Iron decreases the nuclear but not the cytosolic content of the neurohormone melatonin in several tissues in chicks

This paper describes the influence of iron on both nuclear and cytosolic melatonin contents in several tissues of chicks. The neurohormone melatonin was estimated by means of radioimmunoassay. Iron, administered as FeCl₃, decreased the nuclear melatonin level in a variety of tissues, including brain, heart, lung, kidney, and erythrocytes (nucleated cells in chicks) but was not seen in either the liver or gut. All variations related with iron were seen in the nuclear fraction, while only in the pineal gland did the melatonin content of the cytosol change as a result of iron treatment. We also observed a day-night rhythm in the nuclear melatonin: high nuclear levels of melatonin at night and low levels during the light period. This is the first report of nuclear localization of melatonin in any avian cell. © 1996 Wiley-Liss, Inc.     

Distribution and Significance of Nitric Oxide Synthase in Brain Tissues of Rats Exposed to Deltamethrin Insecticide

The distribution of nitric oxide synthase(NOS)in brain tissues of rats exposed to deltamethrininsecticide has been examined by histochemical NADPH-diaphorase staining techniques on frozen sec-tions.After injection of deltamethrin(12.5mg/kg,i.p.),a reproducible sequence of toxic signs ofhyperexcitability were elicited.The observation and image analysis showed that,within brain sec-tions of rats exposed to deltamethrin,the numbers and the total staining areas of the NOS positiveneurons were greatly increased,especially in cerebral cortex,hippocampal formation and paraventric-ular nucleus.In addition,the density of single neuron and the processes were also increased.The re-sults suggested that deltamethrin may induce the NOS expression or activate the NOS activity.TheNOS activation may involve in the chains responsible for the excitatory neurotoxicities induced bydeltamethrin.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

促肾上腺皮质激素引起大鼠肾上腺c-fos原癌基因表达的免疫组化研究

采用冰冻切片及免疫组化法观察了注射促肾上腺皮质激素(ACTH,75U/kg)、胰岛素及正常对照大鼠中肾上腺各部分c-fos原癌基因表达产物Fos蛋白的出现和分布特点。结果表明注射ACTH后90min,大鼠肾上腺皮质网状带出现Fos蛋白染色阳性细胞,阳性染色物集中于细胞核,肾上腺皮质束状带仅见少数Fos蛋白染色阳性细胞,肾上腺髓质则未见Fos蛋白染色阳性细胞。与注射ACTH相反,注射胰岛素引起肾上腺髓质出现Fos蛋白染色阳性细胞。注射生理盐水对照组动物肾上腺皮质和髓质均未见Fos蛋白染色阳性细胞。上述结果表明,注射ACTH或胰岛素可以引起大鼠肾上腺不同部位c-fos原癌基因表达。     

Immunocytochemical investigation on the distribution of small chondroitin sulfate-dermatan sulfate proteoglycan in the human

Polyclonal antibodies against the core protein of the small chondroitin sulfate-dermatan sulfate proteoglycan from human skin fibroblast secretions were used, after affinity-purification, as a probe to study localization of crossreactive material in several human tissues by indirect immunocytochemistry. In contrast to skin, kidney, and the adventitial layer of aorta, positive staining of brain, liver, cartilage, and intimal and medial layers of aorta required pre-treatment of tissue sections with chondroitin ABC lyase. In all tissues investigated, antigenic material was present in the interstitial space. Filamentous structures were perpendicularly oriented towards basement membranes. In liver, specific staining was seen along the sinusoidal walls. Reticular fibers with or without focal condensations were seen in cerebral cortex and cerebellum. The results suggest a role of small chondroitin sulfate-dermatan sulfate proteoglycan in cell-matrix interactions.     

松果体及其褪黑素对大鼠胸腺细胞凋亡的影响

目的探讨松果体及其褪黑素对胸腺细胞凋亡的影响以及Caspase-3的表达。方法选用清洁级SD大鼠,分为正常对照组、假手术对照组、松果体摘除组、松果体摘除 褪黑素腹腔注射7.5mg/kg/d组和松果体摘除 褪黑素腹腔注射15mg/kg/d组。术后4、8周取材。运用TUNEL法检测胸腺细胞的凋亡程度,用ABC法染胸腺Caspase-3阳性细胞,计算机图像分析仪测量阳性细胞面积及其染色强度。以RT-PCR法检测褪黑素干预原代培养胸腺细胞Caspase-3的表达。结果松果体摘除后8周时胸腺细胞凋亡显著增加,补充褪黑素则能明显减少胸腺细胞的凋亡。Caspase-3阳性细胞主要见于胸腺皮质,松果体摘除后胸腺皮质Caspase-3阳性细胞面积增加明显,补充褪黑素则使其下降。褪黑素能上调培养胸腺细胞Caspase-3的表达水平。结论松果体能调控大鼠胸腺细胞的凋亡,松果体摘除促进胸腺细胞的凋亡,补充褪黑素能缓解相关影响。     

Detection of bikunin mRNA in limited portions of rat brain.

Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney, testis and adrenal gland, expressed bikunin mRNA. Although signals of bikunin mRNA were faint in the whole brain of rats and mice, distinct signals were found in limited portions of rat brain, such as the hippocampus, cerebral cortex and pituitary, but undetectable in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain and choroid plexus. In three distinct types of cells, such as neurons, astrocytes and meningeal cells, in primary cultures isolated from the cerebral cortex and meninges of 1-day-old newborn rats, only neurons positively expressed bikunin mRNA. These results suggest that, in addition to peripheral tissues, neurons in the hippocampus and cerebral cortex produce bikunin, suggesting a potential role of bikunin/IalphaI family in these brain regions.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

Differential daily rhythms of melatonin in the pineal gland and gut of goldfish Carassius auratus in response to light

The objectives of this study were to test the effects of light on melatonin rhythms in the pineal gland and gut of goldfish Carassius auratus and to investigate whether melatonin function differed in these two tissues, which are photosensitive and non-photosensitive respectively. Rhythms were evaluated by measuring arylalkylamine N-acetyltransferase (AANAT2) and melatonin receptor 1 (MT-R1) mRNA expression and melatonin concentration in the pineal gland, gut (in vivo), and cell cultures of the two tissues (in vitro). Compared to control, pineal gland melatonin secretion was higher at night, whereas the 24-h dark and ophthalmectomy groups maintained higher AANAT2 and MT-R1 mRNA expression during the day. Melatonin levels and AANAT2 and MT-R1 mRNA expression in the gut were also the highest at night, but the 24-h light, dark, and ophthalmectomy groups did not significantly differ from control. Furthermore, we measured AANAT2 and MT-R1 mRNA expression in high temperature water (30 °C) to investigate differences in the antioxidant capacity of pineal gland vs. gut melatonin. Melatonin and H₂O₂ levels, as well as AANAT2 and MT-R1 mRNA expression, were all higher in the two tissues under thermal stress, compared with their levels at 22 °C. Taken together, our results suggest that light has no effect on melatonin patterns in the gut, which appears to exhibit its own circadian rhythm, but both gut and pineal gland melatonin exhibit similar antioxidant function.     

Effects of pinealectomy and exogenous melatonin on ghrelin and peptide YY in gastrointestinal system and neuropeptide Y in hypothalamic arcuate nucleus: immunohistochemical studies in male rats

It is reported that the pineal gland and its main hormone melatonin may have a role in the regulation of ghrelin synthesis in the brain. Stomach is the place where ghrelin is predominantly expressed and secreted. One aim of this study was to investigate possible effects of pinealectomy and melatonin treatment on gastric ghrelin amount. The studies on the effects of the pineal gland on leptin and ghrelin arises the question whether the pineal gland has also effects on the other energy-regulatory peptides such as peptide YY (PYY) and neuropeptide Y (NPY). Therefore, we also aimed to investigate the changes in the immunohistochemical staining of intestinal PYY and hypothalamic NPY following pinealectomy and melatonin treatment. Serum PYY levels were also investigated. Sprague-Dawley rats were divided into four groups as sham-operated (SHAM), sham-operated with melatonin treatment (SHAM-MT), pinealectomised (PNX) and melatonin-treated PNX (PNX-MT) groups. The cells immunostained for ghrelin were abundant throughout the gastric mucosa in all the groups. Neither pinealectomy nor exogenous melatonin affected significantly immunohistochemical staining of ghrelin in stomach. Pinealectomy resulted in a significant increase in immunohistochemical staining of PYY in ileum. The results of serum PYY measurement corresponded closely to the data obtained by immunohistochemical analysis of PYY in ileum, being significantly lower and higher in SHAM and PNX groups, respectively. Pinealectomy caused a decrease in NPY synthesis in ARC as understood from low immunohistochemical staining of NPY. Melatonin treatment increased NPY synthesis in SHAM rats and restored reduction in NPY synthesis caused by pinealectomy. In conclusion, the pineal gland and its main hormone melatonin can be suggested to have a role in the regulation of NPY synthesis in ARC and PYY in gastrointestinal system.     

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

Abstract

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

Melatonin inhibits cholangiocyte hyperplasia in cholestatic rats by interaction with MT1 but not MT2 melatonin receptors

In bile duct-ligated (BDL) rats, large cholangiocytes proliferate by activation of cAMP-dependent signaling. Melatonin, which is secreted from pineal gland as well as extrapineal tissues, regulates cell mitosis by interacting with melatonin receptors (MT1 and MT2) modulating cAMP and clock genes. In the liver, melatonin suppresses oxidative damage and ameliorates fibrosis. No information exists regarding the role of melatonin in the regulation of biliary hyperplasia. We evaluated the mechanisms of action by which melatonin regulates the growth of cholangiocytes. In normal and BDL rats, we determined the hepatic distribution of MT1, MT2, and the clock genes, CLOCK, BMAL1, CRY1, and PER1. Normal and BDL (immediately after BDL) rats were treated in vivo with melatonin before evaluating 1) serum levels of melatonin, bilirubin, and transaminases; 2) intrahepatic bile duct mass (IBDM) in liver sections; and 3) the expression of MT1 and MT2, clock genes, and PKA phosphorylation. In vitro, large cholangiocytes were stimulated with melatonin in the absence/presence of luzindole (MT1/MT2 antagonist) and 4-phenyl-2-propionamidotetralin (MT2 antagonist) before evaluating cell proliferation, cAMP levels, and PKA phosphorylation. Cholangiocytes express MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 that were all upregulated following BDL. Administration of melatonin to BDL rats decreased IBDM, serum bilirubin and transaminases levels, the expression of all clock genes, cAMP levels, and PKA phosphorylation in cholangiocytes. In vitro, melatonin decreased the proliferation, cAMP levels, and PKA phosphorylation, decreases that were blocked by luzindole. Melatonin may be important in the management of biliary hyperplasia in human cholangiopathies.     

The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infused intracerebroventricularly with beta-amyloid 1-40

Intraventricular infusion of rats with beta-amyloid for 14 days resulted in memory deficit in the water maze as well as decreases in choline acetyltransferase activities and somatostatin levels in the cerebral cortex and hippocampus. These changes were not altered by daily intraperitoneal injection of 20 mg/Kg melatonin. Orally administered Ginkgo biloba extract, however, partially reversed the memory deficit and the decrease in choline actyltransferase activities in the hippocampus. The latter treatment failed to reverse the decrease in somatostatin levels. The results indicate that orally administered Ginkgo biloba extract can protect the brain against beta-amyloid from changes leading to memory deficit through its effect on the cholinergic system.     

Fliv-1: a surface membrane differentiation antigen of fetal rat hepatocytes

A monoclonal antibody of IgG1 subclass was raised against 15 day fetal hepatocytes. The antibody appears to recognize a surface membrane-associated fetal hepatocyte antigen. Preliminary experiments suggest that the antibody recognizes a protein epitope. The tissue distribution of the antigen, designated Fliv-1, was studied by immunoperoxidase staining on whole body sections from fetal rats of different gestational ages. In 15 day fetal rats the antigen was detected on Rathke's pouch, brain infundibulum, tongue, liver, pancreatic acini, adrenal gland, vertebral myotome, and some developing muscle tissue. All normal adult tissues that were examined, two rat hepatoma cell lines, and hepatomas induced in rats by a single injection of diethylnitrosamine, were negative for the Fliv-1 antigen. On the basis of its tissue distribution, it seems unlikely that Fliv-1 is a previously described fetal hepatocyte marker.     

Whole-body autoradiographic distribution of exogenously administered renal renin in rats

We studied, by whole-body autoradiography, the distribution of exogenously administered renal renin in rat. Rat renal renin was completely purified and labeled with 125I ([125I]-renin) and was then injected into the tail veins of conscious rats at a dose of 30 microCi, 430 ng. After various intervals, rats were killed by an overdose of ether, the whole body rapidly frozen in acetone-dry ice, and autoradiography performed on sagittal whole-body sections. To remove breakdown products ([125I]-tyrosine and free 125I) from [125I]-renin, sections were treated with perchloric acid solution. The main accumulation of [125I]-renin acid-insoluble radioactivity was observed in liver and renal cortex. The accumulation in these organs was already evident 2 min after the injection, reached a maximum level by 15 min, then gradually decreased. A small amount of [125I]-renin was also evident in spleen, bone marrow, and adrenal gland. Thirty min after the injection, radioactivity began to appear in the thyroid gland, stomach, and small intestine, but disappeared with acid treatment, except in the thyroid. Radioactivity was negligible in other organs including brain, submaxillary gland, lung, heart, and testis. These autoradiographs clearly demonstrate that exogenously administered renal renin is distributed mainly in the liver and renal cortex.     

Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats

Reduction of cerebral edema, an early symptom of ischemia, is one of the most important remedies for reducing subsequent chronic neural damage in stroke. Melatonin, a metabolite of tryptophan released from the pineal gland, has been found to be effective against neurotoxicity in vitro. The present study was aimed to demonstrate the effectiveness of melatonin in vivo in reducing ischemia-induced edema using magnetic resonance imaging (MRI). Rats were subjected to middle cerebral artery (MCA) occlusion/reperfusion surgery. Melatonin was administered twice (6.0 mg/kg, p.o.): just prior to 1 h MCA occlusion and 1 day after the surgery. T2-weighted multislice spin-echo images were acquired 1 day after the surgery. Increases in T2-weighted signals in ischemic sites of the brain were clearly observed after MCA occlusion. The signal increase was found mainly in the striatum and in the cerebral cortex in saline-treated control rats. In the melatonin-treated group, the total volume of cerebral edema was reduced by 45.3% compared to control group (P < 0.01). The protective effect of melatonin against cerebral edema was more clearly observed in the cerebral cortex (reduced by 56.1%, P < 0.01), while the reduction of edema volume in the striatum was weak (reduced by 23.0%). The present MRI study clearly demonstrated that melatonin is effective in reducing edema formation in ischemic animals in vivo, especially in the cerebral cortex. Melatonin may be highly useful in preventing cortical dysfunctions such as motor, sensory, memory, and psychological impairments.     

Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin

Previous work in our laboratory has shown that daily injection of large doses of the pineal hormone melatonin entrains the free-running locomotor rhythms of rats held in constant darkness and synchronizes the disrupted patterns of rats maintained in constant bright light. The present experiments determined the dose-response characteristics of entrainment to daily melatonin injections and made preliminary biochemical estimates of blood melatonin levels and half-lives after two critical doses of the hormone. The data indicated that the median effective dose for melatonin as an entraining agent in free-running rats was 5.45 +/- 1.33 micrograms/kg, considerably lower than doses previously employed and lower than doses employed in reproductive and metabolic studies in rats and hamsters. The data further indicated that the response to melatonin was quantal; rats either entrained to melatonin or they did not. No "partial entrainment" was evident, nor were there differences in phase angle, activity, or period among all effective doses. Biochemical estimates of blood melatonin after either 1 mg/kg or 1 microgram/kg of melatonin indicated that all effective doses resulted in supraphysiological levels of blood melatonin, although doses of 1 microgram/kg resulted in blood levels that were within one order of magnitude of normal nighttime values. Together, the data suggest that the rat circadian system is sensitive to the pineal hormone melatonin at or below doses required to effect rodent reproduction. Whether this sensitivity reflects a role for the pineal gland in rat circadian organization, however, still remains to be determined.     

Nuclear and cytoplasmic RNA in visual cortex neurons of adult rats following visual deprivation and photic stimulation

It has been shown by two-wavelength cytospectrophotometry of gallocyanin-chrome alum-stained sections that visual deprivation in adult rats kept in a complete darkness for 30 days resulted in an accumulation of cytoplasmic RNA by layer V neurons of the visual cerebral cortex and by the cells of the perineuronal neuroglia of this layer. The nuclear RNA content remained unchanged. Stimulation of intact rats with a flickering or constant light induced an increase in the cytoplasmic RNA in these neurons rather than in the nuclear RNA as well as in RNA in their glial satellite cells. Similar light stimulation of the deprived animals gave rise to a complete return of the neuronal RNA to normal with only a slight decrease in the deprivation-induced RNA accumulation by the neuroglial cells. Neither visual deprivation nor light stimulation affected the RNA content in the neurons and neuroglia of layer V of the motor cerebral cortex. Compartmentation of RNA metabolism within the neuronal-neuroglial unit is discussed.     

The Effects of Middle Cerebral Artery Occlusion on Central Nervous System Apoptotic Events in Normal and Diabetic Rats

Apoptosis and neural degeneration are characteristics of cerebral ischemia and brain damage. Diabetes is associated with worsening of brain damage following ischemic events. In this study, the authors characterize the influence of focal cerebral ischemia, induced by middle cerebral artery occlusion, on 2 indexes of apoptosis,TUNEL(terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate nick end-labeling) staining and caspase- 3 immunohistochemistry. Diabetes was induced in normal rats using streptozotocin and maintained for 5 to 6 weeks. The middle cerebral artery of both normal and diabetic rats was occluded and maintained from 24 or 48 hours. Sham-operated normal and diabetic animals served as controls. Following 24 to 48 hours of occlusion, the animals were sacrificed and the brains were removed, sectioned, and processed for TUNEL staining or caspase-3 immunohistochemistry. Middle cerebral artery occlusion in normal rats was associated with an increase in the number of both TUNEL-positive and caspase-3– positive cells in selected brain regions (hypothalamic preoptic area, piriform cortex, and parietal cortex) when compared to nonoccluded controls. Diabetic rats without occlusion showed significant increases in both TUNEL-positive and caspase-3–positive cells compared to normal controls. Middle cerebral artery occlusion in diabetic rats resulted in increases in TUNEL-positive as well as caspase-3–positive cells in selected regions, above those seen in nonoccluded diabetic rats. Both TUNEL staining and caspase-3 immunohistochemistry revealed that the number of apoptotic cells in diabetic animals tended to be greatest in the preoptic area and parietal cortex. The authors conclude that focal cerebral ischemia is associated with a significant increase in apoptosis in nondiabetic rats, and that diabetes alone or diabetes plus focal ischemia are associated with significant increases in apoptotic cells.