Involvement of L-arginine-nitric oxide system in the response of isolated trachea to reactive oxygen species.

Abstract

The aim of this study was to investigate the effect of reactive oxygen species (ROS), generated by electrolysis of Krebs-Henseleit solution (GE-KHS), on isolated guinea pig trachea and to assess the possible involvement of nitric oxide (NO) in the observed effects. The isolated trachea was superfused in GE-KHS, generating H2O2 and hypochlorous acid (HOCl), both of which slowly increased in the organ bath and reached final stable concentrations of 42 and 63 microM, respectively, at the rate of 20 ml/min(-1), and 261 and 245 microM, respectively, at the rate of 5 ml/min(-1). ROS GE-KHS-induced relaxation of tracheal rings was preceded by a small transient contraction in 40% and 65% of experiments when tracheal rings were superfused at the rate of 20 ml/min(-1) and 5 ml/min(-1), respectively. Removal of tracheal epithelium abolished the relaxation of the trachea induced by ROS GE-KHS and unmasked or potentiated trachealis contraction. The ROS GE-KHS-induced changes in trachealis tension were accompanied by an increase in thiobarbituric acid reactive substances (TBARS) and a decrease in nonprotein (NP) thiols in the trachea. These changes were inhibited by treatment with the antioxidant N-acetylcysteine (100 microM). Pretreatment of tracheal rings with the inhibitor of NO synthase (NOS) N(omega)-nitro-L-arginine (L-NOARG; 100 microM) for 20 min prior to exposure to ROS GE-KHS decreased the ROS GE-KHS-induced relaxation. When L-NOARG (100 microM) was present in the superfusing solution, not only 20 min before but also during superfusion with ROS GE-KHS, the evoked trachealis relaxation was reduced in the first 15 min but was enhanced in the 30th min. This late enhancement of relaxation was accompanied by a 12-fold increase in nitric oxide metabolites (NO(x)). ROS GE-KHS-induced elevation of TBARS levels in the trachea was decreased to 63% by pretreatment with L-NOARG (100 microM). Elevation of TBARS levels induced by incubation of brain liposomes with a hydroxyl radical generating system was decreased to 90% by L-NOARG (10, 100 microM), while the antioxidant stobadine (100 microM) nearly completely inhibited the evoked lipid peroxidation. In comparison with Trolox, L-NOARG exerted a slight scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl radical. The presence of L-arginine and D-arginine in the superfusion fluid for 15-20 min before and during exposure of the trachea to ROS GE-KHS inhibited trachealis relaxation. Results indicate that epithelium derived NO may participate in the response of guinea pig trachea to ROS GE-KHS. The presence of L-NOARG in the bathing fluid during superfusion with ROS GE-KHS gave rise to NO(x), with relaxing activity. L- and D-arginine induced an inhibition of the relaxatory response to ROS GE-KHS and partially prevented a ROS-induced decrease in NP thiols. The involvement of the small antioxidant effects of L-NOARG and L- and D-arginine in the above mentioned actions of L-NOARG and L-arginine requires additional investigation.