Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-epsilon to RACK2 in adult rat and mouse.
Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A(1) receptors (A(1)R) on the adrenergic signaling pathway. An indirect A(1)R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-epsilon to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A(1)R activation of PKC-epsilon induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A(1)R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-epsilon and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-epsilon to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A(1)R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-epsilon co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-epsilon with RACK2 by 61%. With rat cardiomyocytes, the beta-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A(1)R stimulation of the heart is associated with PLC-initiated PKC-epsilon translocation and association with RACK2.
Fenton
Richard A
RA
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. richard.fenton@umassmed.edu
Komatsu
Satoshi
S
Ikebe
Mitsuo
M
Shea
Lynne G
LG
Dobson
James G
JG
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D000367:Age Factors; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002478:Cells, Cultured; D020756:Coatomer Protein / Q000276:immunology / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008297:Male; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043682:Receptor, Adenosine A1 / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014970:Xanthines / Q000494:pharmacology
D058907:Adenosine A1 Receptor Agonists; D058916:Adenosine A1 Receptor Antagonists; D000906:Antibodies; D020756:Coatomer Protein; C523489:Copb1 protein, mouse; C543179:Copb1 protein, rat; D004963:Estrenes; D008565:Membrane Proteins; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D043682:Receptor, Adenosine A1; D014970:Xanthines; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C058394:2-chloro-N(6)cyclopentyladenosine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D051744:Protein Kinase C-epsilon; D010738:Type C Phospholipases; D000241:Adenosine