Reactive oxygen species potentiate the negative inotropic effect of cardiac M-2-muscarinic receptor stimulation

The aim of the present study was to investigate the influence of reactive o xygen species (ROS) on the contractile responses of rat isolated left atria to muscarinic receptor stimulation. ROS were generated by means of electro lysis (30 mA, 75 s) of the organ bath fluid. Twenty minutes after the elect rolysis period, the electrically paced atria (3 Hz) were stimulated with th e adenylyl cyclase activator forskolin (I muM). Subsequently, cumulative ac etylcholine concentration-response curves were constructed (0.01 nM-10 muM) . In addition, phosphoinositide turnover and adenylyl cyclase activity unde r basal and stimulated conditions were measured. For these biochemical expe riments we used the stable acetylcholine analogue carbachol. The atria exposed to reactive oxygen species were influenced more potently (pD(2) control: 6.2 vs. 7.1 for electrolysis-treated atria, P <0.05) and mo re effectively (E-max control: 40% vs. 90% reduction of the initial amplitu de, P <0.05) by acetylcholine. In contrast. ROS exposure did not alter the responses to adenosine, whose receptor is also coupled via a G(i)-protein t o adenylyl cyclase The basal (-40% vs. control, P <0.05) as well as the carbachol-stimulated ( -85% vs. control, P <0.05) inositolphosphate formation was reduced in atria exposed to ROS. The forskolin-stimulated adenylyl cyclase activity was ide ntical in both groups but carbachol stimulation induced a more pronounced r eduction in adenylyl cyclase activity in the electrolysis-treated atria. Accordingly we may conclude that ROS enhance the negative inotropic respons e of isolated rat atria to acetylcholine by both a reduction of the positiv e (inositide turnover) and increase of the negative (adenylyl cyclase inhib ition) inotropic components of cardiac muscarinic receptor stimulation. Thi s phenomenon is most likely M-2-receptor specific, since the negative inotr opic response to adenosine is unaltered by ROS exposure.