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This Article Full Text Full Text (PDF) All Versions of this Article: 297/2/H718    most recent 00247.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Fenton, R. A. Articles by Dobson, J. G. PubMed PubMed Citation Articles by Fenton, R. A. Articles by Dobson, J. G., Jr.

Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC- to RACK2 in adult rat and mouse

Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts

Submitted 11 March 2009 ; accepted in final form 9 June 2009

Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A₁ receptors (A₁R) on the adrenergic signaling pathway. An indirect A₁R-induced attenuation of adrenergic responsiveness involves the translocation of PKC- to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A₁R activation of PKC- 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 A₁R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC- and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC- to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A₁R, 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- co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A₁R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC- with RACK2 by 61%. With rat cardiomyocytes, the β-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, A₁R stimulation of the heart is associated with PLC-initiated PKC- translocation and association with RACK2.

adenosine A₁ receptor; protein kinase C-; receptor for activated C kinase 2; rodent