Heitaroh Iwata

Localization of basic fibroblast growth factor-like immunoreactivity in the rat brain.

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the adult rat brain, using a specific antibody against a synthetic bFGF fragment (the N-terminal 12 residues). Widespread but uneven regional localization of bFGF-like immunoreactive neurons and fibers was observed. Ependymal cells were also stained. The immunoreactive neurons were found in the cerebral cortex, olfactory bulb, septum, basal magnocellular nuclei, thalamus, hypothalamus, globus pallidus, hippocampus, amygdala, red nucleus, central gray of the midbrain, cerebellum, dorsal tegmental area, reticular formation, cranial motor nuclei and spinal cord. Immunoreactive fiber bundles and nerve terminals were also detected. These results indicate that bFGF is produced by or present in a specific neuronal cell population of the central nervous system.

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P2) and slices of rat cerebral cortex were investigated. The P2 fraction had a Na+‐dependent high‐affinity uptake system for cysteine sulfinic acid (Km, 12μM), which was restricted to the synaptosomes. High‐affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [14C]cysteine sulfinic acid from preloaded cortical slices or P2 fractions was examined by a superfusion method, which avoided reuptake of released [14C]cysteine sulfinic acid. High K+ (56 mM) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P2 fraction in a partly Ca2+‐dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P2 fraction.

Microassay of cysteine sulfinic acid by an enzymatic cycling method.

Abstract A method for microassay of cysteine sulfinic acid (CSA) was developed. The principle of this method is the enzymatic conversion of CSA to lactate and the subsequent enzymatic cycling of NAD, which is a final by-product of the enzymatic conversion system. Asparatate, pyruvate, and NAD, which cause interference, were eliminated by two procedures. The absorbance of NADH was linearly proportional to the concentration of CSA over the concentration range of 5–50 pmol and CSA could be measured in as little as 50 μg of rat brain tissue. The regional and subcellular distributions of CSA in rat brain were studied.

Degradation of methylmercury by selenium

When methylmercury was incubated in the presence of selenite and reduced glutathione (GSH), the mercury which was extracted into benzene under acidic condition decreased gradually with the elapse of time. This decrease was due to the cleavage of mercury-carbon bond of methylmercury. The reaction did not proceed when selenite or GSH was singly added to the reaction mixture. L-Cysteine, 2-mercaptoethanol and sodium sulfide in place of GSH also were effective for decomposition of methylmercury in combination with selenite, but oxidized glutathione (GSSG) and L-cystine were not. This suggests that reduction of selenite is needed for the degradation of methylmercury. Thus, the effect of reduced metabolites of selenite produced by GSH was investigated. Glutathione selenotrisulfide (GSSeSG) required GSH for the degradation of methylmercury, whereas H2Se possessed a strong activity even in the absence of GSH. This may indicate that H2Se is involved directly in the conversion of methylmercury to inorganic mercury. This phenomenon found in in vitro experiments is discussed in relation to the biotransformation of methylmercury.

Involvement of tissue sulfhydryls in the formation of a complex of methylmercury with selenium.

Albumin-bound methylmercury was converted to a benzene-extractable form by the soluble fraction of rat liver, kidney or brain in the presence of selenite, but not in its absence. The factors in the soluble fraction causing this conversion were investigated by column chromatography. Sephadex G-25 chromatography showed that effective factors were present in non-protein and protein fractions. It was concluded from ion exchange and Sephadex G-200 chromatography that these factors in the non-protein and protein fractions were reduced glutathione (GSH) and protein sulfhydryl groups respectively. Because GSH and the soluble protein could be replaced by sulfhydryl compounds, such as cysteine and 2-mercaptoethanol, as well as by a purified protein with sulfhydryl groups, reduced ribonuclease (RNase), respectively, it was concluded that sulfhydryl groups of GSH and/or proteins in the soluble fraction were needed for selenite-induced conversion of methylmercury to a benzene-soluble form. Among the various selenium compounds tested, only H2Se (the reduced metabolite of selenite) was found to react directly with methylmercury to form a benzene extractable mercury compound in the absence of the soluble fraction. These findings suggest that the conversion of methylmercury to a benzene-soluble form occurs by reaction of methylmercury with selenium (possibly H2Se) reduced by GSH and/or protein sulfhydryl groups in the soluble fraction. Thin-layer chromatography showed that benzene-extractable mercury consists mainly of bis(methylmercuric) selenide (BMS). A minor component, trismethylmercuric selenonium, was also detected by mass spectrography.

L-GLUTAMATE-INDUCED SWELLING OF CULTURED ASTROCYTES IS DEPENDENT ON EXTRACELLULAR CA2+

L-Glutamate (L-Glu)-induced swelling of astrocyte cultures from rat brain was examined by determining [3H]O-methyl-D-glucose ([3H]OMG) uptake. Time-course of the L-Glu (0.5 mM)-induced increase in [3H]OMG space of astrocytes showed two phases; about 30% increase was obtained in 10 min and the increased [3H]OMG space was steady up to 20 min. Then, the [3H]OMG space was further increased during the incubations longer than 30 min. In Ca2(+)-free conditions, while the time-course up to 20 min was similar to that in the normal condition, the increase in [3H]OMG space by further incubations was not shown. The L-Glu-increased [3H]OMG space persisted for 2 h in a L-Glu-free medium and thereafter turned to the normal level in 4 h. In contrast, the incubation in a L-Glu-free medium for 30 min reversed the increased [3H]OMG space in the absence of extracellular Ca2+. These results indicate that swelling of astrocytes induced by L-Glu is characterized by an accompanied influx of Ca2+.

Possible regulation mechanism of microsomal glutathione S-transferase activity in rat liver.

After rats were injected with the reduced glutathione (GSH) depletor phorone (diisopropylidene acetone, 250 mg/kg, i.p.), there was a significant increase in microsomal glutathione S-transferase activity in the liver. The maximum activity was observed 24 hr after injection and was about 2-fold that of the control activity. Diethylmaleate (500 mg/kg, i.p.) had the same effect. Twenty-four hours after phorone injection (250 mg/kg, i.p.), the concentrations of GSH and oxidized glutathione (GSSG) in the liver were increased about 2-fold. Under the same conditions, the level of mixed disulfides with microsomal proteins (GSS-protein) was also increased. Further, the activity of microsomal glutathione S-transferases was increased by the in vitro addition of disulfide compounds such as GSSG, cystine and homocystine, and the activity increased by GSSG was reduced to control levels by incubating with the corresponding sulfhydryl compounds such as GSH, cysteine and homocysteine respectively. Thus, microsomal glutathione S-transferase activity appears to be regulated by the formation and/or cleavage of a mixed disulfide bond between the sulfhydryl group present in the enzyme and GSSG. Therefore, the increase of microsomal glutathione S-transferase activity after phorone injection may be due to the formation of a mixed disulfide bond between the sulfhydryl group in the enzyme and GSSG.

Inhibition by Diazepam and γ-Aminobutyric Acid of Depolarization-Induced Release of [14C]Cysteine Sulfinate and [3H]Glutamate in Rat Hippocampal Slices

Abstract: Effects of diazepam and γ‐aminobutyric acid‐related compounds on the release of [14C]cysteine sulfinate and [3H]glutamate from preloaded hippocampal slices of rat brain were examined by a superfusion method. Diazepam markedly inhibited the release of cysteine sulfinate and glutamate evoked either by high K+ or veratridine without affecting that of other neurotransmitter candidates, e.g., γ‐aminobutyric acid, acetylcholine, noradrenaline, and dopamine; IC50 values for the release of cysteine sulfinate and glutamate were about 20 and 7 μM, respectively. γ‐Aminobutyric acid (1 to 10 μM) and muscimol (100 μM) significantly reduced high K+‐stimulated release of glutamate. Bicuculline, which had no effect on the release at a concentration of 50 μM by itself, antagonized the inhibitory effects of diazepam and γ‐aminobutyric acid on glutamate release. Similar results were obtained with the release of cysteine sulfinate except that a high concentration (100 μM) of γ‐aminobutyric acid was required for the inhibition. These results indicate the modulation by γ‐aminobutyric acid innervation of the release of excitatory amino acids in rat hippocampal formation, and also suggest that some of the pharmacological effects of diazepam may be a consequence of inhibition of excitatory amino acid transmission.

Reversible inhibition of adenylate cyclase activity of rat brain caudate nucleus by oxidized glutathione

Abstract Basal and dopamine-stimulated adenylate cyclase (EC 4.6.1 1.) activities were strongly inhibited by GSSG, but not by GSH. Adenylate cyclase that had been inactivated by GSSG was reactivated by incubation with various sulfhydryl compounds including GSH. Formation of mixed disulfides by reaction between GSSG and protein-SH groups increased on incubation with GSSG and returned to the normal level on subsequent incubation with DTT.

Differential changes of glutathione S-transferase activity by dietary selenium

Dietary selenium deficiency produced increased activity of the glutathione S-transferases in the liver, kidney and duodenal mucosa. In these tissues, the residual activity of total glutathione peroxidase that included selenium-independent activity was considerably higher than that of selenium-dependent glutathione peroxidase. The enhanced activity of glutathione S-transferases was restored to control level 48 hr after an injection of selenite equivalent to the amount of daily selenium intake. Under the same conditions, selenium-dependent glutathione peroxidase activity increased with time and reached 11.9, 11.6 and 46.2% of the activity in the liver, kidney and duodenal mucosa of selenium-supplemented rats, respectively, 48 hr after selenite injection, whereas total glutathione peroxidase activity was not altered except in the kidney. These differential changes of glutathione S-transferase activity were intimately related to those of selenium-dependent glutathione peroxidase activity produced by selenium depletion and repletion, suggesting that the glutathione S-transferase activity was regulated by dietary selenium. Present findings support the idea that glutathione S-transferases having selenium-independent glutathione peroxidase activity function as a substitute for selenium-dependent glutathione peroxidase in selenium-deficient rats.