title: Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.
abstract: We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.
authors.affiliation: Department of Pharmacology, University of Mississippi Medical Center, Jackson, MS, United States of America.
authors.forename: M R
authors.identifier: http://orcid.org/0000-0001-6353-9570
authors.initials: MR
authors.lastname: Pabbidi
authors.affiliation: Department of Physiology, Loyola University Medical Center, Maywood, IL, United States of America.
authors.forename: X
authors.initials: X
authors.lastname: Ji
authors.affiliation: Department of Physiology, Loyola University Medical Center, Maywood, IL, United States of America.
authors.forename: J T
authors.initials: JT
authors.lastname: Maxwell
authors.affiliation: Department of Physiology, Loyola University Medical Center, Maywood, IL, United States of America.
authors.forename: G A
authors.initials: GA
authors.lastname: Mignery
authors.affiliation: Department of Medicine, Loyola University Medical Center, Maywood, IL, United States of America.
authors.forename: A M
authors.initials: AM
authors.lastname: Samarel
authors.affiliation: Department of Physiology, Loyola University Medical Center, Maywood, IL, United States of America.
authors.forename: S L
authors.initials: SL
authors.lastname: Lipsius
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D002227:Carbazoles / Q000494:pharmacology; D002415:Cats; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D054513:Group IV Phospholipases A2 / Q000235:genetics / Q000378:metabolism*; D006325:Heart Atria / Q000166:cytology*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D002227:Carbazoles; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011758:Pyrroles; D018343:Receptors, Adrenergic, beta-2; C057416:KT 5720; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D054513:Group IV Phospholipases A2