S.F. Pang

Pretreatment with melatonin reduces volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model.

Melatonin is a potent scavenger of free radicals and an indirect antioxidant. Recent studies have shown that melatonin possesses beneficial effects in experimental models of brain trauma and global cerebral ischemia. The effects of pretreatment with melatonin on volume of cerebral infarction were investigated in the present study. Adult male Sprague–Dawley rats were anesthetized with sodium pentobarbital to undergo right‐sided endovascular middle cerebral artery occlusion (MCAO) for 3 hr. A single dose of melatonin (1.5, 5, 15, or 50 mg/kg in 1 mL normal saline) or its vehicle was given via an intraperitoneal injection at 0.5 hr before MCAO. Relative infarction volumes on day 3 after MCAO were significantly reduced in the groups treated with melatonin at 5 (mean ± S.E.M., 15.7 ± 2.5%) or 15 (21.4 ± 3.1%) mg/kg but not at 1.5 (30.6 ± 3.5%) or 50 (26.7 ± 2.8%) mg/kg when compared with the vehicle group (33.9 ± 3.5%). There was no significant difference in the arterial blood pressure (BP), heart rate (HR) and relative cerebral blood flow among the experimental groups. These results indicate that pretreatment with melatonin at a dose between 5 and 15 mg/kg protects against focal cerebral ischemia.

Melatonin concentrations in serum and tissues of porcine gastrointestinal tract and their relationship to the intake and passage of food

Abstract: Melatonin concentrations were determined in serum and 10 segments of the gastrointestinal tract (GIT) of 48 pigs (100 kg weight). The animals were fasted for 30 hr and then sacrificed 0, 1,2, 5, 10, and 20 hr after refeeding. Peak amount of gastric digesta (2,428 g) and ileum digesta (850 g) were observed 1 hr and 5 hr, after refeeding, respectively. Conversely, colon content reached a minimal weight (726 g) at 2 hr after refeeding. Serum levels of melatonin increased from 3.4 pg/ml to 15.5 pg/ ml (peak 5 hr after refeeding). Melatonin levels in GIT tissues before refeeding varied from 23.8 pg/g (stomach‐fundus) to 62.1 pg/g (rectum). Increasingly higher levels of melatonin were detected in the distal segments of the GIT. Higher melatonin levels after refeeding were observed in most GIT tissues except the rectum. In most tissues, peak melatonin values were detected 5 hr after refeeding. A significant change in weight of digesta across time (P<0.05) was detected in the stomach, ileum, and cecum. Similar changes in melatonin levels across time were found in most tissues except the esophagus, stomach (cardia and pylorus), and rectum. Adjacent GIT tissues exhibited similar (P<0.05) melatonin levels. The GIT melatonin levels correlated best with the variation of digesta weight in the ileum. In addition, the increase of serum melatonin levels correlated best with the increase of GIT melatonin levels in the distal part of the GIT. Our results suggest that melatonin produced in the ileum, cecum, and colon may contribute significantly to the short‐term increase of serum melatonin levels observed after refeeding.

Fluctuation of Blood Melatonin Concentrations With Age: Result of Changes in Pineal Melatonin Secretion, Body Growth, and Aging

Melatonin in the systemic circulation of rats fluctuates with age, and the causes for such changes were investigated. Male rats (aged 7 days, 16 days, 18 days, 20 days, 30 days, 48 days, 60 days, and > 17 months) were adapted under a lighting regime of 12L: 12D for at least 7 days. Pineals and blood samples from the trunk or confluens sinuum were collected in the dark period. Melatonin in tissues was extracted, identified, and determined by gas chromatography‐mass spectrometry (GC‐MS) and/or radioimmunoassay. Tissue melatonin levels obtained by radioimmunoassay correlated closely with those quantified by GC‐MS. Thus, the melatonin radioimmunoassay used is a reliable assay method for melatonin in the plasma and pineal of the rat.

The effect of food deprivation on brain and gastrointestinal tissue levels of tryptophan, serotonin, 5‐hydroxyindoleacetic acid, and melatonin

Abstract: In order to investigate the effect of food deprivation on the levels of indoles in the brain and the gastrointestinal tissues, we have determined tissue levels of tryptophan (TRP), serotonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐H1AA), and melatonin in the brain and the gastrointestinal tract (GIT) of mice on ad libitum diet as well as in mice deprived of food for 24 and 48 hr. The reduction of food intake 1) had no effect on TRP levels in the brain, but increased TRP concentrations in the stomach and the gut, especially in the colon; 2) decreased 5‐HT levels in the brain, but increased values in the stomach and the intestines; 3) decreased 5‐HIAA levels in the brain, but increased them in the stomach and the intestines; 4) did not change 5‐HT conversion to 5‐HIAA in the brain, stomach, and the jejunum, but increased the conversion in the ileum and colon and; 5) increased melatonin levels in all tissues investigated, particularly in the stomach and the brain. The changes of indole levels induced by food deprivation were compared to their known function in the brain and the individual segments of the GIT. A possible serotonin‐melatonin antagonism in the brain and GIT function is considered.

Persistence of circadian rhythms of melatonin and N-acetylserotonin in the serum of rats after pinealectomy.

To study the effect of pinealectomy (Px) on the rhythms of serum indoles, male rats were adapted to 12L:12D for 2 weeks after which half of them were Px. The animals were decapitated at 4-hour intervals 1 week after operation. Melatonin (Mel) and N-acetylserotonin (NAS) were extracted and quantified by RIA. The concentrations of serum Mel and NAS were significantly reduced after Px, suggesting that the pineal contributes a significant amount of these to the blood. However, circadian rhythms of them were not abolished by Px, indicating the existence of extrapineal sources at least one of which is capable of secreting Mel and NAS wih a diurnal rhythm. Retina is suspected to be an important source.

Circadian variation of portal, arterial and venous blood levels of melatonin in pigs and its relationship to food intake and sleep

Circadian levels of melatonin were determined in the hepatic portal vein, cranial vena cava, and the lower aorta of ten juvenile pigs. Blood was sampled every hour for a total of 24 hr via temporary cannulas introduced into blood vessels under anesthesia. No peak levels of melatonin were found in the mid‐scotophase, but hepatic portal concentrations peaked at 06.00 hr. Overall levels of melatonin were highest in the hepatic portal vein (range 35–65 pg/mL), followed by an artery (range 30–55 pg/mL) and the vena cava (range 25–35 pg/mL). Levels of melatonin exhibit strong variation between individual pigs, but generally the average levels from all three sources follow each others time course. However, on occasion, melatonin levels in the hepatic portal vein varied independently from the levels in the vena cava. Large portal peaks were usually preceded by a feeding period and were associated with a subsequent period of sleep. The data indicate that: 1) there is no clear circadian rhythm of melatonin in the peripheral blood of pigs, 2) relatively little melatonin is metabolized during the first liver passage, 3) food intake may elevate melatonin levels in the hepatic portal vein, and 4) increased levels of melatonin originated in the gastrointestinal tract may induce sleep.

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

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.

Modulation of Blood Glucose by Melatonin: A Direct Action on Melatonin Receptors in Mouse Hepatocytes

Melatonin receptors were studied in isolated mouse hepatocytes using the 2[125I]iodomelatonin binding assay. The binding of 2[125I]iodomelatonin to hepatocytes isolated from the mouse using collagenase was stable, saturable, reversible and of high affinity. The equilibrium dissociation constant (Kd) obtained from saturation studies was 10.0 ± 0.4 pmol/l (n = 16), which was comparable to the Kd obtained from kinetics studies (6.9 ± 1.2 pmol/l, n = 3), and the maximum number of binding sites (Bmax) was 2.9 ± 0.4 fmol/mg protein (n = 16). The relative order of potency of indoles in competing for 2[125I]iodomelatonin binding was 2-iodomelatonin > 2-phenylmelatonin > 6-chloromelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that the binding was mediated by the ML1 receptor subtype. The linear Rosenthal plots, the close proximity of the Hill coefficient to unity and the monophasic competition curves suggest that a single class of 2[125I]iodomelatonin binding sites is present in the mouse hepatocytes. Guanosine 5′-O-(3-thiotriphosphate) dose-dependently inhibited 2[125I]iodomelatonin by lowering the affinity of binding, while no inhibitory effects of adenosine nucleotides were observed, suggesting that the binding sites are G-protein linked. Western immunoblotting was used to identify the melatonin receptor subtype in mouse hepatocytes using anti-Mel1a and anti-Mel1b. Hepatocyte membrane extract reacted with anti-Mel1b but not anti-Mel1a giving a peptide-blockable band of 36 kD, supporting the hypothesis that the melatonin receptors in mouse hepatocytes are of the Mel1b subtype. Melatonin injection and a high plasma glucose level affected 2[125I]iodomelatonin binding in the whole mouse liver homogenates. Plasma glucose was elevated by mid-light intraperitoneal injection of melatonin (4 and 40 mg/kg body weight) in a dose-dependent manner with maximum elevation achieved 1 h after injection. 2[125I]Iodomelatonin binding at this time showed increased Kd with no changes in Bmax. When the plasma glucose returned to normal within 2 h, the binding remained lowered with increased Kd but no changes in Bmax. Elevation of plasma glucose by 2-deoxyglucose injection (500 mg/kg), on the other hand, decreased the binding by decreasing the Bmax without affecting the Kd. Suppression of plasma glucose by insulin injection (3 IU/kg) did not change the binding. Thus, melatonin may act directly on the liver to elevate the plasma glucose level, and changes in plasma glucose level itself may in turn affect hepatic melatonin binding.

High affinity specific binding of 2-[125I]iodomelatonin by spleen membrane preparations of chicken

Abstract: The binding sites for 2‐[125I]iodomelatonin in chicken spleens were characterized. The binding was rapid, stable, saturable, reversible, and of high affinity. Both melatonin and 6‐chloromelatonin strongly inhibited the binding. The dissociation constant (Kd) obtained from the Scatchard analysis was 31.4 ± 5.19 pmol/1 (3‐weeks old, n = 4), which was in good agreement with the Kd (50.6 pmol/1) calculated from the kinetic study. The maximum number of binding sites (Bmax) was 1.09 ± 0.11 fmol/mg protein (3‐weeks old, n = 4). Twelve 11‐week‐old chicks were killed in two groups at mid‐light or mid‐dark. Saturation studies indicated no significant difference (P > 0.05) in the Kd between mid‐light (42.1 ± 3.9 pmol/1) and mid‐dark (31.6 ± 4.9 pmol/1). The maximum number of binding sites (Bmax) at mid‐light and mid‐dark were 1.52 ± 0.16 and 1.35 ± 0.08 fmol/mg protein, respectively, with no significant variation (P > 0.05) recorded. However, when the whole spleen was taken into consideration, the Bmax per spleen protein of the mid‐light samples (253 ± 36 fmol/spleen protein) was significantly greater than that (129 ± 16 fmol/spleen protein) of the mid‐dark samples (P < 0.05). This indicated that in our study a diurnal rhythm of the total number of 2‐[125I]iodomelatonin binding sites might exist in the chicken spleen.

2-[125l]lodomelatonin Binding Sites in the Lung and Heart: A Link between the Photoperiodic Signal, Melatonin, and the Cardiopulmonary System

The pineal gland plays an important role in seasonal adaptation including variation in energy requirement. Animals exhibiting seasonal changes in their energy expenditure would be benefited if their cardiac and pulmonary systems respond to the pineal photoperiodic signal, melatonin, appropriately. Thus, we would like to hypothesize that melatonin receptors are present in the heart and lung. Using a specific labeled melatonin agonist, 2-[125I]iodomelatonin, binding sites were demonstrated in the lung and heart of birds and other animals. In the chicken lung, there were high affinity (equilibrium dissociation constant, Kd = 9.11 +/- 0.73 pmol/l) and low density (maximum number of binding sites, Bmax = 1.29 +/- 0.16 fmol/mg protein) 2-[125I]iodomelatonin binding sites that were highly specific to melatonin. Similar binding with lower density was demonstrated in the quail and frog lungs. In the duck heart, specific 2-[125I]iodomelatonin binding sites with a Kd of 30.5 +/- 3.5 pmol/l and a Bmax of 0.46 +/- 0.1 fmol/mg protein (n = 4) were demonstrated. Competitive studies suggested that these binding sites were specific to melatonin. Thus, saturable and reversible 2-[125I]iodomelatonin binding was present in the lung and heart membrane preparations of birds and possibly other animals. The picomolar affinity, femtomolar density and highly specific pharmacological profile of these binding sites suggest that they can be classified as ML-1 melatonin receptors. The 2-[125I]iodomelatonin binding sites described in the lung and heart as well as those binding sites demonstrated in other peripheral tissues suggest the ubiquitous direct action of melatonin on peripheral tissues.