Peters S L, Sand C, Batinik H D, Pfaffendorf M, van Zwieten P A
Department of Pharmacotherapy, Academic Medical Center, University of Amsterdam, The Netherlands.
Naunyn Schmiedebergs Arch Pharmacol. 2001 Aug;364(2):166-71. doi: 10.1007/s002100100442.
The aim of the present study was to investigate the influence of reactive oxygen species (ROS) on the contractile responses of rat isolated left atria to muscarinic receptor stimulation. ROS were generated by means of electrolysis (30 mA, 75 s) of the organ bath fluid. Twenty minutes after the electrolysis period, the electrically paced atria (3 Hz) were stimulated with the adenylyl cyclase activator forskolin (1 microM). Subsequently, cumulative acetylcholine concentration-response curves were constructed (0.01 nM-10 microM). In addition, phosphoinositide turnover and adenylyl cyclase activity under basal and stimulated conditions were measured. For these biochemical experiments we used the stable acetylcholine analogue carbachol. The atria exposed to reactive oxygen species were influenced more potently (pD2 control: 6.2 vs. 7.1 for electrolysis-treated atria, P<0.05) and more effectively (Emax control: 40% vs. 90% reduction of the initial amplitude, P<0.05) by acetylcholine. In contrast, ROS exposure did not alter the responses to adenosine, whose receptor is also coupled via a Gi-protein to adenylyl cyclase. The basal (40% vs. control, P<0.05) as well as the carbachol-stimulated (-85% vs. control, P<0.05) inositol-phosphate formation was reduced in atria exposed to ROS. The forskolin-stimulated adenylyl cyclase activity was identical in both groups but carbachol stimulation induced a more pronounced reduction in adenylyl cyclase activity in the electrolysis-treated atria. Accordingly we may conclude that ROS enhance the negative inotropic response of isolated rat atria to acetylcholine by both a reduction of the positive (inositide turnover) and increase of the negative (adenylyl cyclase inhibition) inotropic components of cardiac muscarinic receptor stimulation. This phenomenon is most likely M2-receptor specific, since the negative inotropic response to adenosine is unaltered by ROS exposure.
本研究的目的是探讨活性氧（ROS）对大鼠离体左心房毒蕈碱受体刺激收缩反应的影响。通过对器官浴液进行电解（30 mA，75 s）来产生ROS。电解期20分钟后，用腺苷酸环化酶激活剂福斯高林（1 microM）刺激电起搏的心房（3 Hz）。随后，构建累积乙酰胆碱浓度-反应曲线（0.01 nM - 10 microM）。此外，还测量了基础和刺激条件下的磷酸肌醇周转率和腺苷酸环化酶活性。对于这些生化实验，我们使用了稳定的乙酰胆碱类似物卡巴胆碱。暴露于活性氧的心房对乙酰胆碱的反应更敏感（pD2对照：6.2，电解处理的心房为7.1，P<0.05）且更有效（Emax对照：初始振幅降低40%，电解处理的心房降低90%，P<0.05）。相反，ROS暴露并未改变对腺苷的反应，腺苷受体也通过Gi蛋白与腺苷酸环化酶偶联。暴露于ROS的心房中，基础（40% vs.对照，P<0.05）以及卡巴胆碱刺激的（-85% vs.对照，P<0.05）肌醇磷酸形成均降低。两组中福斯高林刺激的腺苷酸环化酶活性相同，但卡巴胆碱刺激在电解处理的心房中诱导腺苷酸环化酶活性更明显降低。因此，我们可以得出结论，ROS通过降低心脏毒蕈碱受体刺激的正性（肌醇磷酸周转率）和增加负性（腺苷酸环化酶抑制）变力成分，增强了离体大鼠心房对乙酰胆碱的负性变力反应。这种现象很可能是M2受体特异性的，因为ROS暴露对腺苷的负性变力反应没有影响。
