Machová J, Stefek M, Kukan M, Sinský M, Ondriás K, Racková L, Navarová J, Bauer V
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovakia.
Methods Find Exp Clin Pharmacol. 2003 May;25(4):287-96. doi: 10.1358/mf.2003.25.4.769677.
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.
本研究的目的是探讨通过电解克雷布斯 - 亨泽莱特溶液（GE - KHS）产生的活性氧（ROS）对豚鼠离体气管的影响，并评估一氧化氮（NO）在观察到的效应中可能的参与情况。将离体气管在GE - KHS中进行灌流，产生过氧化氢（H₂O₂）和次氯酸（HOCl），二者在器官浴液中均缓慢增加，当灌流速率为20 ml/min⁻¹时，最终稳定浓度分别达到42和63 μM，当灌流速率为5 ml/min⁻¹时，分别达到261和245 μM。当气管环分别以20 ml/min⁻¹和5 ml/min⁻¹的速率灌流时，在40%和65%的实验中，ROS GE - KHS诱导的气管环舒张之前会出现短暂的小收缩。去除气管上皮消除了ROS GE - KHS诱导的气管舒张，并使气管平滑肌收缩暴露或增强。ROS GE - KHS诱导的气管平滑肌张力变化伴随着气管中硫代巴比妥酸反应性物质（TBARS）增加和非蛋白（NP）硫醇减少。这些变化可被抗氧化剂N - 乙酰半胱氨酸（100 μM）处理所抑制。在暴露于ROS GE - KHS之前，用一氧化氮合酶（NOS）抑制剂Nⁿ - 硝基 - L - 精氨酸（L - NOARG；100 μM）预处理气管环20分钟，可降低ROS GE - KHS诱导的舒张。当在灌流溶液中存在L - NOARG（100 μM）时，不仅在暴露于ROS GE - KHS前20分钟，而且在灌流过程中，诱发的气管平滑肌舒张在前15分钟减少，但在第30分钟增强。这种舒张的后期增强伴随着一氧化氮代谢产物（NOₓ）增加12倍。用L - NOARG（100 μM）预处理可使ROS GE - KHS诱导的气管中TBARS水平升高降低至63%。用羟自由基产生系统孵育脑脂质体诱导的TBARS水平升高，L - NOARG（10、100 μM）可使其降低至90%，而抗氧化剂司他丁（100 μM）几乎完全抑制诱发的脂质过氧化。与曲洛昔芬相比，L - NOARG对1,1 - 二苯基 - 2 - 苦基肼自由基有轻微的清除作用。在气管暴露于ROS GE - KHS之前和期间，在灌流液中存在L - 精氨酸和D - 精氨酸15 - 20分钟，可抑制气管平滑肌舒张。结果表明，上皮来源的NO可能参与豚鼠气管对ROS GE - KHS的反应。在与ROS GE - KHS灌流期间，浴液中存在L - NOARG会产生具有舒张活性的NOₓ。L - 和D - 精氨酸诱导对ROS GE - KHS舒张反应的抑制，并部分防止ROS诱导的NP硫醇减少。L - NOARG以及L - 和D - 精氨酸的小抗氧化作用在上述L - NOARG和L - 精氨酸作用中的参与情况需要进一步研究。
