title: G protein-coupled receptor internalization signaling is required for cardioprotection in ischemic preconditioning.
abstract: The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gbetagamma-sequestering peptide, betaARKct (TG betaARKct mice), to test whether the protection of PC is Gbetagamma-dependent. To test the role of G(i) protein, we used wild-type mice pretreated with the G(i) inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG betaARKct mice. To determine the mechanism of Gbetagamma-induced protection in PC, we investigated one of the downstream targets of Gbetagamma, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG betaARKct hearts; therefore, we investigated other targets of Gbetagamma. Recent studies suggest a role for Gbetagamma in GPCR internalization. We found that betaARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A1, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kgamma, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kgamma blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Tong
authors.forename: Howard A
authors.initials: HA
authors.lastname: Rockman
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Koch
authors.forename: Charles
authors.initials: C
authors.lastname: Steenbergen
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D058544:Class Ib Phosphatidylinositol 3-Kinase; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000502:physiology; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D044387:GTP-Binding Protein beta Subunits / Q000037:antagonists & inhibitors / Q000502:physiology*; D044388:GTP-Binding Protein gamma Subunits / Q000037:antagonists & inhibitors / Q000502:physiology*; D019157:Ischemic Preconditioning, Myocardial; D007527:Isoenzymes / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000502:physiology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009203:Myocardial Infarction / Q000473:pathology / Q000503:physiopathology; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D000077191:Wortmannin; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D000730:Androstadienes; D058544:Class Ib Phosphatidylinositol 3-Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D000077191:Wortmannin; D051552:beta-Adrenergic Receptor Kinases; C118981:G-protein Beta gamma; C546764:Pik3cg protein, mouse