Oxidative stress and adenosine A1 receptor activation differentially modulate subcellular cardiomyocyte MAPKs.
The mechanism by which distinct stimuli activate the same mitogen-activated protein kinases (MAPKs) is unclear. We examined compartmentalized MAPK signaling and altered redox state as possible mechanisms. Adult rat cardiomyocytes were exposed to the adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 500 nM) or H(2)O(2) (100 microM) for 15 min. Nuclear/myofilament, cytosolic, Triton-soluble membrane, and Triton-insoluble membrane fractions were generated. CCPA and H(2)O(2) activated p38 MAPK and p44/p42 ERKs in cytosolic fractions. In Triton-soluble membrane fractions, H(2)O(2) activated p38 MAPK and p42 ERK, whereas CCPA had no effect on MAPK activation in this fraction. The greatest difference between H(2)O(2) and CCPA was in the Triton-insoluble membrane fraction, where H(2)O(2) increased p38 and p42 activation and CCPA reduced MAPK activation. CCPA also increased protein phosphatase 2A activity in the Triton-insoluble membrane fraction, suggesting that the activation of this phosphatase may mediate CCPA effects in this fraction. The Triton-insoluble membrane fraction was enriched in the caveolae marker caveolin-3, and >85% of p38 MAPK and p42 ERK was bound to this scaffolding protein in these membranes, suggesting that caveolae may play a role in the divergence of MAPK signals from different stimuli. The antioxidant N-2-mercaptopropionyl glycine (300 microM) reduced H(2)O(2)-mediated MAPK activation but failed to attenuate CCPA-induced MAPK activation. H(2)O(2) but not CCPA increased reactive oxygen species (ROS). Thus the adenosine A(1) receptor and oxidative stress differentially modulate subcellular MAPKs, with the main site of divergence being the Triton-insoluble membrane fraction. However, the adenosine A(1) receptor-mediated MAPK activation does not involve ROS formation.
Ballard-Croft
Cherry
C
Department of Surgery, University of Kentucky, MN265 Chandler Medical Center, 800 Rose St., Lexington, KY 40536-0298, USa. ccrof2@uky.edu
Locklar
Adam C
AC
Keith
Byron J
BJ
Mentzer
Robert M
RM
Lasley
Robert D
RD
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D051244:Caveolin 3 / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D003902:Detergents; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D066298:In Vitro Techniques; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048308:Mitogen-Activated Protein Kinase 14 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D017830:Octoxynol; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D054648:Protein Phosphatase 2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D043682:Receptor, Adenosine A1 / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D008625:Tiopronin / Q000494:pharmacology
D058907:Adenosine A1 Receptor Agonists; D000975:Antioxidants; C495549:Cav3 protein, rat; D051244:Caveolin 3; D003902:Detergents; D016877:Oxidants; D017382:Reactive Oxygen Species; D043682:Receptor, Adenosine A1; C058394:2-chloro-N(6)cyclopentyladenosine; D017830:Octoxynol; D006861:Hydrogen Peroxide; D008625:Tiopronin; D019950:Mitogen-Activated Protein Kinase 1; D048308:Mitogen-Activated Protein Kinase 14; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D054648:Protein Phosphatase 2; D000241:Adenosine