Motohisa Suzuki

Concurrent formation of peroxynitrite with the expression of inducible nitric oxide synthase in the brain during middle cerebral artery occlusion and reperfusion in rats.

Peroxynitrite is assumed to play a crucial role in brain damage associated with the overproduction of nitric oxide (NO). The purpose of this study is to examine time-dependent changes of nitrite and nitrate (NOx) concentration in the circulation, and peroxynitrite formation as well as the expression of inducible nitric oxide synthase (iNOS) in the penumbra of rat brains during transient middle cerebral artery occlusion (MCAO) of Wistar rat for 2 h and reperfusion for 4-70 h. NOx concentration in the circulation was continuously monitored at the right jugular vein by microdialysis. The expression of iNOS was detected at 22-70 h after reperfusion in vascular walls and the cortex. Nitrotyrosine, a marker of peroxynitrite, appeared 4 h after reperfusion in the cortex, increasing substantially at 22-46 h in vascular walls. NOx level in dialysate increased immediately after MCAO. After a gradual decrease, the level increased again 4 h after reperfusion, reaching a maximum at 46 h. Brain myeloperoxidase activity, a marker of neutrophil infiltration, was not detected 4 h after reperfusion, but greatly increased at 22 h and then decreased. These results suggest that a marked increase of NOx level in the circulation might reflect the expression of iNOS, while neuronal NOS may contribute to peroxynitrite formation in the cortex observed at an earlier phase of reperfusion. This study indicates that monitoring NOx level in the circulation serves to assess the progress of stroke, and to determine appropriate therapeutic measures.

Mechanism of pathophysiological effects of diesel exhaust particles on endothelial cells.

The suspension of diesel exhaust particles (DEP) inhibited endothelium-dependent relaxation (EDR). The mechanism of the impairment of EDR by DEP was investigated with cultured porcine endothelial cells (PEC) and NO synthase (NOS) cell free system. Incubation of PEC with DEP (50-150 μg/ml) for 10-30 min did not induce cell damage. Bradykinin-induced endothelium-dependent relaxing factor (EDRF) release from PEC was bioassayed by cyclic GMP formation in RFL-6 cells. A 10-min preincubation of PEC with DEP (0.1-100 μg/ml) inhibited EDRF release. NOS activity from rat cerebellum cytosol was measured either by the conversion of 3H-l-arginine to (3)H-l-citrulline or the NO(2)(-) formation. A 10-min preincubation of NOS with DEP (0.1-100 μg/ml) did not affect the formation of (3)H-l-citrulline. In contrast, it inhibited NO(2)(-) formation. These results suggest that DEP neither induced cell damage nor inhibited EDRF release from PEC, but DEP scavenged NO to block its physiological action.

Fluctuation of serum NOx concentration at stroke onset in a rat spontaneous stroke model (M-SHRSP): Peroxynitrite formation in brain lesions

Malignant stroke-prone spontaneously hypertensive rats (M-SHRSP) develop hypertension and stroke at earlier ages than do nonmalignant SHRSP. Our previous findings suggested that reactive oxygen species were involved in the development of stroke in this strain. Nitric oxide (NO) which is more released at ischemia, might play a crucial role in stroke development by producing peroxynitrite, a neurotoxic substance. This study investigated whether the development of cerebrovascular lesion in M-SHRSP could be assessed by the fluctuation of serum NO(x) concentration, and whether peroxynitrite is associated with brain damage. Serum NO(x) levels were examined using an automated NO detector. Stroke-onset was temporally assessed according to a known method: changes in body weight, water intake, and neurologic symptoms. Cerebral lesions were confirmed by magnetic resonance imaging (MRI), and Evans blue extravasation at autopsy. MRI taken just after estimated stroke onset disclosed brain lesions. The baseline serum NO(x) level remained at 15-18 micromol/l, but the level gradually increased prior to stroke, and significantly at stroke onset. A marked rise in serum NO(x) occurred subsequently at poststroke. Immunohistochemical staining of nitrotyrosine, a peroxynitrite marker, was detected around vessels, neuronal cells and parenchyma in cerebral lesions. Stroke occurred in 50% of male M-SHRSP at 80 days of age. In conclusion, this study provides the first evidence for fluctuation of serum NO(x) at the onset of spontaneous stroke accompanying the appearance of peroxynitrite in brain lesions. Monitoring serum NO(x) would serve to assess the development of brain lesions at least in spontaneous stroke model.