understood before the physiological importance of magnetic fields for circadian time keeping mechanisms may be realized.

The role of serotonin and melatonin in gastrointestinal physiology: Ontogeny, regulation of food intake, and mutual serotonin‐melatonin feedback

Abstract: Bubenik GA, Pang SF. The role of serotonin and megaton in gastrointestinal physiology: Ontogeny, regulation of food intake, and mutual serotonin-melatonin feedback. J. Pineal Res. 1994: 16: 91–99. Abstract Average levels of melatonin in the brain and the gastrointestinal (GIT) tissues of newborn mice declined dramatically during the first week postnatally. Food consumption increased considerably in mice bearing subcutaneous serotonin (5-HT) implants (2 mg). Melatonin implants (2 mg) also increased overall consumption but to a lesser degree. Both 5-HT and melatonin implants (2 mg) increased water content of mice fecal pellets, albeit the melatonin effect was less pronounced. Serotonin implants (2,4,6 mg/mouse) increased melatonin levels in brain, jejunum, ileum, and colon, but the effect was not dose-dependent. Intraperitoneally administered melatonin (5, 20 and 200 ug/mouse) elevated melatonin levels in brain and GIT tissues more than 100 times that of the controls, but the effect was not dose-dependent. In contrast, intraperitoneal administration of melatonin (5, 50, and 200 ug) in mice bearing a 5-HT implant (2 mg) resulted in only 3-7 times higher melatonin levels in the GIT as compared to controls, and the brain levels of melatonin were actually lower. A feedback system between 5–HT and melatonin is proposed that regulates appetite and digestive processes by endocrine as well as paracrine effects in both the brain and the GIT.

Different patterns of light exposure in relation to melatonin and cortisol rhythms and sleep of night workers

Abstract: There is strong evidence to suggest that circadian psychophysiological adaptation processes are modified by light, depending on its intensity and timing. To characterize such modifications and determine whether they are associated with an alteration in the day/night pattern of melatonin excretion, measurements were obtained around the clock in 14 permanent night workers, each studied over a 48 hr period in the field. The light exposure behavior of these workers was studied with a newly developed light dosimetry by measuring light intensity at eye level. Physical activity was continuously registered and sleep indices were obtained by sleep logs and activity markings. Circadian rhythms of melatonin and cortisol were analysed from salivary samples collected for 24 hr at 2 hr intervals. The interindividual variation of melatonin acrophase determined by cosinor analysis was greater than 180 degrees (from around midnight to noon) and that of cortisol was about 135 degrees (from early morning to afternoon). Hormonal phase positions coincided significantly with light exposure: the more bright light pulses in the morning (maximum lux between 0600 and 0900), the less were the melatonin and cortisol acrophases shifted into the day. There was also a negative correlation between melatonin acrophase shift and duration of the overall light exposure above 1500 lux. Morning light maximum and sleep onset correlated highly significantly. Night workers were divided into those with less than ('non-shifters', n = 9) and more than 6 hr deviation from midnight ('shifters', n = 5) of the melatonin acrophase. The group comparison revealed a marked difference of the mean melatonin concentrations at night, and at 0700.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and quantification of melatonin in a dinoflagellate, Gonyaulax polyedra: Solutions to the problem of methoxyindole destruction in non-vertebrate material

Abstract: Poeggeler B, Hardeland R. Detection and quantification of melatonin in a dinoflagellate, Gonyaulax polyedra: Solutions to the problem of methoxyindole destruction in non-vertebrate material. J. Pineal Res. 1994: 17: 1–10. Abstract Preservative procedures are described for the extraction and quantification of melatonin in cell material from the dinoflagellate Gonyaulax polyedra, an organism in which this indoleamine is rapidly degraded due to interaction with free oxygen radicals and photooxida-tion. Cells were shock-frozen in liquid nitrogen and pulverized. Various extraction methods were applied to the powder, and rates of recovery were compared. For the determination of melatonin by high performance liquid chromatography (HPLC), extractions with acetone or perchloric acid were more suitable than the use of other solvents. For purposes of radioimmunoassay (RIA), extraction with acetone gave the best results. Several other inorganic solvents, which are often applied in melatonin research, such as chloroform, dichloromethane, and diethyl ether, led to considerable losses of the indoleamine. The procedures developed for HPLC with either electrochemical or fluorescence detection also allow the quantification of other indolic compounds, in particular, tryptophan and 5-methoxytryptamine. The methods described may be of value in the further search for melatonin and related indoleamines in non-vertebrate material, especially, from unicells, multicellular plants, and invertebrates.

Melatonin modulates growth factor activity in MCF-7 human breast cancer cells.

Abstract: Melatonin has been shown to have direct oncostatic actions on estrogen-responsive, MCF-7 human breast cancer cells in culture. In the present study, we examined whether these inhibitory actions on cell growth may be mediated through actions on bioassayable growth factor activity. In order to test this hypothesis, we estimated the growth factor activity of conditioned medium (CM) from estradiol (E2), or melatonin-treated cells, in the presence or absence of melatonin on MCF-7 cell growth. We also determined whether melatonin inhibits the action of epidermal growth factor (EGF) action in the absence of E2. The addition of melatonin (10(-9) M) to the cultures of MCF-7 cells with CM from E2 (10(-8) M)-treated cells significantly inhibited the growth stimulatory activity of CM, suggesting that melatonin inhibited cell proliferation by blocking the action of E2-induced autocrine growth stimulatory factors. Conditioned medium from melatonin-treated cells significantly inhibited cell proliferation, while an additional supply of melatonin to these cultures had an even greater inhibitory effect. Melatonin was also active in the complete absence of serum as long as cell growth was stimulated by EGF, an E2-inducible growth factor. The inhibitory effect of melatonin increased as the dose of EGF increased. This non-antiestrogenic inhibitory effect of melatonin was reversed by E2, but not by EGF itself, suggesting that melatonin requires accessible estrogen receptor sites for its inhibitory activity on the growth stimulating action of EGF. Taken together, these findings suggest that melatonin may inhibit the action and/or release of growth stimulatory factors as well as stimulate the release of growth inhibitory factors in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Melatonin rhythms in Arctic urban residents

Abstract: Stokkan K-A, Reiter RJ. Melatonin rhythms in Arctic urban residents. J. Pineal Res. 1994: 16: 33–36. Abstract The 24-hr rhythm of salivary melatonin was measured in persons living in the city of Tromso (70°N) at the following times of the year: in January at a day length of 2 hr of twilight, in June under continuous sunshine, and in March and September at about 12 hr light and 12 hr darkness. The hormone patterns varied widely between individuals, but, in general, they were consistent within most individuals between the seasons. Highest peak values occurred in January when the mean level was also significantly higher than at any other time of year. The lowest mean levels occurred in June. Although individual rhythms were not always apparent, the mean patterns showed significantly elevated melatonin concentrations during the night at all seasons. The June melatonin peak was similar to that in March and September, but appeared to be phase-delayed with increased melatonin concentrations from midnight until 0900. It is assumed that the delayed melatonin peak in June may be associated with a tendency among people to shift their activity/rest rhythm and that the pineal sensitivity to light is reduced in the morning in summer.

Effects of melatonin and the melatonin receptor agonist S-20098 on the vigilance states, EEG spectra, and cortical temperature in the rat

Abstract: Tobler I, Jaggi K, Borbely AA. Effects of melatonin and the melatonin receptor agonist S-20098 on the vigilance states, EEG spectra, and cortical temperature in the rat. J. Pineal Res. 1994: 16: 26–32. Abstract The effects of melatonin (3 mg/kg i.p.) and the melatonin receptor agonist S-20098 (3 mg/kg i.p.) on the vigilance states, electroencephalogram power spectra (0.25–25.0 Hz), and cortical temperature were determined in eight rats in the first 6-hr interval of the 12-hr light period. Compared to the vehicle injection both compounds reduced the power density in non-rapid eye movement sleep in the low frequency range (1–8 Hz) but did not affect the vigilance states and brain temperature. The present findings do not indicate that the stimulation of the melatonin receptor exerts a hypnotic effect at doses that had been shown to affect the circadian rest-activity rhythm.

Inhibitory effect of melatonin on production of IFNγ or TNFα in peripheral blood mononuclear cells of some blood donors

Abstract: Melatonin, the main pineal hormone, has been shown to influence many biological functions, including the immune response and cancer growth. The purpose of this study was to examine the effect of melatonin on the production of interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha) by peripheral blood mononuclear cells (PBMC) in culture. Melatonin at physiological concentrations fails to induce production of IFN or TNF by PBMC in culture but causes a dose-related inhibition of production of both cytokines if the PBMC are stimulated with phytohaemagglutinin. This inhibitory effect occurs in only 22% of cases (melatonin-sensitive) but disappears when the cells are stored at 4 degrees C for 4 days. The effect of melatonin appears not to be mediated by opiates nor to be correlated with the age, sex, or blood group of donors, but seems to be influenced by the seasonal time of blood collection. These results provide further evidence for an interaction between melatonin and the immune system and suggest that the effect of melatonin on production of IFN and TNF may be mediated by various factors not yet fully understood.

Pharmacological effects of melatonin treatment on both locomotor activity and brain serotonin release in rats.

Abstract: Chuang J-I, Lin M-T. Pharmacological effects of melatonin treatment on both locomotor activity and brain serotonin release in rats. J. Pineal Res. 1994: 17: 11–16. Abstract The effects of intraperitoneal administration of pharmacological doses of melatonin (60 mg/kg) on both locomotor activity and brain monoamine release were assessed in rats. The spontaneous levels of either horizontal motion, vertical motion, or total distance traveled were decreased following melatonin injection. On the other hand, the spontaneous levels of postural freezing increased after treatment. External heat exposure (36°C) produced increases in locomotion (including horizontal motion, vertical motion, and total distance traveled) as well as decreases of postural freezing in rats. The heat-induced increases of horizontal motion and total distance traveled as well as decreases of postural freezing were attenuated by melatonin treatment. In addition, cold exposure (4 C) produced increases of vertical motion as well as decreases of postural freezing. Again, the cold-induced behavioral responses were attenuated by melatonin treatment. Biochemical data revealed that the serum levels of melatonin were decreased by both heat and cold exposure in rats. Furthermore, voltammetric data revealed that intraperitoneal administration of melatonin (60 mg/kg) decreased serotonin, but not the dopamine, release in the hypothalamus, the corpus striatum or nucleus accumbens of rat brain. Neither the locomotor activity responses to thermal stress nor brain monoamine release was affected by a smaller dose of melatonin (30 mg/kg, i. p.). The results suggest that systemic administration of melatonin, at pharmacological doses, inhibits brain serotonin release and results in a reduction in both the spontaneous locomotion and the thermal stress-induced locomotor activity responses in rats.

Are the annual reproductive and body weight rhythms in the male European hamster (Cricetus cricetus) dependent upon a photoperiodically entrained circannual clock

Abstract: Most of the data obtained so far on the European hamster (Cricetus cricetus) suggest direct photoperiodically driven seasonal changes in sexual activity and body weight. The results of the present long-term study support the hypothesis that these annual changes are the expression of photoperiodically driven endogenous circannual rhythms. When subjected following capture (April-May) to constant conditions of long photoperiod (LP) and constant temperature a large number of the European hamsters present, in September-December, complete gonadal atrophy associated with a decrease in body weight. A sexual reactivation as well as an increase in body weight are observed in the same animals between January and April. Of the six animals that survived long enough, two only presented partial gonadal atrophy during the second year. These observations clearly demonstrate that the decline in sexual activity in subjective autumn does not require a decrease in photoperiod, at least in the first year. Theoretically, the observed rhythms, if circannual in nature, would be generated by a self-sustained annual oscillator (circannual clock) able to function in the absence of a photoperiodic input. Pinealectomy makes animals unable to detect or measure photoperiodic information. Of the six European hamsters tested (pinealectomized in June and then kept continuously under LP), five showed clear annual rhythms in body weight and reproductive capacities for two consecutive years. Clearly endogenous annual rhythms were expressed in these conditions. To be entrained to a 1-year period, such a circannual clock should, however, be able to react to either LP and/or to short-photoperiod (SP), at least at certain periods of the annual cycle. In animals exposed to LP in August or October, after gonadal atrophy had been established by exposure to natural SP, gonadal regrowth started in December or January, about 2 to 3 months earlier than in animals kept outside or in experimental SP. With the same experimental conditions, exactly the same results were obtained in pinealectomized animals; thus stimulatory effect of LP or LP-induced phase advance of the circannual clock can be excluded. The absence of the SP information would then induce such reaction. In animals kept under constant LP and temperature following capture, however, pinealectomy in January--when all animals are sexually active--induces gonadal atrophy within--weeks. This clearly demonstrates that LP is stimulatory at this time of the subjective year.

Calcium, calcification, and melatonin biosynthesis in the human pineal gland: A postmortem study into age‐related factors

Abstract: It is believed that pineal calcification may be age-associated and that the well-demonstrated age-related decline in melatonin biosynthesis may be an expression of an alteration in calcium homeostasis in the pinealocyte. Prior correlations of melatonin to calcium deposition and age were made on the basis of radiological or semiquantitative analysis. In this postmortem study of 33 subjects (age range 3 months to 65 years) calcium deposits measured by atomic absorption spectrometry correlated positively with age in day and night samples (day: r = 0.56, P < 0.05; night: r = 0.818, P < 0.001). Nighttime (2200 h to 0800 h) pineal melatonin content (HPLC fluorometry) was higher than daytime melatonin levels (nighttime 3.80 +/- 0.3 vs. daytime 0.85 +/- 0.4 ng/mg protein). Nighttime calcium levels in the supernatant correlated negatively with melatonin content (r = -0.59, P < 0.05).

Effect of pinealectomy upon the nonspecific immune response of the ring-dove (Streptopelia risoria)

Abstract: The different stages of the phagocytic process by granulocytes of pinealectomized or sham-pinealectomized ring doves (Streptopelia risoria) as well as hematological parameters (total white blood cells, smear, and total protein) and serum hormone levels (triiodothyronine (T3), thyroxine (T4), and corticosterone) were studied. A number of immunological parameters of the phagocytosis process, including adherence capacity, mobility rate, the phagocytosis capacity for inert particles and the digestion capacity of ingested material, were studied. Adult male ring doves were injected intravenously with either 0.1 ml of normal sheep serum (NSS) or saline (SS). Blood samples were collected before injection, and 1 hr, 3 hr, 24 hr, and 4 days afterwards. The results indicate that pinealectomy produces a significant increase in the number of total white blood cells and total protein concentration in plasma in addition to altering different stages of the phagocytic process. During the immunization study, a decrease in the percentage of leukocytes and lymphocytes and an increase in the percentage of heterophils accompanied by an increase in the concentration of serum corticosterone were observed 3 hr following treatment. For the immunological parameters, adherence capacity and latex bead ingestion were increased 3 hr after NSS injection and the NBT reduction test 3 and 24 hr after NSS treatment. In addition, the administration of NSS produced a significant increase in serum T3 and T4 concentrations 4 days following injection. These results show that pinealectomy has a marked effect on both the number and function of immune cells.

Reproductive and no reproductive responsiveness to photoperiod in laboratory rats

Abstract: Laboratory rats (Rattus norvegicus) have been traditionally considered nonphotoperiodic because reproductive function is unaffected by day length. However, at least three experimental manipulations of rats--perinatal androgen injection, peripubertal androgen implants, and peripubertal olfactory bulbectomy--have been reported to unmask reproductive responsiveness to photoperiod. The physiological means by which early testosterone treatment or olfactory bulbectomy affect the expression of photoperiodism were hypothesized to operate through similar underlying mechanism(s) that involved gonadotropin and prolactin blood levels. Short day lengths reduce blood levels of gonadotropins in so-called photoperiodic rodent species. Reduced prolactin levels result in virtually all reproductively photoperiodic species housed in short day lengths. In Experiment 1, male weanling rats either were olfactory-bulbectomized or received a sham-procedure and housed for 10 weeks in long (LD 16:8) or short (LD 8:16) days. Short-day rats reduced body mass, testicular sperm counts, and the size of their reproductive systems; olfactory bulbectomy amplified this inhibitory effect for some parameters including testicular and epididymal sperm counts. However, neither short days nor olfactory bulbectomy affected blood titers of follicle stimulating hormone (FSH) or prolactin. Pelage density was also unaffected by photoperiod, but rats retained their juvenile fur color; i.e., short-day rats remained white, but long-day rats became yellowish. In Experiment 2, male rats were injected with testosterone at 3 days of age, then housed in long or short days until 10 weeks of age. Day length alone did not affect any experimental parameter measured in Experiment 2 except fur color; again, short-day rats retained their juvenile fur color.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple circadian oscillators in the photosensitive pike pineal gland: A study using organ and cell culture

Abstract: Bolliet V, Begay V, Ravault J-P, Ali MA, Collin J-P, Falcon J. Multiple circadian oscillators in the photosensitive pike pineal gland: A study using organ and cell culture. J. Pineal Res. 1994: 16: 77–84. Abstract The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production. In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light: dark cycle. In the present study, it is shown that the endogenous rhythm in melatonin release of super fused pike pineals maintained under constant darkness is expressed at temperatures of 19°C, 20°C, 25°C, and 30°C (period > 24 hr), but not at temperatures of 10°C and 15°C. Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs. Dissociated pike pineal cells, cultured statically at 20°C, expressed a high amplitude rhythm in melatonin secretion under a light: dark cycle. Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed. This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks. It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock. Damping of the overall rhythm under constant darkness might reflect the resynchronization (uncoupling) between these clocks and/or damping of individual oscillators.

Reliable analysis of individual human megaton profiles by complex cosinor analysis

Abstract: Lerchl A, Partsch C-J. Reliable analysis of individual human melatonin profiles by complex cosinor analysis. J. Pineal Res. 1994: 16: 85–90. Abstract The analysis of diurnal secretion patterns of the pineal hormone melatonin should provide information about magnitude of peak concentration, time of peak (acrophase), and duration of elevated hormone levels. We report here on a method for analysis of human melatonin secretary rhythms which fulfills these requirements with considerable precision. Blood samples were obtained from 10 healthy male volunteers at 30 min intervals throughout a 24 hr period, three times (the initial blood sampling was repeated after 2 weeks and 3 months). These sets of 48 plasma samples per volunteer were analyzed for melatonin by radio immunoassay. The mean intra-assay variation was 10. 3%, and inter-assay variations were 15. 8% at 21 pg/ml, 11% at 28 pg/ml, and 9. 4% at 48. 6 pg/ml. Data were subjected to single cosinor analysis (SCA) or analyzed by a complex cosinor analysis (CCA) with the fundamental and the first harmonic as parameters. Both methods provided essentially the same information about the macrophages, whereas the CCA had a clear advantage in terms of better regression coefficients between the original data points and the calculated curve (CCA: r = 0.952 ± 0.018; SCA: r = 0.867 ± 0.039 [means ± SD]; P < 0.001). As a consequence, maximum and minimum values and the times of onset and cessation of melatonin production could be estimated easily and reliably without the need for rough and/or subjective measures. By comparing the three sampling sessions, the secretary rhythm of each individual was clearly reproducible (mean coefficient of variation 8. 4%), thus confirming earlier work. Interindividual differences, however, were quite pronounced, especially with respect to amplitudes (more than 25% coefficient of variation). The present method for describing the secretary pattern of emblazoning may also be useful for other study protocols since it requires only slightly more effort than the commonly used single cosinor analysis.

Interaction between melatonin and estradiol on morphological and morphometric features of MCF-7 human breast cancer cells.

Abstract: Melatonin has been shown to have a direct inhibitory effect on the proliferation of estrogen-responsive MCF-7 human breast cancer cells, involving an interaction with estradiol. The anti-proliferative effect of melatonin is reversed by the addition of estradiol to the culture. In the present study, we examined whether inhibition by melatonin and subsequent estrogen rescue of MCF-7 cells are correlated with morphological and morphometric changes in these cells. After 4 days of exposure to melatonin, MCF-7 cells showed significantly smaller cell and nuclear sizes than other groups. These morphometric results were closely related to the ultrastructural features observed in these cells. While control and estradiol-treated cells showed increased tumor characteristics, melatonin-treated cells presented greater differentiation, in keeping with their epithelial origin (presence of cytokeratin filament bundles, conspicuous rough endoplasmic reticulum, and Golgi cisternae together with the presence of prominent nucleoli at the nuclear level). Additionally, some melatonin-treated cells displayed degenerative features (mitochondrial swelling with disruption of cristae, cytoplasmic vacuolation, nuclear chromatin disgregation and cell lysis). The addition of estradiol to cells previously incubated with melatonin reversed the changes induced by the latter and these cells showed the same ultrastructural features as the control cells. Our results support the notion that melatonin exerts its antitumor effect through a cell-cycle-specific mechanism by delaying the entry of MCF-7 cells into mitosis. This allows the tumor cells to achieve greater differentiation. The fact that the morphometric and morphological effects induced by melatonin are counteracted by estrogens suggests a cell-cycle acceleration induced by estradiol.

Effects of single doses and daily melatonin treatments on serotonin metabolism in rat brain regions

Abstract: The acute effects of two doses (0.5 and 1 mg/kg) of melatonin on the levels of tryptophan, serotonin, and 5-hydroxyindoleacetic acid in several rat brain regions were studied. Tryptophan content in the brain regions was unchanged by the treatments. Melatonin at a dosage of 0.5 mg/kg increased medial hypothalamic serotonin levels at 60 and 90 min after the injection. However, the dose of 1 mg/kg increased the levels of this amine or its metabolite in the preoptic area-anterior hypothalamus, medial and posterior hypothalamus, amygdala, and midbrain. These results suggest a specific regional sensitivity to melatonin as well as a dose-dependent response. The stimulatory melatonin effect on the serotoninergic system was also observed after a daily treatment with this hormone (0.5 mg/kg, twice daily during 10 days) in both intact or pinealectomized rats. In intact rats, melatonin treatment increased the levels of 5-hydroxyindole-3-acetic acid in the preoptic area-anterior hypothalamus and medial hypothalamus, while in pinealectomized rats melatonin increased the serotonin content in the medial hypothalamic region. The data support the idea that melatonin has a selective action on serotonin metabolism in regions that contain serotoninergic terminals, especially at medial hypothalamic level.

Melatonin and farm animals: endogenous rhythms and exogenous applications.

Abstract: Studies on farm animals have contributed significantly to our increased understanding of basic melatonin-related physiological mechanisms, as well as to the regulation of reproduction and pelage in individual domestic species. This review concentrates on recent work on the role of melatonin in the regulation of porcine reproduction, cervine endocrine, and behavioral cycles and wool and cashmere production which has added to this knowledge base. Early studies of the domestic pig indicated that melatonin secretion in this species differed markedly from that in other domestic and laboratory animals. There is now clear evidence that this is not the case and that the domestic pig uses a circadian rhythm of melatonin release for the transduction of photoperiodic information. Apparent inconsistencies among reports may be due, in part, to differences in the conditions under which the experiments were performed and to the assay systems employed to measure circulating melatonin, the concentrations of which are much lower than in other domestic species. Appropriately administered exogenous melatonin advances the onset of puberty in gilts, and may prove to be effective in overcoming seasonal infertility in female pigs. Appropriately timed melatonin and/or photoperiod treatments, administered to ruminants in utero, influence the reproductive physiology of the offspring, indicating that even in species which don't develop an endogenous melatonin rhythm till some weeks postnatally, awareness of photoenvironment, presumably via maternal melatonin, predates birth. Pre- or early postnatal melatonin-related treatments also influence the development/cycle frequency of pelage. Areas requiring further investigation include the hormonal/growth factors involved, reasons for the transient nature of the effects in sheep and goats, and the reason for similar effects on pelage of augmenting or inactivating melatonin. Aspects of endogenous melatonin rhythms in farm species which require further study include: the significance of the abolition of the nocturnal melatonin peak in the sheep by prolonged short day exposure; the increased pineal bloodflow in sheep bred to produce high wool yields; the presence of high daytime melatonin levels immediately prior to the rut in the fallow buck; and the low amplitude of the rhythm in the domestic pig.

Effects of febrile and epileptic convulsions on daily variations in plasma melatonin concentration in children.

Abstract: Plasma melatonin was measured in 118 infants and children (39 controls, 28 with epileptic convulsions, and 51 with febrile convulsions). The control group displayed a typical circadian rhythm, with melatonin peaking between 0200 and 0400. This normal daily variation significantly changed in the epileptic group, which showed a characteristic phase-advance, with the nocturnal melatonin peak appearing between 2400 and 0200. Febrile convulsions were associated with the disappearance of the normal circadian rhythm of melatonin, which was replaced by melatonin bursts throughout the light:dark cycle. In both febrile and epileptic children, melatonin levels were significantly increased in comparison with normal children.

Localization and characterization of [125I]iodomelatonin binding sites in duck gonads

Abstract: Ayre EA, Wang ZP, Brown GM, Pang SF. Localization and characterization of [125I]iodomelatonin binding sites in duck gonads. J. Pineal Res. 1994: 17: 39–47. Abstract The characterization and localization of [125I]iodomelatonin binding sites in the gonads advances the understanding of possible regulatory sites of melatonin action. With the availability of [125I]iodomelatonin as a biologically active radioligand, our study utilized a combined approach of autoradiography for anatomical resolution together with an established radioligand binding assay to assess mid-light [125I]iodomelatonin binding in the testes and ovaries of ducks. In the autoradiography study, specific [125I]iodomelatonin binding was shown to be homogeneous throughout the testes, while in the ovaries, specific [125I]iodomelatonin binding appeared to be concentrated around the follicle. Radioligand binding assay results indicated a single class of binding sites with the maximum number of [125I]iodomelatonin binding sites measured at 1. 91 ± 0. 70 fmol/mg protein in testicular membrane and 4. 54 ± 0. 64 fmol/mg protein in ovarian membrane. [125I]Iodomelatonin binding affinity, characterized by equilibrium dissociation constants of 29 ± 6 pmol/L in testicular membrane and 53 ± 9 pmol/L in ovarian membrane, was in accordance with circulating melatonin levels, suggesting an appropriate concentration for eliciting a physiological response. [I]Iodomelatonin binding in duck gonads satisfied all the criteria for a binding site, being rapid, stable, saturable, reversible, specific, and of high affinity, and may indicate a direct pineal-gonadal connection.

Melatonin is involved in cholecystokinin-induced changes of ileal motility in rats.

Abstract: Benouali-Pellisier S. Melatonin is involved in cholecystokinin-induced changes of ileal motility in rats. J. Pineal Res. 1994: 17: 79–85. ©Munksgaard 1994 Abstract The aim of this study was to determine, in the rat, the interaction between melatonin and cholecystokinin in the regulation of the ileal interdigestive motility. This was analyzed by the chronic electromyography technique. Ileal motility was defined by the presence of intermittent spike bursts corresponding to the contractile activity of the organe. In control rats, these spike bursts were organized in cyclic myoelectrical complexes. Each complex is characterized by two successive spiking activity phases: the irregular phase (ISA) followed by the regular phase (RSA). Pinealectomy suppressed the RSA phase so ileal motility was constituted only by the ISA phase. When melatonin (1 mg/kg i. v.) was injected into pinealectomized rats, RSA phases were immediately and definitively restored. RSA phases were also re-established when the “alimentary” type of cholecystokinin receptors (CCKA) were blocked by selective antagonists such as L364,718 or SR27897 (1 mg/kg i. v.). The latter had better brain accessibility than L364,718. Unlike the effects of melatonin, the effect of these antagonists was neither immediate (the latency is longer for L364,718 than for SR27897) nor definitive. In control rats, cholecystokinin (5 |μg/kg i. v.) induced a characteristic long-lasting (29 |Mp 2 min) excitomotor effect on the ileum. This effect was suppressed in pinealectomized rats and was restored after melatonin treatment. These results suggest that, via the central nervous system, melatonin is involved in the modulation of cholecystokinin action on ileal motility.

Direct influence of melatonin on steroid, Nona peptide hormones, and cyclic nucleotide secretion by granulosa cells isolated from porcine ovaries

Abstract: Sirotkin AV. Direct influence of melatonin on steroid, nonapeptide hormones, and cyclic nucleotide secretion by granulosa cells from porcine ovaries. J. Pineal Res. 1994; 17: 112–117. Abstract The release of progesterone, estradiol-173, oxytocin, arginine-vasopressin, cAMP, and cGMP by cultured granulosa cells isolated from porcine ovaries without and in the presence of melatonin (0. 001, 0. 01, 0. 1, 1, 10, and 100 ng/ml medium) was analyzed. It was found that melatonin is able to inhibit progesterone and stimulate estuarial secretion. Melatonin treatments significantly inhibited oxytocin release. Some inhibition of vasopressin and cAMP and significant stimulation of cGMP also resulted from melatonin treatment. The present observations suggest a direct effect of melatonin on the steroid, nonapeptide hormone, and cyclic nucleotide release from porcine ovarian cells.

Differential regulation of melatonin receptors by short- versus long-term pinealectomy in the rat suprachiasmatic nuclei and pars tuberalis.

Abstract: Gauer F, Masson-Pevet M, Pevet P. Differential regulation of melatonin receptors by short- versus long-term pinealectomy in the rat suprachiasmatic nuclei and pars tuberalis. J. Pineal Res. 1994: 16: 73–76. Abstract We have investigated the effects of short- and long-term pinealectomy on the density of melatonin receptors in both the suprachiasmatic nuclei (SCN) and pars tubercles (PT) of the rat. Short-term pinealectomy (conducted 3-5 days before sacrifice) induced a significant increase in the density of melatonin receptors in both structures. This increase can be interpreted as an up-regulation of melatonin receptors following the absence of plasma melanoma. In contrast, 7 days after pinelectomy, the receptor density began to decrease, reaching a low point 1. 5 months after pinealectomy. At this time, the decrease represents a 40-45% reduction in binding sites as compared with values measured 3–5 days after pinealectomy. This slow decrease could be related to a slowdown of melatonin receptor synthesis. These results suggest that emblazoning has a stimulatory effect on its own receptor synthesis. However, at 1. 5 months after pinealectomy a novel equilibrium between synthesis and hydrolysis seems to be reached. Melanoma does not appear to be indispensable for the synthesis of its receptors.

Effects of melatonin treatment on the gonadotropin-releasing hormone neuronal system and on gonadotropin secretion in male mink, Mustela vison, in the presence or absence of testosterone feedback.

Abstract: NToumi F, Martinet L, Mondain-Monval M. Effects of melatonin treatment on the gonadotropin-releasing hormone neuronal system and on gonadotropin secretion in male mink, Mustela vison, in the presence or absence of testosterone feedback. J. Pineal Res. 1994: 16: 18–25. Abstract The effects of subcutaneous melatonin capsules on the gonadotropin-releasing hormone (GnRH) immunoreactive (ir) system and the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) have been tested in intact, castrated, and castrated adult male mink supplemented with testosterone. Animals were transferred in July, i.e., during the period of sexual rest, under a daily light: dark cycle of 16-hr light and 8-hr darkness and studied over 13 weeks. GnRH (ir) perikarya, visualized by immunocytochemistry, were counted on serial coronal sections from the diagonal band of Broca to the infundibulum. Serum FSH and LH concentrations were measured by radioimmunoassay. In intact mink, melatonin induced a significant increase in the number of (ir) perikarya and in FSH and LH concentrations 3 and 8 weeks, respectively, after melatonin capsule implantation. In castrated mink, the number of perikarya and the concentrations of FSH, which had increased within 2 weeks after castration, did not change during melatonin treatment. In contrast, the concentrations of LH, which were not altered by castration, increased 3–6 weeks after the onset of melatonin administration, suggesting a stimulation of GnRH release. In castrated testosterone-treated mink, the number of perikarya was increased as in castrated males, but the elevation of FSH in response to castration was prevented. Within 2 weeks after melatonin capsule implantation, the concentrations of FSH decreased while those of LH remained low, indicating an inhibition of GnRH release. These results show that testosterone modulates the effect of melatonin on the activity of the GnRH–gonadotropin system.

Photoperiod and melatonin affect testicular growth in the marsh rice rat (Oryzomys palustris)

Abstract: Edmonds KE, Stetson MH. Photoperiod and melatonin affect testicular growth in the marsh rice rat (Orgzonys palustris). J. Pineal Res. 1994: 17: 86–93. ©Munksgaard 1994 Abstract Reproduction in rice rats is subject to photoperiodic control and the pineal gland mediates this effect. We examined the effects of the pineal gland hormone melatonin on testicular weight when administered via implants, injections, and infusions. Testicular weight was modified by photoperiod and the size of the melatonin implant. Twenty-millimeter implants suppressed testicular weight in rice rats housed on 12- and 16-hr photoperiods, while those housed on a 14-hr photoperiod were more sensitive to melatonin; in these animals 10- and 20-mm implants inhibited testicular weight. Melatonin implants also prevented rice rats from responding to a change in photoperiod with the appropriate alteration of testicular growth. Melatonin injections inhibited testicular growth when administered before lights out on LD 14: 10, but not on LD 16: 8. Morning injections had no effect on either photoperiod. Finally, 12-hr duration melatonin infusions inhibited testicular growth in pinealectomized rice rats on LD 16: 8, while 6-hr duration infusions were without effect. These data show that the pineal, through the secretion of melatonin, is a phototransducing organ intimately involved in testicular growth in rice rats.

Calbindin‐D28k, calretinin, and recovering immunoreactivities in developing chick pineal gland

Abstract: Calbindin-D28k, calretinin, and recoverin, three intracellular calcium-binding proteins belonging to the troponin C/calmodulin superfamily, were immunohistochemically localized in chick pineal during development [from embryonic day 16 (E16) to postnatal day 14 (P14)]. At E18, only calretinin immunoreactivity could be detected in nuclei from follicular pinealocytes. With development, calretinin immunoreactivity expanded from nucleus to cytoplasm, and calretinin immuno-positive cell number increased. At P14 almost al pinealocytes were calretinin positive. Calbindin-D28k immunoreactivity was not detected before E20. During development, many follicular and parafollicular pinealocytes became strongly calbindin-D28k positive, reaching a peak both in intensity and in number at P7; thereafter their number decreased. In addition to pinealocytes, neuron-like cells appeared calbindin-D28k positive at E20 and calretinin positive at P7. Recoverin, a myristoylated protein isolated from vertebrate photoreceptor and which might participate in the inactivation of the phototransduction cascade, was transiently expressed in follicular and parafollicular pinealocytes from P1 to P14 with a maximal expression at P7. This transitory expression may coincide with a transitory light sensitivity period in chick pinealocytes, before complete maturity of the pineal gland.