In vitro antioxidant neuroprotective activity of BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation.

BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation, prevents in vivo brain ischemic/reperfusion injury. In the present study, BN 80933 was shown to protect neurons from hypoxia-induced cell death in primary cultures of cortical neurons. BN 80933 prevented lactate dehydrogenase activity elevation induced by hypoxia, displaying an IC50 value of 0.15 +/- 0.05 microM. This effect was likely due to the antioxidant properties of BN 80933 because Trolox, but not NG-nitro-L-arginine, also elicited protection. The antioxidant property of BN 80933 was then further investigated on HT-22 cells subjected to buthionine sulfoximine- or glutamate-induced glutathione depletion. The relative order of potency of the various compounds to inhibit oxidative stress-induced neuronal death (BN 80933 > U104067 > butylated hydroxytoluene > 17beta-estradiol > Trolox > vitamin E) correlated with their ability to inhibit brain membrane lipid peroxidation (correlation coefficient = 0.939). BN 80933 afforded protection even when added 6 h after glutamate exposure. BN 80933 did not reverse intracellular glutathione depletion but prevented elevation of the level of beta-epiprostaglandin F2alpha (8-isoprostane), which appeared to be a delayed phenomenon. In conclusion, BN 80933 induces a potent cytoprotection that may be mediated by inhibition of delayed lipid peroxidation.

BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation: a promising neuroprotective strategy.

Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [(S)-N-[4-[4-[(3,4-dihydro-6-hydroxy-2, 5,7, 8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2- thiophenecarboximidamide], a compound that combines potent antioxidant and selective nNOS inhibitory properties in vitro, affords remarkable neuronal protection in vivo. Intravenous administration of BN 80933 significantly reduced brain damage induced by head trauma in mice, global ischemia in gerbils, and transient focal ischemia in rats. Treatment with BN 80933 (0.3-10 mg/kg) significantly reduced infarct volume (>60% protection) and enhanced behavioral recovery in rats subjected to transient (2-h) middle cerebral artery occlusion and 48-h or 7-day reperfusion. Furthermore, treatment with BN 80933 commencing up to 8 h after the onset of ischemia resulted in a significant improvement of neurological outcome. All these results indicate that BN 80933 represents a class of potentially useful therapeutic agents for the treatment of stroke or trauma and possibly neurodegenerative disorders that involve both NO and ROS.

Endothelin receptor subtypes A and B are up-regulated in an experimental model of acute renal failure.

The two endothelin (ET) receptor subtypes (ETA and ETB) have been characterized in rat kidney from normal rats and rats with acute renal failure induced by hypertonic glycerol administration. In control rats, the total number of ET receptors in kidney cortex and medulla was 155 and 386 fmol/mg of protein, respectively. The ratio of ETA to ETB receptors was 54:46 in renal cortex and 35:65 in renal medulla. Treatment of rats with 10 ml/kg glycerol (50%, w/v) intramuscularly resulted in severe renal dysfunction; the serum urea concentration increased from 0.46 to 2.65 g/liter and the creatinine clearance decreased from 1.06 to 0.30 ml/min. Ligand binding studies showed that glycerol-induced acute renal failure was associated with a marked up-regulation of ETA and ETB receptor subtypes in both cortex and medulla. In glycerol-treated rats, the total ET receptor density in kidney cortex and medulla was increased to 294 and 1172 fmol/mg of protein, with ETA/ETB ratios of 52:48 and 31:69, respectively. The upregulatory effect of glycerol treatment was significantly more pronounced in renal medulla than renal cortex and affected ETB receptors preferentially, compared with ETA receptors. Subsequently, ETA and ETB receptor mRNA levels were markedly increased by glycerol administration in both kidney cortex and medulla, as assessed by polymerase chain reaction coupled to reverse transcription. These results suggest that up-regulation of renal ET receptors, particularly ETB receptors in kidney medulla, may account for or contribute to renal function impairment induced by glycerol, and they support a pathophysiological role for ET in acute renal failure.

Endothelin receptor regulation by endothelin synthesis in vascular smooth muscle cells: effects of dexamethasone and phosphoramidon.

One of the major biological effects of the endothelium-derived peptide endothelin-1 (ET-1) is its receptor-mediated constrictive action on vascular smooth muscle. In this study, we have examined the effects on the ET-1 pathway of 18 h exposure at 37 degrees C of cultured rat aortic smooth muscle cells to dexamethasone (DEX) and phosphoramidon. ET-1 synthesis was evaluated by radioimmunoassay, ET-1 binding characteristics were determined with [125I]iodo-ET-1, and ET-1-induced intracellular calcium mobilization was measured using fura-2-loaded cells. DEX (100 nM) led to a 2- to 3-fold-increase of ET-1 production, it down-regulated ET-1 receptors and reduced ET-1-stimulated calcium mobilization by 70%. In contrast, phosphoramidon (100 microM) inhibited ET-1 production by 60%, up-regulated ET-1 receptors and potentiated ET-1-induced calcium mobilization by 75%. These results indicate that the regulatory effects of DEX and phosphoramidon on ET-1 receptors are mediated via ET-1 production by the cells. This suggests an autocrine control of ET-1 receptors by endogenous ET-1 synthesis in vascular smooth muscle cells.