Showing papers in "Journal of Pineal Research in 1993"

The Timed Infusion Paradigm for Melatonin Delivery - What has it Taught Us About the Melatonin Signal, its Reception, and the Photoperiodic Control of Seasonal Responses

Abstract: This review summarizes the evidence showing that the duration of the nocturnal secretory profile of pineal melatonin (MEL) is critical for eliciting seasonally appropriate reproductive physiological and behavioral responses in mammals. We review experiments using the timed infusion paradigm (TIP) to deliver MEL either systemically or centrally to pinealectomized hamsters and sheep. In this paradigm, MEL is infused, usually once daily, for a specific number of hours and at a predetermined time of day. This experimental strategy tests most directly those features of the MEL signal that are necessary to trigger photoperiodic responses. The data suggest that the duration of the MEL stimulation is the critical feature of the MEL signal for both inhibitory and stimulatory effects of the hormone on the photoperiodic control of reproductive development in juvenile Siberian hamsters, and for the photoperiodic control of reproductive and metabolic responses in adult Siberian and Syrian hamsters and sheep. The use of the TIP reveals the importance of the frequency of the signal presentation of MEL and suggests the importance of a period of low-to-absent circulating concentrations of the hormone. The TIP also reveals that the characteristics of the MEL signal that regulate male sexual behavior are similar to those that are critical for reproductive and metabolic responses in Syrian hamsters. We summarize the locations of possible functional MEL target sites identified by combining the TIP with traditional brain lesion techniques. Evidence from such studies suggests that the integrity of the suprachiasmatic nucleus (SCN) region in Siberian hamsters and the anterior hypothalamus in Syrian hamsters is necessary for the response to short-day MEL signals. The TIP has been used to deliver MEL to putative target sites for the hormone in the brain of juvenile and adult Siberian hamsters. The results of these preliminary experiments suggest that the regions of specific MEL binding in this species, especially the SCN, are effective sites where MEL may stimulate short-day-type responses. In contrast, results from intracranial application of MEL in sheep suggest the medial basal hypothalamus as a critical site of action. Finally, we also discuss potential applications of the TIP for identification of brain MEL target sites, understanding of other photoperiodic phenomena and responses, and resolution of the cellular/molecular basis underlying the reception and interpretation of MEL signals.(ABSTRACT TRUNCATED AT 400 WORDS)

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MIIO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as gamma-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.

Melatonin, hydroxyl radical‐mediated oxidative damage, and aging: A hypothesis

Abstract: Melatonin is a very potent and efficient endogenous radical scavenger. The pineal indolamine reacts with the highly toxic hydroxyl radical and provides on-site protection against oxidative damage to biomolecules within every cellular compartment. Melatonin acts as a primary non-enzymatic antioxidative defense against the devastating actions of the extremely reactive hydroxyl radical. Melatonin and structurally related tryptophan metabolites are evolutionary conservative molecules principally involved in the prevention of oxidative stress in organisms as different as algae and rats. The rate of aging and the time of onset of age-related diseases in rodents can be retarded by the administration of melatonin or treatments that preserve the endogenous rhythm of melatonin formation. The release of excitatory amino acids such as glutamate enhances endogenous hydroxyl radical formation. The activation of central excitatory amino acid receptors suppress melatonin synthesis and is therefore accompanied by a reduced detoxification rate of hydroxyl radicals. Aged animals and humans are melatonin-deficient and more sensitive to oxidative stress. Experiments investigating the effects of endogenous excitatory amino acid antagonists and stimulants of melatonin biosynthesis such as magnesium may finally lead to novel therapeutic approaches for the prevention of degeneration and dysdifferentiation associated with diseases related to premature aging.

Distribution of melatonin in mammalian tissues: The relative importance of nuclear versus cytosolic localization

Abstract: Besides its presence in the pineal gland, melatonin has been found in a variety of other tissues as well. The indoleamine also has been identified in invertebrates including an unicellular organism where it exhibits a diurnal rhythm. Although melatonin is mainly known for its effects on seasonal reproduction and endocrine physiology, there is evidence showing that this ubiquitously acting hormone is also a potent free radical scavenger, thereby providing protection from oxidative attack to DNA and other biomolecules. Through the years, melatonin was thought to be exclusively cytosolic. However, careful examination of some of these pioneering reports revealed a nuclear localization of melatonin in different tissues including the retina and Harderian glands. Using a very sensitive immunocytochemical method, we have also found that melatonin is located in the nucleus of many cells where it may bind to nuclear components. The use of cell fractionation studies followed by radioimmunoassay confirmed these results. The administration of exogenous melatonin resulted in a marked increase in the nuclear melatonin content without a concomitant change in the cytosolic fraction. In addition to its ability to scavenge free radicals, its location in the nucleus suggests possible genomic actions.

Melatonin and sleep in humans.

Abstract: Early studies on the physiological effects of melatonin typically reported hypnotic 'side-effects'. Later studies, specifically addressing this action, failed to reliably replicate hypnotic effects using standard polysomnography. This difference may be related to differences in the basic physiological action of melatonin compared with more conventional hypnotics. It is suggested that melatonin exerts a hypnotic effect through thermoregulatory mechanisms. By lowering core body temperature, melatonin reduces arousal and increases sleep-propensity. Thus, in humans, one role of melatonin is to transduce the light-dark cycle and define a window-of-opportunity in which sleep-propensity is enhanced. As such, melatonin is likely to be an effective hypnotic agent for sleep disruption associated with elevated temperature due to low circulating melatonin levels. The combined circadian and hypnotic effects of melatonin suggest a synergistic action in the treatment of sleep disorders related to the inappropriate timing of sleep and wakefulness. Adjuvant melatonin may also improve sleep disruption caused by drugs known to alter normal melatonin production (e.g., beta-blockers and benzodiazepines). If melatonin is to be developed as a successful clinical treatment, differences between the pharmacological profile following exogenous administration and the normal endogenous rhythm should be minimized. Continued development as a useful clinical tool requires control of both the amplitude and duration of the exogenous melatonin pulse. There is a need to develop novel drug delivery systems that can reliably produce a square-wave pulse of melatonin at physiological levels for 8-10 hr duration.

ELF magnetic fields, breast cancer, and melatonin: 60 Hz fields block melatonin's oncostatic action on ER+ breast cancer cell proliferation

Abstract: In this study we investigated whether a 60 Hz magnetic field can act at the cellular level to influence the growth of human estrogen-dependent breast cancer cells. Our experimental design assessed cell proliferation of a human breast cancer cell line, MCF-7, in the absence or the presence of melatonin which inhibits growth at a physiological concentration of 10(-9) M. In three experiments, continuous exposure to average sinusoidal 60 Hz magnetic fields of 1.90 +/- 0.01, 2.40 +/- 0.70, and 2.53 +/- 0.50 mG, or simultaneous exposure in matched incubators to average 60 Hz magnetic fields of 10.4 +/- 2.12, 11.95 +/- 2.73, and 11.95 +/- 3.28 mG, respectively, had no effect on cell proliferation in the absence of melatonin. When MCF-7 cells were cultured in the presence of 10(-9) M melatonin, an 18% inhibition of growth was observed for cells in a 2.40 +/- 0.70 mG field. This effect was blocked by a 60 Hz magnetic field of 11.95 +/- 2.75 mG. In a second experiment, a 27% inhibition of MCF-7 cell growth was observed for cells in a 2.53 +/- 0.50 mG magnetic field, and this was blocked by a 60 Hz magnetic field of 11.95 +/- 3.28 mG. These results provide the first evidence that ELF frequency magnetic fields can act at the cellular levels to enhance breast cancer cell proliferation by blocking melatonin's natural oncostatic action. In addition, there appears to be a dose threshold between 2 and 12 mG. The mechanism(s) of action is unknown and may involve modulation of signal transduction events associated with melatonin's regulation of cell growth.

The circadian nature of melatonin secretion in Japanese quail (Coturnix coturnix japonica).

Abstract: Plasma melatonin concentrations were measured in Japanese quail held under different photoperiods and constant darkness ( 12 hr) photoperiods Importantly, between night lengths of 4 to 18 hr (ie, LD 20:4 to LD 6:18) a linear relationship existed between the duration of night-length and secretion of melatonin with the duration increasing by about 08 hr for each additional hour of darkness If quail were released into darkness following a short (LD 6:18) or long (LD 20:4) day schedule, the rhythm persisted for at least two cycles with peaks occurring at about 24 hr intervals In those quail coming into darkness from long days (LD 20:4), the rhythm of melatonin secretion decompressed rapidly on both sides of the peak, indicating that both the onset and offset of melatonin secretion were suppressed under long days The endogenous nature of melatonin secretion was tested further by exposing birds to LD 6:30 for 4 cycles and then releasing into darkness The rhythm in melatonin secretion persisted for at least three cycles before beginning to damp-out The circadian nature of the rhythm in melatonin secretion was also examined by subjecting quail to T-cycles and then releasing into darkness Both under the T-cycles and darkness following T-cycle treatments, the phase of the melatonin rhythm was advanced by > 3 hr under T = 27 hr cycles (LD 3:24) compared with T = 24 hr cycles (LD 3:21) This property is consistent with the melatonin oscillator being a circadian rhythm

New sensitive serum melatonin radioimmunoassay employing the Kennaway G280 antibody : Syrian hamster morning adrenergic response

Abstract: : A new procedure with the G280 antibody of Kennaway provides an assay for circulating melatonin (aMT) with a sample volume (200 ul), an analytic (0.33 pg/ml) and functional (0.62-0.80 pg/mi) detectability, a 50% displacement dose (6.4 pg/mi), a Kd (0.657 pM), and measured circulating daytime levels lower than reported for previous procedures, and 100% assay recovery. The non-nal daytime range in adult human and Syrian hamster serum was 0.4-4 pg/ml. The pattern of fall of the nocturnal surge of Syrian hamster serum aMT near the time of lights- on was unaltered by extended darkness. Isoproterenol (ISO) injection 1 hr after lights-on, when aMT had reached daytime levels, raised serum and pineal aMT dramatically 2 hr postinjection. The same dose of ISO injected 4 hr into light produced only a small detectable increase. Novel extension of nocturnal darkness did not affect the responses to ISO. Thus, when they are allowed to occur at the usual time on a 10-hr dark schedule, both the fall from the nocturnal aMT surge and the subsequent loss of pineal beta-adrenergic responsiveness in this species occur endogenously (probably entrained) rather than from gating by acute effects of morning light. Changes in daytime serum aMT consistent with concomitant changes in the pineal can be measured with a sufficiently sensitive radioimmunoassay.

One-hour exposure to moderate illuminance (500 lux) shifts the human melatonin rhythm.

Abstract: Salivary melatonin levels were measured in 12 healthy volunteers in order to determine whether a moderate light intensity, which suppresses the nocturnal rise of melatonin, was able to shift the melatonin rhythm The samples were collected at 1-hr intervals under lighting of < 100 lux (experiment 1) or < 10 lux (experiment 2) The control melatonin profiles were determined during the first night In the second night the subjects were exposed to light of 500 lux for 60 min during the rising phase of melatonin synthesis The third series of samples was collected during the third night The mean decrease of melatonin levels by the exposure to light was 56% of the prelight concentrations The melatonin onset times were delayed significantly (about 30 min) the night after the exposure to light The melatonin offset times tended to be delayed in experiment 2 The shifts of the melatonin offset correlated positively with the amount of the melatonin suppression The results suggest that a relatively small and short lasting light-induced interruption of melatonin synthesis may affect the melatonin rhythm in humans

Melatonin and human puberty: Current perspectives

Abstract: Many studies of melatonin in human puberty are difficult to interpret in light of methodological considerations such as the use of single blood samples collected either during the day or at night; a small number of observations; the failure to include the temporal characteristics of melatonin secretion; the definition of puberty by the use of broad clinical features without use of hormonal markers of puberty; the lack of control for the actual duration and intensity of light exposure during the days preceding the study; and the cross sectional nature of most studies. The few studies that have examined the plasma melatonin rhythm in humans by multiple blood sampling overnight or over 24 hr suggest that normal pubertal development (as well as normal ovarian function) are not linked to alterations in the plasma melatonin profile. There is, however, some evidence to suggest that disorders of the hypothalamic-pituitary-gonadal axis (delayed puberty, precocious puberty, hypothalamic amenorrhea) may be linked to altered plasma melatonin profile, at least in some cases. These findings, taken together with strong evidence for the role of the pineal gland in the reproductive function of other vertebrate species, render unlikely the inference that the pineal gland has no role in the development and function of the human reproductive axis. Thus, one may speculate that a pineal-puberty relation does exist in humans and that the research techniques applied to date have been inadequate to uncover this relation.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiproliferative effect of pineal indoles on cultured tumor cell lines.

Abstract: The in vitro antiproliferative action of pineal indoles on several tumor cell lines including melanoma (B16), sarcoma (S180), macrophage-like cell line (PU5), fibroblasts (3T3), and choriocarcinoma (JAr) was examined by measuring the incorporation of 3H-thymidine by the tumor cells, and, in the case of melanoma cells, by also measuring the incorporation of 3H-leucine and 3H-uridine. Uptake of crystal violet was used to assess the viability of the tumor cells. The order of inhibitory potency of the indoles was found to be methoxytryptamine > melatonin, methoxytryptophol, hydroxytryptophol, and methoxyindoleacetic acid > serotonin and hydroxyindoleactic acid. The possibility of an adverse effect of the indoles on the viability of normal cells was also investigated by employing a primary culture of rat hepatocytes. The release of glutamate-oxaloacetate transaminase by hepatocytes was not affected by the indoles, although the release of glutamate-pyruvate transaminase was increased to a small extent and the uptake of crystal violet was slightly inhibited

In vivo and in vitro effects of the pineal gland and melatonin on [Ca2++ Mg2+]‐dependent ATPase in cardiac sarcolemma

Abstract: The possible diurnal variation in cardiac [Ca(2+) + Mg2+]-dependent ATPase (Ca2+ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemmal membrane [Ca(2+) + Mg2+]-dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time-dependent changes in [Ca(2+) + Mg2+]-dependent ATPase activity did not occur in either pinealectomized or light-exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca(2+) + Mg2+]-dependent ATPase activity, In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca2+ pump. The half-maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca(2+) + Mg2+]-dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.

The role of the pineal in the control of the daily patterns of neurohypophysial hormone secretion

Abstract: Plasma concentrations of neurohypophysial hormones show clear rhythms over 24 hr which can be suppressed by exposure to constant light, an observation consistent with pineal involvement. A study has therefore been performed on the changes in the hormone levels in the hypothalamus, posterior pituitary, and plasma over 24 hr in control, pinealectomised, and sham pinealectomised animals to determine if the pineal could play a role. Water intake, urine excretion, packed cell volume, plasma osmolality, and electrolytes were also monitored. Pinealectomy had little effect on fluid balance, but after 8 weeks for oxytocin and 2 weeks for vasopressin the morning values (0700-0800) for the circulating concentrations of the hormones were significantly higher in the pinealectomized group compared with the combined sham operated and unoperated groups (pineal intact). By contrast, the pituitary vasopressin was significantly lower in the pinealectomised group. The increase in plasma oxytocin and vasopressin seen over the hours of daylight and accompanying fall in plasma osmolality seen in the pineal intact group were absent in the pinealectomised group. Similarly, the evening fall in pituitary hormone concentrations and increase in hypothalamic hormone content were absent in the pinealectomised animals. After 10 days of exposure to constant light, the fall in plasma osmolality in the pineal-intact animals over the day was no longer significant; instead a significant increase in plasma osmolality and sodium was seen in the pinealectomised group. Exposure to constant light, while altering the patterns of neurohypophysial activity in the pineal intact group, had little effect on the pinealectomised animals.

Melatonin binding sites in the Harderian gland of Syrian hamsters: sexual differences and effect of castration.

Abstract: The presence of specific melatonin binding sites in the Harderian gland of Syrian hamsters was studied using [125I]melatonin. Saturation binding experiments conducted with [125I]melatonin at 37 degrees C using Harderian glands of both male and female Syrian hamsters revealed a single nanomolar-affinity site. The dissociation constants (Kd) were 6.47 and 6.94 nM for males and females, respectively. The concentration of the binding sites was 7.58 fmol/mg protein for males and 13.50 fmol/mg protein for females. Castration of male hamsters resulted in a significant increase in [125I]melatonin binding sites while chronic melatonin administration did not modify the binding properties. The results confirm the presence of melatonin binding sites in the Harderian glands of rodents. The gender-associated differences found together with the effects of castration in male hamsters suggest an androgenic control in [125I]melatonin binding sites of the Syrian hamster Harderian gland.

Inhibitory effect of melatonin on platelet activation induced by collagen and arachidonic acid

Abstract: Melatonin, an indolamine synthesized in the pineal gland, is known to have antiprostanoid activity. The inhibition of platelet aggregation induced by melatonin has been proposed to take place through the cyclooxygenase pathway. In the present study, we found that melatonin has a marked inhibitory effect on collagen, arachidonic acid (AA), adenosine diphosphate (ADP), epinephrine, and A23187-induced aggregation in platelet-rich plasma. On the other hand, using metrizamide-filtered platelets resuspended in Tyrode's buffer, melatonin fails to suppress AA-induced platelet aggregation and 14C-5-HT release. Under the same conditions, melatonin inhibits collagen-induced platelet activation; however, the addition of threshold doses of AA (0.3 mM) abrogates this effect. These studies suggest that melatonin also inhibits platelet function at a stage preceding the cyclooxygenase-dependent pathway.

Melatonin and hormonal changes in disturbed sleep during late pregnancy

Abstract: Although sleep disturbance is commonly reported in pregnancy, there have been surprisingly few studies on the etiology of this condition. Since most hormones show circadian rhythmicity and maintain specific phase relationships with that of the sleep?wake cycle, it was of interest to establish whether sleep disturbances covaried with endocrine changes. This overnight study of pregnant women compared melatonin, cortisol, and prolactin secretion rhythms in six good sleepers and six poor sleepers. The groups were compared by ratios of the areas under the various hormonal curves. Significant differences in the cortisol/melatonin ratio were found between the poor sleeper group (lower values) and the good sleeper group (higher values). Nonsignificant trends, which might be expected to become significant with larger sample sizes, were found for decreased amplitude in the cortisol rhythm and increased amplitude in the melatonin rhythm in poor sleepers. The decreased amplitude of the cortisol rhythm in poor sleepers appeared to be due to a suppression of the early morning (0500–0800) rise. Prolactin levels were high and showed no rhythmicity in both groups. These differences may reflect changes in the circadian pacemaker system of poor sleepers, with increases in melatonin release being a response to counteract poor sleep. Copyright © 1993, Wiley Blackwell. All rights reserved

Immunohistochemical study of the pineal astrocytes in the postnatal development of the cat and dog pineal gland

Abstract: The expression of glial antigens vimentin (VIM) and glial fibrillary acidic protein (GFAP) is described in the pineal gland of cats and dogs from the first postnatal days to adulthood. VIM immunopositive cells were observed from the first postnatal days in both species. GFAP expression starts from the second postnatal week. In adults, a notable population of stellate cells immunopositive for GFAP and VIM was found dispersed throughout the gland. According to their immunocytochemical profile, these cells could be identified as astrocytes.

Pineal modulation of the rat caudate‐putamen spontaneous neuronal activity: Roles of melatonin and vasotocin

Abstract: The effects of microiontophoretic application of melatonin and melatonin plus vasotocin on spontaneously active neurons of caudate-putamen in sham-operated and pinealectomized rats were studied. Extracellular unit recordings showed that in sham-pinealectomized rats, melatonin ejection primarily produced inhibition of the responsive neurons (74.1%), whereas only 24.9% of the neurons were excited. Iontophoretic ejection of vasotocin or melatonin + vasotocin produced, in both cases, an inhibition of 100% of the responsive neurons. In pinealectomized rats, iontophoretic melatonin ejection produced a similar percentage of inhibition (46.1%) and excitation (53.8%) of the responsive neurons. The simultaneous ejection of melatonin + vasotocin further increased the percentage of inhibition (88.8%) compared with the melatonin only treated group. Moreover, iontophoretic ejection of vasotocin inhibited 100% of the responsive neurons in pinealectomized rats. The actions of melatonin and vasotocin seem to be specific, because their effects are dependent on the amount of these compounds ejected, i.e., the intensity of the ejection current. These results indicate that the pineal compounds melatonin and vasotocin are neuromodulators of spontaneous neuronal activity of the rat caudate-putamen.

Vasopressin and vasoactive intestinal peptide infused in the paraventricular nucleus of the hypothalamus elevate plasma melatonin levels

Abstract: The connection between the suprachiasmatic nucleus (SCN) and the paraventricular nucleus of the hypothalamus (PVN) forms an important component of the melatonin rhythm-generating system. However, the chemical identity of this projection is not known. To test the possible implication of the SCN peptides vasopressin (VP) and vasoactive intestinal peptide (VIP) in this projection, we performed microinfusions in the PVN during the first half of the dark period and subsequently monitored resulting plasma melatonin levels. Infusions for 7 hr of either VP or VIP, but not oxytocin, caused increased plasma melatonin levels in the middle of the dark period. These observations confirm the role of the PVN in the melatonin rhythm-generating pathway and indicate that both VP and VIP released at the level of the PVN, and probably derived from the SCN, are able to influence peripheral plasma melatonin levels.

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: attempts to define mechanisms.

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm2) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status--the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.

Characteristics of 2‐[125I]iodomelatonin binding sites in the pigeon spleen and modulation of binding by guanine nucleotides

Abstract: 2-[125]Iodomelatonin binding sites in membrane preparations of pigeon spleen have been characterized. The binding was stable, saturable, reversible, and of high affinity. Rosenthal and Hill analyses showed that the radioligand-receptor interaction involved a single class of binding sites. Analysis of the binding results of spleens collected during mid-light revealed an equilibrium dissociation constant (Kd) of 36.6 +/- 4.8 pmol/l (mean +/- sem, n = 10) and a maximum density (Bmax) of 2.3 +/- 0.2 fmol/mg protein. There was no significant difference in the Kd (46.9 +/- 5.0 pmol/l) or the Bmax values (2.4 +/- 0.3 fmol/mg protein) for spleens collected during mid-dark (n = 9), although the mid-dark serum and pineal melatonin levels were significantly higher (P < 0.05) than the corresponding mid-light values. Kinetic analysis showed a Kd of 8.6 +/- 2.0 pmol/l (n +/- 4), in agreement with that derived from the saturation studies. Except for inhibition by 2-iodomelatonin, melatonin, 6-chloromelatonin, 6-hydroxymelatonin and N-acetylserotonin, the other indoles or neurotransmitters tested have little inhibition on the binding. In addition, guanosine 5'-O-(3-thiophosphate) (GTP gamma S), a nonhydrolysable analog of GTP, was found to inhibit the binding in a dose-dependent manner. Saturation studies revealed that this is due to a decrease in both the affinity and density of the binding sites. These data suggest that a single type of melatonin receptor is found in the pigeon spleen and that the site is coupled to a guinine nucleotide binding protein (G-protein). Our findings support a direct pineal melatonin action on the immune system.

Immunohistochemical study of the pineal glial cells in the postnatal development of the rat pineal gland.

Abstract: The developmental expression of the glial antigens, vimentin (VIM), glial fibrillary acidic protein (GFAP), and S-100 protein is described in the rat pineal gland from the first postnatal day to adulthood. Thick VIM immunopositive cell cords forming a network throughout the pineal gland were observed from the first postnatal days. These cords progressively disappeared during the first postnatal month as their cells dispersed into the pineal parenchyma. From 20 to 25 postnatal days, pineal glial cells appeared as isolated star-shaped VIM immunopositive cells. Immunostaining for GFAP and S-100 protein showed a similar developmental expression pattern. Both antigens appeared later than VIM (15–20 postnatal days) and were restricted to the pineal glial cells located in the proximal third of the gland, close to the pineal stalk.

The effect of exogenous melatonin on the hypothalamic‐pituitary‐adrenal axis in intact and pinealectomized rats under basal and stressed conditions

Abstract: The effect of exogenous melatonin on the adrenocortical axis was studied by determining serum corticosteroid and adrenocorticotrophic hormone (ACTH) levels under basal and stressed conditions in intact, pinealectomized, and adrenalectomized mature male rats. A direct interrelationship between pineal/melatonin and the adrenal cortex, dissociated from the hypothalamic-pituitary axis, was established. Intraperitoneal injection of 100 micrograms melatonin brought about an elevated adrenocortical response recorded in the serum corticosteroid levels 15 hr afterwards. This effect could be demonstrated only at the beginning of the photophase, when physiological levels of corticosteroids and ACTH are at their lowest daily levels. The pattern of this reaction is different from those caused by stressors such as ether or hypoglycemic reagents, and did not occur in pinealectomized animals although the daily rhythm of corticosteroids and ACTH persisted. No effect on ACTH was evident in intact or adrenalectomized rats, where the serum levels remained unchanged. Moreover, the corticotrophin releasing factor (CRF) content of the median eminence was not affected by melatonin.

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one‐week‐and two‐month‐old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week- and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1/1000 displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increased during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreactivity.

Hydroxyindole-O-methyltransferase activity in ocular and brain structures of rabbit and hen.

Abstract: Relative activities of hydroxyindole-O-methyltransferase (HIOMT) of some brain and ocular structures of the rabbit and hen were analyzed using different 5-hydroxyindoles, i.e., N-acetylserotonin (NAS), 5-hydroxytryptophol (HTOL), 5-hydroxytryptophan (HTP), 5-hydroxytryptamine (HT), and 5-hydroxy-3-indoleacetic acid (HIAA), as enzyme substrates. Pineal glands of both species, as well as hen retina, are capable of producing, to varying degrees, melatonin, 5-methoxytryptophol, and 5-methoxytryptamine. Hen choroid and iris-ciliary body O-methylated NAS and HTOL, whereas rabbit choroid and, to a much lesser extent, hypothalamus and cerebral cortex all O-methylated only NAS. No measurable HIOMT activity was found in hen brain. NAS was a preferred substrate for HIOMT in the hen tissues, whereas in the rabbit pineal gland NAS and HTOL were equally good substrates for HIOMT. Other tested 5-hydroxyindoles, i.e., HTP, HT, and HIAA, were poor methyl acceptors. Of the tissues examined, the highest HIOMT activity was found in the hen pineal gland, followed by the rabbit pineal gland and hen retina. No significant differences between day and nighttime enzyme activities were observed in the pineal gland and retina of either species. The data suggest that in vertebrates some nervous and ocular tissues possess the potential to produce 5-methoxyindole compounds; however, the HIOMT-catalyzed process shows remarkable substrate-, tissue- and species-dependent variations.

A non-invasive simple method for measurement of urinary excretion of melatonin in undisturbed mice.

Abstract: Melatonin is a hormone involved in neuroendocrine responses; its plasma concentrations display a circadian pattern which is modified by stress. Studies for determining the effects of stressors on melatonin levels in laboratory animals present the difficulty that the procedures for blood sampling are by themselves potential stressors capable of influencing the levels of the hormone measured. A simple non-stressful method for measuring urinary excretion of melatonin has been consequently developed. The method is applicable to single undisturbed mice kept in conventional cages, and consists of urine collection on chromatographic paper followed by extraction and melatonin assay by radioimmunoassay. The use of this method with BD2F1 mice indicates nocturnal excretion of melatonin significantly higher than during the day; nighttime melatonin levels were shown to be suppressed by constant illumination. A significant increase in nocturnal melatonin excretion was caused by the application of rotational stress applied as a mild experimental stressor.

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/l) and magnesium (1.34 mmol/l) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/l) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.

An immunohistochemical study of neuropeptide Y in the bovine pineal gland

Abstract: An immunohistochemical study of the bovine pineal gland was performed using rabbit polyclonal antibodies raised against neuropeptide Y (NPY) or against the C-terminal flanking peptide of proNPY (CPON). A large number of NPY/CPON-immunoreactive (IR) nerve fibers were demonstrated throughout bovine pineal gland. The IR-fibers were located in the capsule of the gland, usually piercing into the gland together with blood vessels. In the gland itself, the fibers wen also located intraparenchymally between the pinealocytes. Within the rostral and caudal areas of the pineal stalk, NPY-IR fibers were also observed, and these fibers could be followed not only into the gland bul also to the habenular and posterior commissures. The morphological localization of the NPY-IR nerve fibers in the bovine pineal gland indicate that the majority of fibers originate from the superior cervical ganglion. However, some fibers probably originate from the brain itself.

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland.

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.