Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS. In the rabbit heart, bradykinin and ACh trigger preconditioning by a mechanism involving ATP-sensitive potassium channel-dependent production of reactive oxygen species (ROS). Recent evidence indicates that the pathway by which bradykinin causes ROS generation includes nitric oxide synthase (NOS) and protein kinase G (PKG). On the other hand, Akt was shown to be essential for ACh to

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... erate ROS. This study determines whether these two G-coupled receptor agonists indeed have similar signaling targets, i.e., whether Akt is involved in bradykinin's pathway and whether NOS is involved in ACh's pathway. Isolated adult rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 M) caused a 51.4 Ϯ 14.8% and 39.8 Ϯ 11.7% increase, respectively, in ROS production (P Ͻ 0.005). Coincubation of either agonist with Akt inhibitor (20 M) or infection of cells with an adenovirus containing dominant negative Akt abolished this increase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP, 1 M) also increased the ROS signal by 40.8 Ϯ 15.7%, but this increase was unaffected by Akt inhibitor (39.0 Ϯ 6.4%), implying that Akt is upstream of NOS. ACh-induced ROS production could be abolished by either of the NOS inhibitors N -monomethyl-L-arginine monoacetate (100 M) and L-N 5 -(1-iminoethyl)ornithine hydrochloride (L-NIO, 5 M). L-NIO also blocked the anti-infarct effect of ACh (550 M) in isolated rabbit hearts exposed to 30 min of regional ischemia. We conclude that both bradykinin and ACh trigger ROS generation by sequentially activating Akt and NOS