Met5-enkephalin-induced cardioprotection occurs via transactivation of EGFR and activation of PI3K
American Journal of Physiology. Heart and Circulatory Physiology
Cao, Zhiping, Lijuan Liu, and Donna M. Van Winkle. Met 5enkephalin-induced cardioprotection occurs via transactivation of EGFR and activation of PI3K. Our previous studies indicated that opioid-induced cardioprotection occurs via activation of mitochondrial ATP-sensitive K ϩ (KATP) channels. However, other elements of the Met 5 -enkephalin (ME) cardioprotection pathway are not fully characterized. In the present study, we investigated the role of tyrosine kinase, MAPK, and phosphatidylinositol
... osphatidylinositol 3-kinase (PI3K) signaling in ME-induced protection. Ca 2ϩ -tolerant, adult rabbit cardiomyocytes were isolated by collagenase digestion and subjected to simulated ischemia for 180 min. ME was administered 15 min before the 180 min of simulated ischemia; blockers were administered 15 min before ME. Cell death was assessed by trypan blue as a function of time. The epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 (250 nM) blocked ME-induced protection, but the inactive analog AG-9 (100 M) did not. Treatment with herbimycin (1 M) completely eliminated ME-induced protection. To verify that ME activates EGFR and to determine the involvement of Src, Western blotting of EGFR was performed after ME administration with and without herbimycin A. ME resulted in herbimycin-sensitive robust phosphorylation of EGFR at Tyr 992 and Tyr 1068 . Administration of the selective MAPK inhibitor PD-98059 (10 nM) and the specific MEK1/2 inhibitor U-0126 (10 M) also inhibited ME-induced cardioprotection. ME-induced ERK1/2 phosphorylation was significantly reduced by PD-98059, the EGFR kinase inhibitor PD-153035 (10 M), and chelerythrine (2 M). The PI3K inhibitor LY-294002 (20 M) abrogated ME-induced protection, and ME-induced Akt phosphorylation at Ser 473 was suppressed by LY-294002, PD-153035, and chelerythrine. We conclude that ME-induced cardioprotection is mediated via Src-dependent EGFR transactivation and activation of the PI3K and MAPK pathways. peptides; opioid; opioid receptor; ischemic preconditioning; signaling PRECONDITIONING DUE TO ACTIVATION of opioid receptors has been reported to be mediated by a kinase cascade involving protein kinase C (PKC) (20, 21, 24, 35) and via opening of the ATP-sensitive K ϩ (K ATP ) channels (4, 16, 18, 43, 45) . However, the details of the signal pathway involved in opioid peptide-induced cardioprotection have remained unclear. Opioid receptors belong to the superfamily of G protein-coupled receptors (GPCRs), and GPCR-induced epidermal growth factor (EGF) receptor (EGFR) signal transactivation occurs in a variety of cell types (8, 13, 55) . In cardiomyocytes, EGFR signal transactivation has previously been shown to be dependent on PKC activity (30, 47, 49) .