Activation of Akt is essential for acetylcholine to trigger generation of oxygen free radicals

Objectives: Acetylcholine (ACh) receptor activation in the heart causes mitochondrial production of reactive oxygen species (ROS) that is dependent on mitochondrial K-ATP channel opening. Recent data show that Akt (also known as protein kinase B) is phosphorylated at its activation site following exposure to ACh. However, since no reliable Akt inhibitor is available, it has not been possible to determine whether Akt activation is an actual step in the protective pathway. Methods: Cultured rat vascular smooth muscle cells (A7r5) were transiently transfected with a dominant negative Akt (Akt-AAA), thus inhibiting the ability of ACh in these cells to phosphorylate Akt. Transfected cells were identified by co-transfection of enhanced green fluorescent protein (EGFP). ROS production was determined by incubating the cells for 15 min with 1 mM reduced MitoTracker Red which becomes fluorescent only after reacting with ROS. Cells were then triple-washed to remove any voltage-dependent pool of dye and single cell fluorescence was measured. Results: ACh exposure (2 mM) led to a 1.64 +/- 0.15-fold increase in the average fluorescence over that seen in untreated cells (P=0.002). A similar increase in ROS production occurred after treatment with either the K-ATP channel opener diazoxide (DIAZ) or the potassium ionophore valinomycin (VAL). Akt-AAA transfection abolished ACh-induced ROS production, but not increased ROS production after treatment with either DIAZ or VAL. Conclusion: Thus, at least in the smooth muscle cell model, Akt phosphorylation is an important step in the signal transduction pathway leading from ACh receptor activation to the generation of ROS. The experiments reveal that Akt is positioned between the receptor and the K-ATP channel in this model. (C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.