Osamu Inanami

Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats

Focal electrical stimulation of the magnocellular nucleus of the basal forebrain (nucleus basalis of Meynert; NBM) or a microinjection of L-glutamate (50 nmol) into the NBM increased cerebral cortical blood flow in the parietal lobe in urethane-anesthetized rats. The vasodilative responses were elicited only ipsilateral to the site of stimulation. Most of the vasodilative responses were abolished by intravenous administrations of muscarinic and nicotinic cholinergic blocking agents (atropine 0.5 mg/kg and mecamylamine 2 mg/kg). This suggests that the cholinergic projecting system sending fibers from the NBM to the parietal lobe contributes to the vasodilation of the cortex by activating muscarinic and nicotinic cholinergic receptors.

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase.

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes.

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum‐opsonized zymosan (sOZ) induced the activation of p38 mitogen‐activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3‐K) and sOZ‐induced O2 − production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3‐K). They caused significant attenuation of sOZ‐induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3‐K participated in both signaling pathways of NADPH oxidase activation (O2 − production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.

Oral administration of (-)catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil

The effect of ad libitum oral-administration of (-)catechin solution on ischemia-reperfusion-induced cell death of hippocampal CA1 in the gerbil was histologically examined. When (-)catechin solution instead of drinking water was orally administered ad libitum for 2 weeks, dose-dependent protection against neuronal death following by transient ischemia and reperfusion was observed. To evaluate the involvement of reduction of reactive-oxygen-species (ROIs) by the antioxidant activity of (-)catechin in this protection, the superoxide scavenging activity of the brain in catechin-treated gerbils was measured by ESR and spin-trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The superoxide scavenging activities of the brains obtained from catechin-treated gerbils were significantly higher than those of catechin-untreated animals. From these results, it was suggested that orally administered (-)catechin was absorbed, passed through the blood-brain barrier and that delayed neuronal death of hippocampal CA1 after ischemia-reperfusion was prevented due to its antioxidant activities.

Synthesis and characterization of a practically better DEPMPO-type spin trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO)

5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), a new cyclic DEPMPO-type nitrone was evaluated for spin-trapping capabilities toward hydroxyl and superoxide radicals. CYPMPO is colorless crystalline and freely soluble in water. Both the solid and diluted aqueous solution did not develop electron spin resonance (ESR) signal for at least 1 month at ambient conditions. CYPMPO can spin-trap superoxide and hydroxyl radicals in both chemical and biological systems, and the ESR spectra are readily assignable. Half life for the superoxide adduct of CYPMPO produced in UV-illuminated hydrogen peroxide solution was approximately 15 min, and in biological systems such as hypoxanthine (HX)/xanthine oxidase (XOD) the half-life of the superoxide adduct was approximately 50 min. In UV-illuminated hydrogen peroxide solution, there was no conversion from the superoxide adduct to the hydroxyl adduct. Although overall spin-trapping capabilities of CYPMPO are similar to DEPMPO, its high melting point, low hygroscopic property, and the long shelf-life would be highly advantageous for the practical use.

Nitric oxide (NO) is involved in increased cerebral cortical blood flow following stimulation of the nucleus basalis of Meynert in anesthetized rats

The effects of i.v. administration of a nitric oxide (NO) synthase inhibitor, L-NG-nitroarginine (L-NOArg), on the increase in cerebral cortical blood flow (cortical BF), following either electrical stimulation of the nucleus basalis of Meynert (NBM), whose cholinergic fibers project to the cortex, or hypercapnia with 10% CO2 inhalation, were studied in anesthetized rats. Cortical BF was measured using laser Doppler flowmetry. The threshold intensity of electrical stimulation of the NBM (0.5 ms, 50 Hz for 10 s) that induced an increase in regional cortical BF was defined as 1T. The cortical BF was increased on a stimulus intensity dependent manner at 1T-5T intensities tested. L-NOArg was administered cumulatively i.v. starting from 0.3 mg/kg, then 3 mg/kg, and 30 mg/kg. Time interval between each cumulative administration of L-NOArg was approximately 40 min. Three and 30 mg/kg of L-NOArg significant reduced the NBM stimulation-induced increase of cortical BF at intensities of 2T and 3T. The response at an intensity of 5T was reduced only by 30 mg/kg of L-NOArg to about half the control response. The reduced responses at 2T, 3T, and 5T were reversed following the i.v. administration of a physiological precursor of NO, L-arg (300 mg/kg). Inhalation of 10% CO2 for 15 s induced an increase in cortical BF which was not influenced by L-NOArg and L-Arg. These results suggest that NO is a necessary factor in the vasodilation of the cortical BF that is brought about by cholinergic fibers originating in the NBM.

Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells.

Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl-L-cysteine (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X(L) and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6h after X irradiation. Furthermore, the O(2)(-*) production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO-OOH was detected. This NADH/succinate-dependent O(2)(-*) production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O(2)(-*) production from mitochondria to trigger cytochrome c release in A549 cells.

Neuroprotective effect of α-phenyl-N-tert-butylnitrone in gerbil hippocampus is mediated by the mitogen-activated protein kinase pathway and heat shock proteins

α-Phenyl-N-tert-butylnitrone (PBN), a spin trap, is known as a protective agent against delayed-neuronal death after ischemia-reperfusion. To investigate this neuroprotective effect of PBN, we examined the effect of PBN on the mitogen-activated protein kinase (MAPK) signaling pathway and the expression of heat shock proteins (HSPs) in the gerbil hippocampus following transient (5 min) ischemia. Immunoblot analysis revealed that intraperitoneal (i.p.) injection of PBN (200 mg/kg) enhanced the activation of extracellular-response kinase (ERK) and suppressed the activation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (p38) at 6 h after ischemia. Elevated levels of HSP27 and HSP70 were seen at the same period. These data suggest that PBN protects against delayed-neuronal death not only by its inherent radical-trapping activity but also by regulating the MAPK pathway and up-regulating HSPs.

Attenuation of caspase-3-dependent apoptosis by Trolox post-treatment of X-irradiated MOLT-4 cells

PURPOSE The relationship between post-irradiation treatment with Trolox, an antioxidant that inhibits lipid peroxidation, and X-ray-induced apoptosis, with regard to signal transduction pathways, was examined in MOLT-4, a human leukaemia cell line. MATERIALS AND METHODS In MOLT-4 cells treated with Trolox after X-irradiation, viability, DNA fragmentation, expression of p53, BCL-2, BAX, active SAPK/JNK, active caspase-3 and the cleavage of PARP were measured by the trypan blue exclusion test, agarose gel electrophoresis and Western blotting. RESULTS Stained cells and ladder-like DNA cleavage were observed after X-irradiation. Cell death and DNA fragmentation were significantly inhibited by the post-irradiation treatment with Trolox. The expression of p53 and active SAPK/JNK was increased after X-irradiation, and fragments of PARP and the activated fragment of caspase-3 were produced. Post-irradiation treatment with Trolox attenuated the X-irradiation-induced expression, fragmentation or activation of these apoptosis-related biomolecules. The expression of BCL-2 and BAX, which would occur downstream from p53, was not changed by irradiation and Trolox treatment. Furthermore, cell death was associated with caspase-3 because the ladder-like DNA cleavage was completely inhibited by Ac-DEVD-CHO but not Ac-YVAD-CHO, TLCK and PMSF. CONCLUSION Post-irradiation events such as membrane damage induce caspase-3-dependent apoptosis, which might be mediated by the activation of SAPK/JNK and be independent of p53.

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.