title: Mitochondrial ROS generation following acetylcholine-induced EGF receptor transactivation requires metalloproteinase cleavage of proHB-EGF.
abstract: Acetylcholine (ACh) mimics ischemic preconditioning by a mechanism dependent on phosphatidylinositol 3-kinase (PI3-kinase) and reactive oxygen species (ROS). In other tissues muscarinic receptors activate a metalloproteinase, which liberates surface-associated heparin-binding epidermal growth factor (HB-EGF) and causes transactivation of epidermal growth factor receptors (EGFRs) with activation of PI3-kinase. We tested whether this pathway is operative in myocardium. Adult rabbit cardiomyocytes were incubated in reduced MitoTracker Red, which fluoresces after ROS exposure. ACh caused a 36 +/- 6% increase in fluorescence (P<0.001) and metalloproteinase inhibitor III (MPI) abolished this increase. Both exogenous EGF as well as HB-EGF caused similar increases in the ROS signal (41 +/- 12%, P=0.005 and 40 +/- 7%, P<0.001, respectively). The ROS burst from HB-EGF was unaffected by MPI (37 +/- 6%, P=0.002), confirming that inhibition of metalloproteinase activity blocked ACh's effect at a site upstream of EGFR. CRM-197, which inhibits HB-EGF activity, also blocked ACh-induced ROS generation, again implicating release of HB-EGF as a necessary step for ROS generation. An HB-EGF-neutralizing antibody also prevented ACh-induced increase in ROS. In isolated, perfused rabbit hearts ACh increased phosphorylation of EGFR by 127.4 +/- 43.7%, and this increase was abolished by MPI. Finally, ACh decreased infarct size from 30.1 +/- 2.9% of the risk zone in control hearts to 13.7 +/- 3.0% (P=0.002), and this protection could be abolished by co-treatment with MPI (28.7 +/- 2.6%, P=n.s. vs. control). Stimulation of a second Gi-protein-coupled receptor by the delta-opioid agonist [D-Ala2, D-Leu5]-enkephalin acetate (DADLE) also protected the heart (9.1 +/- 2.0% infarction, P<0.005 vs. control), and this protection was similarly blocked by MPI (28.9 +/- 2.3% infarction). We conclude that ACh-induced ROS generation in myocytes is mediated via transactivation of EGFR through metalloproteinase-dependent release of HB-EGF, and that this pathway is also operative in the intact heart and is required for ACh's cardioprotection.
authors.affiliation: Department of Physiology, College of Medicine, University of South Alabama, MSB 3074, Mobile, AL 36688, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Krieg
authors.forename: Lin
authors.initials: L
authors.lastname: Cui
authors.forename: Qining
authors.initials: Q
authors.lastname: Qin
authors.forename: Michael V
authors.initials: MV
authors.lastname: Cohen
authors.forename: James M
authors.initials: JM
authors.lastname: Downey
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D019157:Ischemic Preconditioning, Myocardial; D045726:Metalloproteases / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D009203:Myocardial Infarction / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000201:enzymology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000494:pharmacology; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D017382:Reactive Oxygen Species; D011956:Receptors, Cell Surface; D011976:Receptors, Muscarinic; D015842:Serine Proteinase Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D045726:Metalloproteases; D000109:Acetylcholine