Essential activation of PKC-δ in opioid-initiated cardioprotection

Abstract

Stimulation of the δ1-opioid receptor confers cardioprotection to the ischemic myocardium. We examined the role of protein kinase C (PKC) after δ-opioid receptor stimulation with TAN-67 ord-Ala2-d-Leu5-enkephalin (DADLE) in a rat model of myocardial infarction induced by a 30-min coronary artery occlusion and 2-h reperfusion. Infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of the area at risk (IS/AAR). Control animals, subjected to ischemia and reperfusion, had an IS/AAR of 59.9 ± 1.8. DADLE and TAN-67 administered before ischemia significantly reduced IS/AAR (36.9 ± 3.9 and 36.7 ± 4.7, respectively). The δ1-selective opioid antagonist 7-benzylidenenaltrexone (BNTX) abolished TAN-67-induced cardioprotection (54.4 ± 1.3). Treatment with the PKC antagonist chelerythrine completely abolished DADLE- (61.8 ± 3.2) and TAN-67-induced cardioprotection (55.4 ± 4.0). Similarly, the PKC antagonist GF 109203X completely abolished TAN-67-induced cardioprotection (54.6 ± 6.6). Immunofluorescent staining with antibodies directed against specific PKC isoforms was performed in myocardial biopsies obtained after 15 min of treatment with saline, chelerythrine, BNTX, or TAN-67 and chelerythrine or BNTX in the presence of TAN-67. TAN-67 induced the translocation of PKC-α to the sarcolemma, PKC-β1 to the nucleus, PKC-δ to the mitochondria, and PKC-ε to the intercalated disk and mitochondria. PKC translocation was abolished by chelerythrine and BNTX in TAN-67-treated rats. To more closely examine the role of these isoforms in cardioprotection, we utilized the PKC-δ selective antagonist rottlerin. Rottlerin abolished opioid-induced cardioprotection (48.9 ± 4.8) and PKC-δ translocation without affecting the translocation of PKC-α, -β1, or -ε. These results suggest that PKC-δ is a key second messenger in the cardioprotective effects of δ1-opioid receptor stimulation in rats.