Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-{varepsilon} 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 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- 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- 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- 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 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- 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- 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, A 1 R stimulation of the heart is associated with PLC-initiated PKC- translocation and association with RACK2. adenosine A 1 receptor; protein kinase C- ; receptor for activated C kinase 2; rodent Address for reprint requests and other correspondence: R. A. Fenton, Dept. of Physiology, Univ. of Massachusetts Medical School, 55 Lake Ave., North, Worcester, MA 01655 (e-mail: richard.fenton{at}umassmed.edu )