Balance of Superoxide and Nitric Oxide in Hyperoxic Lung Injury

• Project/Area Number: 10670560
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Respiratory organ internal medicine
• Research Institution: Tokai University
• Principal Investigator: TANIGAKI Toshimori Tokai University, School of Medicine, Assistant Professor, 医学部, 講師 (90246091)
• Co-Investigator(Kenkyū-buntansha): TSUJI Chizuko Tokai University, School of Medicine, Assistant Professor, 医学部, 講師 (80130079)
• Project Period (FY): 1998 – 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 1999: ¥400,000 (Direct Cost: ¥400,000); Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
• Keywords: hyperoxia / oxygen toxicity / acute lung injury / superoxide / nitric oxide synthase / selective inhibitor / L-NMMA / L-NIL / _L-NIL / _L-NMMA / 酸化窒素

Research Abstract

EXPERIMENT 1: To study the interaction of superoxide (OィイD22ィエD2-) and nitric oxide (NO) production in the pathogenesis of acute lung injury, we controlled NO production in the isolated lung exposed to OィイD22ィエD2- by alienation of the L-arginine concentration in the perfusate following in vivo exposure to lipopolysaccharide (LPS). Rats were divided lo three groups (n=6 in each): (G1) saline+ィイD2LィエD2-Arginine (3mM), (G2) LPS+ィイD2LィエD2-Arginine (3mM), (G3) LPS+ィイD2LィエD2-Arginine (0.1mM). The animals in G2 and G3 received LPS 6 hr before isolation of the lungs. Xathine oxidase (0.1 U/ml) was infused to generate OィイD22ィエD2- and 2-methyl-6-(p-methoxyphenyl)-3,7-dihydro-imiduo (1,2-a) pyrazin-3-one (MCLA; 6mM), OィイD22ィエD2-specific chemiluminescence probe was added to the perfusate. We estimated lung injury by measuring the concentration of lactate dehydrogenase (LDH) in the perfusate. Nitrite and nitrate in the perfusate were assayed using the Griess reaction. LPS administration increased O ィイD22ィエD2- production only slightly. Both NO production and LDH concentration increased significantly in G2 but not in G3 compared with G1. In conclusion, excess production of NO may exacerbate ROS mediated acute lung injury.
EXPERIMENT 2: The role of nitric oxide (NO) in pathogenesis of oxidant lung injury remains uncertain. We investigated the effect of two NO synthase (NOS) inhibitors, ィイD2LィエD2-NィイD16ィエD1-(1-iminoethyl)lysine (ィイD2LィエD2-NIL), highly selective inhibitor of iNOS, and ィイD2LィエD2-NG-monomethyl arginine (ィイD2LィエD2-NMMA), non-selective NOS inhibitor on hyperoxic lung injury in rats. Animals received ィイD2LィエD2-NIL, ィイD2LィエD2-NMMA or diluent intraperitoneally every 12 hr beginning 12 hr before exposure to 100% oxygen (OィイD22ィエD2) or air for a period of 50 hr. Absorption of the NOS inhibitors was confirmed by measurement of changes in the nitrite/nitrate concentration in serum. We measured wet-to-dry weight ratio (W/D) of lung tissue as an index of pulmonary edema and total protein concentration in bronchoalveolar lavage fluid (BALP) as an index of lung protein flux. We also evaluated hyperoxia-induced alternations in inducible NOS (iNOS) and endothelial NOS (eNOS) mRNA expression in lung tissue. OィイD22ィエD2 exposure increased W/D, BALP and serum nitrite/nitrate concentration. ィイD2LィエD2-NIL and ィイD2LィエD2-NMMA attenuated the OィイD22ィエD2 induced increases in nitrite/nitrate concentration but only ィイD2LィエD2-NMMA produced further significant increases in W/D and BALP above those with OィイD22ィエD2 exposure. Exposure to 100% OィイD22ィエD2 increased the expression of both iNOS and eNOS mRNA and this effect was not altered by ィイD2LィエD2-NIL or ィイD2LィエD2-NMMA, which acts as competitive NOS inhibitors. In conclusion, ィイD2LィエD2-NMMA but not ィイD2LィエD2-NIL accentuates hyperoxic lung injury suggesting that endogenous endothelial NO play a protective role in pulmonary oxygen toxicity.