Masashi Katsura

Nitric oxide-evoked [3H]γ-aminobutyric acid release is mediated by two distinct release mechanisms

Mechanisms underlying the release of [3H] gamma-aminobutyric acid (GABA) evoked by nitric oxide (NO) were investigated by use of primary cultured neurons prepared from the mouse cerebral cortex. NO generators such as sodium nitroprusside (SNP) and S-nitroso-N-a etylpenicillamine (SNAP) increased both [3H]GABA release from the neurons and [45Ca2+] influx into the neurons in a dose-dependent manner, which was significantly diminished by hemoglobin. The removal of Ca2+ significantly reduced the NO-induced [3H]GABA release by about 50%. Nipecotic acid and 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1, 2, 5, 6-tetrahydro-3- pyridinecarboxylic acid (NO-711), GABA uptake inhibitors dose-dependently inhibited the NO-evoked [3H]GABA release in either the presence or absence of Ca2+. The concentration of these GABA uptake inhibitors to suppress the NO-induced release of [3H]GABA was sufficiently lower than that to exhibit the inhibition of [3H]GABA transport into the neurons. In addition, the NO-evoked [3H]GABA release was reduced by approximately 50% when total Na+ in incubation buffer was replaced with equimolar choline, and was also completely abolished by the removal of both Ca2+ and Na+. These results indicate that the release of [3H]GABA evoked by NO is mediated by two release mechanisms, a Ca2+ -dependent release system and the reverse process of the Ca2+ -independent and Na+ -dependent carrier-mediated GABA uptake system.

Participation of peroxynitrite in acetylcholine release induced by nitric oxide generators

Peroxynitrite is a product produced by spontaneous reaction of nitric oxide (NO) with superoxide. Functional roles of peroxynitrite in the release of endogenous acetylcholine evoked by NO generators has been examined using primary-cultured cerebral cortical neurons. NO generators, such as sodium nitroprusside and S-nitroso-N-acetylpenicillamine, dose-dependently increased the release of acetylcholine (ACh); such increase of the release was significantly suppressed by hemoglobin which has the capacity to abolish biological effects of NO. Two types of superoxide scavengers, Cu2+, Zn2+ superoxide dismutase and ceruloplasmin, significantly reduced the NO-evoked ACh release. These results indicate that NO requires superoxide to evoke the release of ACh. Synthesized peroxynitrite evoked the release of ACh from cerebral cortical neurons in a dose-dependent manner. Thus, it is indicated that the NO-evoked ACh release is mediated, at least in part, by peroxynitrite produced by the reaction of NO with superoxide.

Involvement of peroxynitrite in N-methyl-D-aspartate- and sodium nitroprusside-induced release of acetylcholine from mouse cerebral cortical neurons.

Functional roles of peroxynitrite in N-methyl-D-aspartate (NMDA)- and sodium nitroprusside (SNP)-evoked releases of acetylcholine (ACh) from cerebral cortical neurons in primary culture have been investigated. NMDA increased the release of ACh in a dose-dependent manner, which was significantly suppressed by (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801), a non-competitive antagonist specific for the NMDA receptor complex, and NO synthase inhibitors. SNP also showed a concentration-dependent increase in ACh release. Hemoglobin significantly abolished the stimulatory effects of both NMDA and SNP on ACh release. In addition, superoxide anion scavengers such as superoxide dismutase and ceruloplasmin significantly reduced the increased ACh release evoked by NMDA and SNP. Synthesized peroxynitrite dose-dependently elevated the release of ACh. These results indicate that the increased release of ACh by NMDA and SNP is mediated through peroxynitrite formed in the reaction of superoxide anion with nitric oxide produced by NMDA receptor activation and liberated from SNP rather than nitric oxide itself.

Presence of (NMDA) receptors in neuroblastoma × glioma hybrid NG108-15 cells-analysis using [45Ca2+]influx and [3H]MK-801 binding as functional measures

In the present study, we have attempted to clarify whether neuroblastoma glioma hybrid NG 108-15 cells (NG cells) possess the NMDA receptor complex using [45Ca2+]influx and [3H]MK-801 binding as functional measures. Glutamate and NMDA dose-dependently increased [45Ca2+]influx and these increases were further enhanced by glycine. Scatchard analysis revealed the presence of a high-affinity binding site for [3H]MK-801 with a KD of 18.8 nM and a Bmax of 0.328 pmol/mg protein. This [3H]MK-801 binding was also increased by NMDA in a dose-dependent manner and this increase was further enhanced by glycine. Both ketamine and MK-801 inhibited glutamate- and NMDA-induced [45Ca2+]influx as well as the increase of [3H]MK-801 binding in a dose-dependent manner. Similarly, Mg2+ and Zn2+ dose-dependently reduced both glutamate-induced [45Ca2+]influx and [3H]MK-801 binding. Spermine, one of the polyamines, showed a biphasic stimulatory effects on glutamate-induced [45Ca2+]influx and [3H]MK-801 binding. These results indicate that NG cells possess a pharmacologically distinct NMDA receptor complex and suggest that these cells may be useful for the analyses on pharmacological and biochemical characteristics of the NMDA receptor complex.

Role of peroxynitrite in [3H]γ-aminobutyric acid release evoked by nitric oxide and its mechanism

Role of peroxynitrite in [3H]γ-aminobutyric acid (GABA) release evoked by N-methyl-d-aspartate (NMDA) and S-nitroso-N-acetyl-penicillamine (SNAP) and mechanisms of [3H]GABA release induced by peroxynitrite in comparison with those induced by NMDA and SNAP were investigated using cerebrocortical neurons. NMDA dose dependently increased [3H]GABA release, which was significantly inhibited by hemoglobin and superoxide scavengers, Cu2+, Zn2+-superoxide dismutase and ceruloplasmin. The NMDA-evoked [3H]GABA release was significantly suppressed by GABA transport inhibitors and inhibitors of voltage-dependent L-typed Ca2+ channel. The SNAP-evoked [3H]GABA release was significantly reduced by Ca2+ withdrawal and by GABA transport inhibitors either in the presence or absence of Ca2+. Similar patterns of [3H]GABA release induced by peroxynitrite were observed. These results indicate that peroxynitrite formed by the reaction of NO with superoxide participates, in part, in the release of [3H]GABA induced by NMDA and SNAP.

Ethanol stimulates diazepam binding inhibitor (DBI) mRNA expression in primary cultured neurons.

Changes in expression of diazepam binding inhibitor (DBI) mRNA in cerebral cortical neurons following long-term ethanol (EtOH) exposure were examined. A significant increase in DBI mRNA expression was observed by the exposure of neurons to 50 mM EtOH for up to 5 days and to EtOH (1-100 mM) for 3 days. These EtOH-induced increases in DBI mRNA expression were further elevated after the additional cultivation of neurons under EtOH-free condition. beta-Actin mRNA expression was not altered by similar EtOH treatments. These results indicate that EtOH possesses the activity to increase the expression of DBI mRNA in cerebral cortical neurons.

Nicotine increases diazepam binding inhibitor (DBI) mRNA in primary cultured neurons

The effect of nicotine on the expression of diazepam binding inhibitor (DBI) mRNA in primary cultured cerebral cortical neurons was examined using Northern blot analysis. Nicotine exposure (0.001-10 microM) for 24 h increased the DBI mRNA level in a dose-dependent manner, whereas the beta-actin mRNA level showed no change. This effect of nicotine was faded out over 48 h of its exposure. Hexamethonium (100 microM) completely abolished the nicotine-induced increase in DBI mRNA expression. These results indicate that nicotine increases the expression of DBI mRNA in cerebral cortical neurons via the activation of nicotinic acetylcholine receptor.

GABAA RECEPTOR STIMULATION ENHANCES NMDA-INDUCED CA2+ INFLUX IN MOUSE CEREBRAL CORTICAL NEURONS IN PRIMARY CULTURE

The effect of GABAA receptor stimulation on N-methyl-D-aspartate(NMDA)-induced [45Ca2+]influx has been examined using primary cultured cerebral cortical neurons. NMDA induced a dose-dependent increase in [45Ca2+]influx, which was blocked by MK-801 in a dose-dependent manner. GABAA receptor agonists significantly enhanced the NMDA-induced [45Ca2+]influx, and this enhancement was dose-dependently inhibited by bicuculline, although picrotoxin and tert-butyl-bicyclo[2.2.2]phosphoro-thionate (TBPS) exhibited no alterations in this stimulatory action of GABAA receptor agonists. Blockers of L-type voltage-dependent calcium channels significantly reduced the NMDA-induced [45Ca2+]influx. The increased [45Ca2+]influx by both NMDA and GABAA receptor agonists was also reduced by verapamil and nifedipine. These results suggest that the enhancement of NMDA-induced [45Ca2+]influx by GABAA receptor stimulation in immature cerebral cortical neurons may be due to the increased opening of voltage-dependent calcium channel by synergestic actions between NMDA and GABAA receptors.

Ethanol-induced alteration in activities of cerebral phosphatidylinositol 4,5-biphosphate-specific and cytosolic phospholipase C in the brain : analysis using NG 108-15 cells and brains from ethanol-inhaled mice

Effect of long-term exposure to ethanol (EtOH) on the phosphatidylinositol 4,5-biphosphate (PIP2)-specific and cytosolic phospholipase C (PLC) activities in neuroblastoma x glioma hybrid (NG 108-15) cells and the brains from EtOH-inhaled mice were investigated. Long-term (2 days) exposure of NG 108-15 cells to EtOH induced significant decrease in PIP2-specific PLC activity dependent on concentration and duration of exposure, although the presence of EtOH in the enzyme assay system induced no alteration in PIP2-specific PLC activity. On the other hand, cytosolic PLC activity in NG 108-15 cells significantly increased by both the long-term exposure of the cells to EtOH and the addition of EtOH into the assay system. These changes in activities of both types of PLC in NG 108-15 cells observed after EtOH exposure recovered rapidly by the removal of EtOH. Moreover, the changes in activities of PIP2-specific and cytosolic PLC in the brain of EtOH-inhaled mice were similar to those found in NG 108-15 cells. These results indicate that EtOH inhibits the activity of PIP2-specific PLC and activates cytosolic PLC in the brain. These changes in cerebral PLC activities are suggested to involve in central action of EtOH and establishment of alcohol dependence.

Hydroxyl radical scavengers enhance nitric oxide-evoked acetylcholine release from mouse cerebral cortical neurons

The effect of hydroxyl radical scavengers on acetylcholine (ACh) release evoked by nitric oxide (NO) generators and N-methyl-D-aspartate (NMDA) was investigated. Dimethylthiourea enhanced dose-dependently NO generators-evoked ACh release. Similarly, uric acid and mannitol significantly facilitated ACh release evoked by NO generators. The NMDA-induced ACh release was also significantly facilitated by hydroxyl radical scavengers. These scavengers themselves showed no effects on ACh release. These results suggest that hydroxyl radicals may modify the mechanism for NO-evoked ACh release.