© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
Activation of Akt is essential for acetylcholine to trigger generation of oxygen free radicals
aDepartment of Physiology, University of South Alabama, Medical Science Building Room 3024, Mobile, AL 36688, USA
bDepartment of Cardiology, Ernst-Moritz-Arndt University, Greifswald, Germany
cDepartment of Pharmacology, University of South Alabama, Mobile, AL, USA
dDepartment of Medicine, University of South Alabama, Mobile, AL, USA
* Corresponding author. Tel.: +1-251-460-6818; fax: +1-251-460-6464. jdowney{at}usouthal.edu
Objectives: Acetylcholine (ACh) receptor activation in the heart causes mitochondrial production of reactive oxygen species (ROS) that is dependent on mitochondrial KATP 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 KATP 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 KATP channel in this model.
KEYWORDS Acetylcholine; Free radicals; K-ATP channel; Protein phosphorylation; Signal transduction; Smooth muscle
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Kinoshita, N. Matsuda, H. Kaba, N. Hatakeyama, T. Azma, K. Nakahata, Y. Kuroda, K. Tange, H. Iranami, and Y. Hatano Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH Oxidase in Adenosine 5'-Triphosphate-Sensitive K+ Channel Function Impaired by High Glucose in the Human Artery Hypertension, September 1, 2008; 52(3): 507 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Koneru, S. V. Penumathsa, M. Thirunavukkarasu, S. M. Samuel, L. Zhan, Z. Han, G. Maulik, D. K. Das, and N. Maulik Redox regulation of ischemic preconditioning is mediated by the differential activation of caveolins and their association with eNOS and GLUT-4 Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2060 - H2072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. V. Cohen, X.-M. Yang, and J. M. Downey Nitric oxide is a preconditioning mimetic and cardioprotectant and is the basis of many available infarct-sparing strategies Cardiovasc Res, May 1, 2006; 70(2): 231 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Hausenloy and D. M. Yellon Survival kinases in ischemic preconditioning and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 240 - 253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Schwartz and C. J. Lagranha Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1011 - H1018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Krieg, S. Philipp, L. Cui, W. R. Dostmann, J. M. Downey, and M. V. Cohen Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1976 - H1981. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Hausenloy, A. Tsang, M. M. Mocanu, and D. M. Yellon Ischemic preconditioning protects by activating prosurvival kinases at reperfusion Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H971 - H976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Krieg, Q. Qin, S. Philipp, M. F. Alexeyev, M. V. Cohen, and J. M. Downey Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2606 - H2611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Murphy Primary and Secondary Signaling Pathways in Early Preconditioning That Converge on the Mitochondria to Produce Cardioprotection Circ. Res., January 9, 2004; 94(1): 7 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fraser, B. M. Leung, X. Yan, H. C. Dan, J. Q. Cheng, and B. K. Tsang p53 Is a Determinant of X-Linked Inhibitor of Apoptosis Protein/Akt-Mediated Chemoresistance in Human Ovarian Cancer Cells Cancer Res., November 1, 2003; 63(21): 7081 - 7088. [Abstract] [Full Text] [PDF]
|
|