Characterization and maturational differences of melatonin binding sites in the masu salmon brain

To obtain a better understanding of the roles of melatonin in the mediation of photoperiodic signaling, we have examined the pharmacological characteristics, guanine nucleotide modulation, and maturational differences of melatonin binding sites in the brain of masu salmon Oncorhynchus masou by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. The 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 sites with an affinity constant (K(d)) of 6.3+/-0.5 pM and a total binding capacity (B(max)) of 15.18+/-0.22 fmol/mg protein in underyearling precocious males in July. Competition experiments revealed that the binding sites are highly specific for melatonin and related analogues. Treatment with guanosine 5(')-O-(3-thiotriphosphate) significantly reduced the specific binding, indicating that melatonin binding sites in the masu salmon brain are coupled to G protein. Significant differences were seen in B(max), but not K(d), among the fish groups differing in maturity. In the underyearling fish in July, the B(max) of precocious males and immature males was significantly higher than that of immature females. Then, the B(max) of precocious males decreased in October, when the fish spermiated. In the 2-year-old fish, B(max) was significantly higher in spermiating males than ovulated females. These results indicate that melatonin plays neuromodulatory roles in the central nervous system through specific receptors. Furthermore, gonadal maturation affects the density of melatonin binding sites in the masu salmon brain by an unknown mechanism.     

In vitro autoradiographical localization of melatonin binding sites in the caprine brain

The recent development of a specific 2-[125I]-iodo-melatonin ligand has led to the identification of 125I-melatonin binding sites in the brains of numerous mammalian species. The present study reports the localization of 125I-melatonin binding sites in the brain of the dairy goat. Six previously untreated female goats, aged 5-7 years, were culled under natural light between 0900 and 1100. Brains and pituitaries were immediately dissected out and frozen on dry ice. Both transverse and sagittal sections of frozen brain were cut 20 microns thick and thaw-mounted onto gelatin-coated slides. Three consecutive sections were cut at intervals throughout the brain, mounted onto three slides, labeled A, B, and C, and thusly treated: (A) incubated for 2 hr at room temperature in a 50 pM solution of 125I-melatonin; (B) incubated for 2 hr at room temperature in a 50 pM solution of 125I-melatonin plus 1 microM cold melatonin; (C) fixed in Clarke's fluid and stained with toluidine blue. After incubation, A (specific) and B (nonspecific) slides were washed three times in ice-cold Tris-HCl buffer (pH 7.7), air-dried, exposed to an X-ray film for 2 weeks at -20 degrees C, and then fixed and stained. Specific 125I-melatonin binding sites were found in the pars tuberalis (PT), the area of the suprachiasmatic nucleus (SCN), preoptic area (POA), fornix/mediolateral septal areas, hippocampus, and the cerebral cortex. 125I-melatonin did not bind in the hindbrain, midbrain, neurohypophysis, pars intermedia or pars distalis of the adenohypophysis, or the pineal.     

A comparative study on androgen metabolism in three invertebrate species

A comparative approach was taken in this study to evaluate androgen (androstenedione and testosterone) metabolism in three invertebrate species: the gastropod Marisa cornuarietis, the amphipod Hyalella azteca, and the echinoderm Paracentrotus lividus. The existence of 17beta/3beta-hydroxysteroid dehydrogenase (HSD) and 5alpha-reductase catalyzed reactions was demonstrated in all three species. Androstenedione was primarily converted to 5alpha-androstanedione in M. cornuarietis, while it was primarily metabolized to testosterone in P. lividus and H. azteca. In addition, and consistent with vertebrate findings, tissue specific pathways and sexual dimorphism in androgen metabolism were observed. Namely, testosterone was metabolized to dihydrotestosterone in P. lividus gonads (via 5alpha-reductase), and metabolized to 4-androstene-3beta,17beta-diol in the digestive tube (via 3beta-hydroxysteroid dehydrogenase). Furthermore, the synthesis of 17beta-reduced metabolites of androstenedione (testosterone and dihydrotestosterone) was 3- to 4-fold higher in males of M. cornuarietis than in females. Organotin compounds, which have been shown to interfere with some aspects of androgen metabolism, had no major effect on testosterone metabolism in any of the three species. Fenarimol enhanced 5alpha-reductase-mediated catalysis in gonads of P. lividus. Overall, results demonstrate the ubiquity of some androgen biotransformation processes in invertebrates and reveals interphyla differences in androgen metabolic pathways, and different sensitivity of these pathways to some xenobiotics.     

Characterization and distribution of receptors for the atrial natriuretic peptides in mammalian brain

Both rat 125I-labeled atrial natriuretic polypeptide [125I-ANP or atrial natriuretic factor fragment ANF-(99-126)] and human 125I-ANP [125I-alpha-ANP or human ANF-(99-126)] bind with high specificity and affinity (Kd = 20-80 pM) to an apparent single class of sites in guinea pig brain. The ligand selectivity pattern demonstrates that ANF-(101-126) greater than ANF-(99-126) greater than ANF-(103-125) greater than ANF-(103-123) on 125I-alpha-ANP binding sites. [International nomenclature starting at the end of the signal peptide of the recently sequenced prepropeptide is used; thus, ANF-(101-126) corresponds to the earlier designation ANF-(8-33), ANF-(103-123) to rat atriopeptin I, and ANF-(103-125) to rat atriopeptin II.] Similar results have been reported in peripheral tissues, which indicate that central and peripheral ANP binding sites have fairly similar structural requirements. In vitro receptor autoradiography shows that in the guinea pig brain, 125I-ANP binding sites are highly concentrated in the external plexiform layer of the olfactory bulb, subfornical organ, various thalamic nuclei, medial geniculate nucleus, and cerebellum. Lower densities are found in the central nucleus of the amygdala, dentate gyrus, hippocampus, and area postrema. Most remaining regions contain much lower densities of sites. In rat brain, 125I-ANP binding sites are differentially distributed, with high densities in the subfornical organ, area postrema, and linings of ventricles but low densities in the thalamus and cerebellum. In monkey brain, 125I-ANP binding sites are concentrated in the cerebellum. The presence of high densities of 125I-ANP binding sites in various brain regions strongly suggests the existence of a family of brain-heart peptides, in analogy to the well-known brain-gut peptides. Moreover, the extensive distribution of 125I-ANP binding sites in mammalian brain suggests that the possible roles of ANP/ANF-like peptides in brain are not restricted to the central regulation of cardiovascular parameters.     

Characterization of melatonin binding sites in the brain and retina of the frog Rana perezi

The aim of this study was to characterize 2-[125I]iodomelatonin binding sites in the neural retina and central nervous system (telencephalon, diencephalon, and optic tectum) of the anuran amphibian Rana perezi. Saturation and kinetic studies and pharmacological characterization revealed the existence of a unique melatonin-binding site that belongs to the Mel 1 receptor subtype. The affinity of this site is similar in all tissues studied (Kd, 10.5-12.8 pM), but the density varied from diencephalon and optic tectum, which exhibit the highest density, to telencephalon with the lowest. Neural retina showed an intermediate receptor density. This melatonin-binding site fulfills the requirements of a real hormone receptor; the binding is saturable, reversible, and inhibited by different melatonin agonists and antagonists. The affinity order of ligands is: 2-phenyl-melatonin = 2-I-melatonin > 6-Cl-melatonin = melatoninz > luzindole. Additionally, specific binding is decreased by non-hydrolysable GTP analogue, sodium, and by pretreatment of membranes with pertussis toxin. All these results suggest the existence of a widely distributed and pharmacologically homogeneous melatonin receptor of the subfamily Mel 1 in the nervous system of Rana perezi coupled to a Gi/o protein.     

Diurnal variations in melatonin binding sites in the hamster brain: impact of melatonin

The distribution of 125I-melatonin binding sites in the male Syrian hamster brain was recorded at 3 times over a 24 h period. The binding in the hypothalamus, hippocampus, medulla-pons and midbrain of the hamsters varied significantly over the 24 h period with different patterns and phases. No such variations were observed in the parietal cortex. Daily morning (10.00 h) or late afternoon (18.00 h) injections of melatonin for 28 days markedly increased the serum concentrations of melatonin at all times recorded. Serum concentrations of testosterone were significantly lower in animals injected with melatonin in the late afternoon than in the untreated controls; no such decrease was observed in animals injected in the morning despite the continuously elevated levels of circulating melatonin. The daily melatonin injections did not significantly affect 125I-melatonin binding in the hypothalamus, parietal cortex and medulla-pons. In the midbrain, 125I-melatonin binding decreased regardless of the time of injection. In the hippocampus, morning melatonin injections caused a marked decrease in 125I-melatonin binding at all times recorded whereas melatonin injected in the late afternoon led to a decrease in 125I-melatonin binding at 10.00 h only. These results indicate diurnal variations in 125I-melatonin binding sites in discrete brain areas of the golden hamster, persisting despite prolonged duration of elevated levels of circulating melatonin. The differential effects of timed melatonin injections on the hippocampal 125I-melatonin binding sites are positively correlated with the counter-antigonadal response produced by morning melatonin injections.     

Binswanger病与Alzheimer病脑电地形图对比研究

本文对照观察了Ｂｉｎｓｗａｎｇｅｒ病（ＢＤ）和Ａｌｚｈｅｉｍｅｒ病（ＡＤ）各３０例患者脑电地形图的变化。结果发现，ＢＤ患者局灶性和阵发性脑电改变的阳性率明显高于ＡＤ患者，并且双侧脑电功率谱不对称，以额、中央、枕区θ波多见，而ＡＤ患者双侧功率谙基本对称，以额、颞、顶区δ波多见。ＢＤ患者智能障碍程度与脑电异常改变有显著相关性，提示脑电地形图检查对ＢＤ和ＡＤ的鉴别诊断有参考价值。     

A comparative study of the inhibitors of fibrinolysis in human, dog and rabbit blood.

Comparison of sera from dog, rabbit and man shows that dog serum contains most fibrinolytic inhibitor and human serum least. The inhibitors are potentiated by a variety of inorganic salts, of which sodium chloride is most effective. Euglobulin fractions precipitated from rabbit and human plasma at pH 5.3 contain small amounts of inhibitory material, whereas little remains in the corresponding dog euglobulin or in euglobulins from all three species when precipitated at pH 6.0. The potentiation of fibrinolytic inhibitors by sodium chloride differs between the three species, suggesting that their composition is not identical. Chloroform extraction removes a proportion of the inhibitory material from each serum.     

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125I-labeled melatonin (125I-Mel), a potent melatonin agonist. 125I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent Kd of 2.3 +/- 1.0 x 10(-11) M and 2.06 +/- 0.43 x 10(-10) M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125I-Mel from its receptor. Furthermore, GTP[gamma S] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radio-activity corresponding to Mr greater than 400,000 and Mr ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[gamma S] before solubilization; only the Mr 110,000 peak was present in GTP[gamma S]-pretreated membranes. The results strongly suggest that 125I-Mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.     

A comparative ex vivo and in vivo study of day and night perception in teleosts species using the melatonin rhythm

The purpose of this study was to determine and compare the light sensitivity of two commercially important, phylogenetically different teleost species in terms of melatonin production. Three series of experiments were performed on both Atlantic salmon and European sea bass. First, a range of light intensities were tested ex vivo on pineal melatonin production in culture during the dark phase. Then, light transmission through the skull was investigated, and finally short-term in vivo light sensitivity trials were performed. Results showed that sea bass pineal gland ex vivo are at least 10 times more sensitive to light than that of the salmon. Light intensity threshold in sea bass appeared to be between 3.8 x 10(-5) and 3.8 x 10(-6) W/m2 in contrast to 3.8 x 10(-4) and 3.8 x 10(-5) W/m2 in salmon. These highlighted species-specific light sensitivities of pineal melatonin production that are likely to be the result of adaptation to particular photic niches. Light transmission results showed that a significantly higher percentage of light penetrates the sea bass pineal window relative to salmon, and confirmed that penetration is directly related to wavelength with higher penetration towards the red end of the visible spectrum. Although results obtained in vivo were comparable, large differences between ex vivo and in vivo were observed in both species. The pineal gland in isolation thus appeared to have different sensitivities as the whole animal, suggesting that retinal and/or deep brain photoreception may contribute, in vivo, to the control of melatonin production.     

Dopamine receptor binding in the corpus striatum of mammalian brain.

Specific binding of [3H]dopamine to membranes from the corpus striatum of rat and calf brain appears to involve the postsynaptic dopamine receptor. Specific [3H]dopamine binding is saturable, wnd with half-maximal binding in calf membranes at 7 nM. Apomorphine is about twice as potent as dopamine in competing for binding sites, whereas (-)norepinephrine is 5% as potent as dopamine and isoproterenol is virtually inactive. The relative potencies of phenothiazines as inhibitors of specific dopamine binding correlates with their clinical potencies and actions on the dopamine-sensitive adenylate cyclase.     

Adaptation of pulmonary stretch receptors in different mammalian species.

A consequence of the adaptation of pulmonary stretch receptors is that their pattern of discharge depends on both lung volume and its rate of change. For adaptation dependent, flow related information to modify the breathing pattern by a feedback process, appreciable receptor adaptation must take place during a single respiratory cycle. Moreover, for flow related information to have the same significance in all animals, the time course of stretch receptor adaptation would have to vary among species, being more rapid in small animals, with high respiratory frequencies. We recorded single fiber action potentials from 84 pulmonary stretch receptors in six species of mammals, ranging from hamster to dog. The response of the receptors to maintained transpulmonary pressures was similar in the different species,as was the time course of receptor adptation following sudden lung inflations to 5 and 10 cm H2O. These findings show that the behavior of pulmonary stretch receptors is similar in animals with widely differing respiratory frequencies, and that the significance of receptor adaptation for regulation of the breathing pattern must vary interspecifically.     

Aging of collagen: comparative rates in four mammalian species

Resistance to collagenase digestion was used to compare rates of collagen aging in rat, dog, macaque, and man. The apparent rates were different for each species. Rat collagen was uniformly and readily digested, even at low calcium ion concentration where human collagen is digested slowly. Dog, macaque and human collagens all showed evidence of a progressive, age-related, increased stabilization. Some older dog specimens showed a much greater extent of stabilization than human specimens of the same chronological age, but similar if relative lifespans are used. These results demonstrate the problems caused by uncritically extrapolating an age-related observation from a short-lived species such as rat to a long-lived species such as human.     

Characterization of melatonin receptors in the ram pars tuberalis: influence of light

The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20 degrees C, but unstable at 28 or 37 degrees C; 3. melatonin and [127I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 10(10) l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25 +/- 0.30 vs 1.01 +/- 0.17 fmol/mm2 pars tuberalis, p less than 0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.