Youji Kitamura

Release of vesicular Zn2+ in a rat transient middle cerebral artery occlusion model

In the brain, Zn(2+) is stored in synaptic vesicles of a subgroup of glutamatergic nerve terminals. Although it has been reported that this Zn(2+) is released upon the excitation of nerves in vitro, there has been little study of the release of Zn(2+) during ischemia in vivo. Here, using brain microdialysis, the release of vesicular Zn(2+) was investigated in vivo. When the vesicular Zn(2+) was released into the synaptic cleft by a depolarizing stimulation achieved by perfusion with Ringers solution containing high K(+) (100mM KCl), a significant increase in the extracellular concentration of Zn(2+) could be detected by microdialysis. Then, we investigated the release of vesicular Zn(2+) in a rat transient middle cerebral artery occlusion model using microdialysis. Consequently, the extracellular Zn(2+) level in the cortex increased within 15 min of the start of occlusion and reached a peak at 30 min, which was about twice the basal level. After 30 min, it declined with time returning to the basal level 15 min after reperfusion, which was performed after 60 min of occlusion. The results suggest that vesicular Zn(2+) would be released into the synaptic cleft during brain ischemia in vivo.

5-Iodo-A-85380, a specific ligand for α4β2 nicotinic acetylcholine receptors, prevents glutamate neurotoxicity in rat cortical cultured neurons

5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380, 5IA) has very high affinity and selectivity to nicotinic acetylcholine receptor (nAChR) alpha 4 beta 2 subtype, and a relative safe profile. To assess whether 5IA has neuroprotective properties, we examined the effect of 5IA on glutamate (Glu)-induced neurotoxicity using primary cultures of rat cortical neurons. A 10-min exposure of cultures to Glu followed by 2-h incubation with drug-free medium caused a marked loss of viability, as determined by trypan blue exclusion method. Glu-induced neurotoxicity was prevented by 5IA both in a time- and concentration-dependent manner. 5IA-induced neuroprotection required pretreatment of 5IA prior to Glu exposure with an optimal concentration of 10 nM and an optimal pretreatment time of 2 h. Treatment after Glu exposure could not rescue the cultured cells. The neuroprotective effect of 5IA was antagonized by mecamylamine, a nAChR antagonist, but not by scopolamine, a muscarinic acetylcholine receptor antagonist. Dihydro-beta-erythroidine, an alpha 4 beta 2 nAChR antagonist, completely inhibited 5IA-induced neuroprotection, whereas alpha-bungarotoxin, an alpha 7 nAChR antagonist, had no effect. Furthermore, 5IA did not show neuroprotective effects in the absence of extracellular Ca2+. These results suggest that the neuroprotective effects of 5IA are produced by activation of alpha 4 beta 2 nAChRs followed by the influx of extracellular Ca2+. In conclusion, 5IA is possibly not only useful for the treatment and prevention of glutamate neurotoxicity, but also as an available tool for elucidating the mechanism of neuroprotection associated with alpha 4 beta 2 nAChRs.

Peroxidase-like catalytic activity of Mn- and Fe-tetrakis(4-carboxyphenyl) porphines bound to aminopropyl-glass bead in oxidative reaction of heterocyclic amines

Fe- or Mn-tetrakis(4-carboxyphenyl)porphine (Fe- and Mn-TCPP) bound to aminopropyl-glass bead (Fe- and Mn-TCPP(g)s) was examined for the peroxidase (POD)-like function in order to develop a solid catalyst which can exhibit POD-like activity without adsorbing heterocyclic amines (HCAs). Mn-TCPP in aqueous solution had only a slight POD-like catalytic activity on HCAs (IQ and MeIQ). As for Fe-TCPP, it was impossible to examine the POD-like activity since it reacted with hydrogen peroxide in a liquid reaction system. However, both Fe- and Mn-TCPP when immobilized on aminopropyl-glass bead via peptide bond (Fe- and Mn-TCPP(g)s), catalyzed the oxidative reaction of mutagenic HCAs with hydrogen peroxide. The catalytic activity of Fe- and Mn-TCPP(g)s was investigated in more detail using as a substrate IQ and MeIQ which were oxidized more rapidly among the tested HCAs. Consequently, the optimal conditions for the oxidative reaction catalyzed by Fe- and Mn-TCPP(g)s were determined. In addition, ESI-mass and absorption spectra of oxidation products of IQ and MeIQ showed that they are dimers. Thus, it was demonstrated that a solid catalyst with POD-like activity can be obtained by immobilizing Fe- and Mn-TCPPs on aminopropyl-glass beads.

Peroxidase-like catalytic activity of Mn3+-octabromo-tetrakis(4-sulfophenyl)porphine on linoleate hydroperoxide and its analytical application

To reveal an enzyme-like catalytic activity of metal-octabromo-tetrakis(sulfophenyl)porphines (M-OBPSs), their peroxidease-like catalytic activity on linoleate hydroperoxide (LOOH) were evaluated on the basis of dye-formation in the coloring reaction between N,N-diethylaniline and 4-aminoantipyrine that yields a quinoid-type dye. Among M-OBPSs tested, Mn(3+)-OBPS allowed to produce the largest amount of dye. The optimal conditions of the coloring reaction catalyzed by Mn(3+)-OBPS for the determination of LOOH were determined. A good linear calibration curve was obtained in the concentration range of 0.025-0.4mumole LOOH with good reproducibility (coefficient of variance=1.23%), suggesting that Mn(3+)-OBPS is a good artificial mimesis of the peroxidase for LOOH. In addition, Mn(3+)-OBPS was highly specific for LOOH even in the presence of cumene hydroxyperoxide or hydrogen peroxide. It was revealed that the peroxidase-like activity of Mn(3+)-OBTP is attributable to the redox cycle of Mn(3+)<-->Mn(4+).

Effect of octabromination of a tetrakis(4-carboxyphenyl)porphine derivative bound to silica gels on HPLC retention behaviors of polyaromatic hydrocarbons

The effect of bromination of Cu-porphyrin-derivative-immobilized silica gels (Cu-TCPP(D)) was examined by comparing the retention behaviors of polyaromatic hydrocarbons (PAHs) on Cu-TCPP(D) and Cu-octabromotetrakis(4-carboxyphenyl)porphine-derivative-immobilized silica gels (Cu-OBTCPP(D)) columns. It was revealed that bromination affects strongly the pi-pi electron interactions caused from hydration energy in a polar eluent (80% methanol) possibly as a result of a destruction of planar structure of porphine-ring by bromination. It was also revealed that bromination enhances pi-d interactions as well as the pi-pi electron interactions in a broad sense (e.g., dispersion forces) in a non-polar eluent (n-hexane). However, the bromination did not exert much influence on electrostatic interactions caused from polarization of mono-halogenated benzenes.