Takuro Suzuki

Melatonin signal transduction in the goldfish, Carassius auratus.

Generation and reception of melatonin signals in the goldfish, Carassius auratus, are reviewed. The photoreceptive pineal gland of the goldfish generates circulating melatonin rhythms according to a given photoperiod under light-dark cycles and in a circadian manner under continuous dark conditions. Melatonin is also produced in the retina in a similar fashion. Melatonin produced in the pineal gland and retina is considered to act as internal zeitgeber in the brain and retina, respectively, controlling various physiological events via specific melatonin binding sites that are coupled with G protein. The goldfish exhibit clear diurnal locomotor activity rhythms under light-dark cycles and free-running rhythms under constant conditions. However, the relationship between melatonin and locomotor activity rhythms in the goldfish remains unclear. Further studies should be required to demonstrate the roles of melatonin in the circadian system in this species.

Administration of Melatonin and Related Indoles Prevents Exercise-Induced Cellular Oxidative Changes in Rats

In an attempt to define the role of the pineal hormone melatonin and two analogues (5-methoxytryptamine, 5MT, and 6-hydroxymelatonin, 6HM) in limiting oxidative stress, the present study investigated the changes in glutathione, lipid peroxidation, and the activity of the antioxidant enzyme glutathione peroxidase after exercise (swimming for 60 min) with or without treatment with the indolamines mentioned. Lipid peroxidation was measured by estimating tissue levels of malondialdehyde and 4-hydroxyalkenals; the experimental animals in these studies were male Sprague-Dawley rats. In the liver, swimming exercise increased the levels of reduced glutathione (GSH) and also significantly increasing oxidized glutathione (GSSG), while decreasing the GSH/GSSG ratio, an index directly related to oxidative stress. When the animals were treated with melatonin, the concentrations of GSH and GSSG were also increased after swimming; however, no reduction in the GSH/GSSG ratio appeared. In the animals treated with 6HM the changes were the same as in those treated with melatonin. In muscle as well, the concentration of GSH and the GSH/GSSG ratio were decreased following 60 min of swimming. Pretreatment of the rats with melatonin prevented these effects. Pretreatment of the rats with both 5MT and 6HM also prevented the changes. Brain GSH/GSSG ratio was not affected by either exercise or indolamine administration. Swimming enhanced lipid peroxidation in the liver, muscle and brain; however, this was prevented in animals treated with melatonin or 6HM before swimming. Glutathione peroxidase was significantly elevated after exercise in the brain but not in the liver and muscle. It is concluded that swimming imposes a severe oxidative stress and suggests that melatonin and, to a lesser degree, 5MT and 6HM confer protection against the oxidative damage associated with swimming for 60 min. This mechanism may be reasonably attributed to their indole structure, which possibly allows these molecules to act as free-radical scavengers.

Melatonin and arylalkylamine N-acetyltransferase activity in the silkworm, Bombyx mori

Melatonin (N-acetyl-5-methoxytryptamine) was identified in the head and hemolymph of the silkworm, Bombyx mori, using reversed-phase high-performance liquid chromatography coupled with fluorometric detection and radioimmunoassay. In addition, evidence of arylakylamine (serotonin) N-acetyltransferase (NAT) a key enzyme controlling the synthesis of melatonin in vertebrates, was found in the head of the silkworm. Melatonin levels in the head and hemolymph and the NAT activity in the head were significantly higher during the dark period than during the light period of a 12-h light/12-h dark cycle. The day-night changes persisted in constant darkness but were suppressed by constant light. The results suggest that the synthesis and release of melatonin in the silkworm head occur as a circadian rhythm that is entrained by environmental light/dark cycles, as it is in the pineal gland of vertebrates. Melatonin in the silkworm head may function as a neurochemical mediator of photoperiodic control of developmental events such as molting, eclosion and diapause.

Characteristics, day-night changes, subcellular distribution and localization of melatonin binding sites in the goldfish brain

Melatonin binding sites in the goldfish brain were characterized by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. Specific binding of 2-[125I]iodomelatonin was rapid, stable, saturable and reversible. Saturation experiments demonstrated that 2-[125I]iodomelatonin binds to a single class of receptor site with an affinity constant (Kd) of 29.8 +/- 0.7 pM and a total binding capacity (Bmax) of 11.47 +/- 0.33 fmol/mg protein at mid-light. At mid-dark, the Bmax value decreased significantly to 7.90 +/- 0.23 fmol/mg protein (P < 0.01) with no significant variation in the Kd value (33.8 +/- 1.5 pM). Competition experiments revealed the following order of pharmacological affinities: 2-iodomelatonin > melatonin > 6-hydroxymelatonin > N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine > 5-methoxytryptophol > 5-methoxyindole-3-acetic acid. 5-Hydroxytryptamine, 5-hydroxytryptophol, 5-hydroxyindole-3-acetic acid, norepinephrine and acetylcholine exhibited no inhibition. Subcellular distribution of melatonin binding sites was demonstrated to be greatest in the P2 and P3 fractions as compared with the P1 fraction. Localization of melatonin binding sites in discrete brain areas was determined to be highest in the optic tectum-thalamus and hypothalamus, intermediate in the telencephalon, cerebellum and medulla oblongata, and lowest in the olfactory bulbs and pituitary gland. These results suggest that characteristics of melatonin receptors are highly conserved during evolution and that in this species melatonin plays neuromodulatory roles in the central nervous system through specific receptors.

Day‐night changes in melatonin levels in different organs of the cricket (Gryllus bimaculatus)

Abstract: Day‐night levels of melatonin (N‐acetyl‐5‐methoxytryptamine) were determined in different organs of adult female crickets (Gryllus bimaculatus) exposed to a 12/12 light/dark cycle, using reversed‐phase high performance liquid chromatography coupled with fluorometric detection. Melatonin levels in the compound eye, brain, and palp were significantly higher during the dark period than during the light period, suggesting that a diurnal rhythm of melatonin levels exists in these organs of crickets, with a peak during the dark period. Conversely, melatonin levels were significantly higher during the light period than the dark period in the cercus, ovipositor, antenna, hind‐leg and ovary. No significant day‐night difference was found in the fore‐ and mid‐legs, Malpighian tube, and digestive tube. Thus, these organs may have different melatonin‐metabolizing systems compared to those found in the compound eye, brain, and palp. Differences in the phasing of the melatonin rhythm in various organs of the cricket suggest possible differences in melatonin function in these organs.

Ocular Melatonin Rhythms in the Goldfish, Carassius auratus

Ocular melatonin rhythms in the goldfish were studied and compared to those in the pineal organ and plasma. Under light:dark (LD) of 12 h light:12 h dark, melatonin contents in the eye as well as the pineal organ and plasma exhibited clear day-night changes with higher levels at mid-dark than at mid-light. However, melatonin contents in the eye at mid-light and mid-dark were approximately 100 and 9 times greater than those in the pineal organ, respectively. Day-night changes of ocular melatonin persisted after pinealectomy, which abolished those in plasma melatonin under LD 12:12. Ocular melatonin contents in the pinealectomized fish at mid-light were significantly higher than those in the sham-operated control. Under constant darkness (DD), circadian melatonin rhythms were observed in the eye but damped on the 3rd day, whereas plasma melatonin rhythms generated by the pineal organ persisted for at least 3 days. Under constant light, ocular melatonin contents exhibited a significant fluctuation with a smaller amplitude than that under DD, whereas plasma melatonin remained at low levels. These results indicate the involvement of LD cycles, a circadian clock, and the pineal organ in the regulation of ocular melatonin rhythms in the goldfish.

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.

Histochemical staining of cadmium with benzothiazolylazonaphthol derivatives

SummaryAs part of the continuous developmental research for a highly sensitive chelating agent for cadmium, benzothiazolylazophenol derivatives (BTAP) were synthesized and their staining properties for cadmium were examined. The compounds synthesized for this study were identified as being BTAP by analysis of their melting points and C,H,N,S content. Benzothiazolylazo-p-methoxyphenol,-p-chlorophenol and -p-cresol stained cadmium with the highest optical density and the most precise localization amongst dyestuffs reported to date. Differential sequestration by pyrophosphate makes it possible to distinguish cadmium masked by the presence of zinc. With the modification of a chelating agent to increase sensitivity, the improvement in molar absorptivity was also considered theoretically.

Comparison of cadmium-binding protein induction among mayfly larvae of heavy metal resistant (Baetis thermicus) and susceptible species (B. yoshinensis and B. sahoensis)

1. The mayfly larvae of a heavy metal resistant species (Baetis thermicus) and two heavy metal susceptible species (B. yoshinensis and B. sahoensis) were exposed to cadmium at a concentration of 10 μg/1 and the comparative induction of cadmium-binding protein was correlated to their susceptibility to the metal. 2. Amounts of cadmium accumulated in the larvae were increased linearly depending on the duration of exposure. However, there was little difference in the cadmium concentration in each species. 3. Cadmium-binding protein was induced only in the larvae of resistant species (B. thermicus). 4. Copper and zinc concentrations in the two susceptible larvae were decreased by cadmium exposure, while zinc but not copper concentrations were increased in the resistant larvae.

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

SummarySnell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.