Adenosine A1 receptors selectively target protein kinase C isoforms to the caveolin-rich plasma membrane in cardiac myocytes

Adenosine is a naturally occurring nucleoside that has been shown to regulate a variety of functions in the cardiovascular system. However, the mechanisms in adenosine receptor signaling are not completely understood. Given that adenosine receptors have been linked to protein kinase C (PKC) in cardioprotection and caveolae is critical for receptor signaling, we sought to determine whether activation of adenosine A1 receptors induces selective translocation of PKC isoforms to the membrane from the cytosol and whether activated PKC is targeted to the caveolin-rich plasma membrane microdomains. The freshly isolated adult rat cardiac myocytes were used to examine PKC isoforms including PKC alpha, PKC beta, PKC epsilon, PKC delta and PKC zeta. Immunoblot analysis revealed that the immunoreactivity for PKC epsilon or PKC delta but not for PKC alpha, PKC beta or PKC zeta increased significantly in the membrane fractions from cells pretreated with the selective adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA, 100 nM) when compared with non-stimulated cells. The effect of CCPA on PKC epsilon or PKC delta translocation was blocked by adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM). When Western blot was performed from the caveolin-enriched plasma membrane fractions, the immunoreactivity for PKC epsilon or PKC delta but not PKC alpha, PKC beta or PKC zeta was enhanced significantly by CCPA. Furthermore, PKC epsilon and PKC delta were detected in the anti-caveolin-3 immunoprecipitates but not in the samples without primary antibody. Immunofluorescence staining further indicates increased colocalization of PKC epsilon or PKC delta with caveolin-3 at cell peripheral region and T-tubular-like structures in response to adenosine A1 receptor activation. In conclusion, we demonstrate that activation of adenosine A1 receptors promotes the selective translocation of PKC epsilon and PKC delta to the caveolin-enriched plasma membrane microdomains in cardiac myocytes. (C) 2009 Elsevier B.V. All rights reserved.