δ-Opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-ε-mediated phosphorylation of connexin 43

Abstract

.-The aim of this study was to examine the hypothesis that ␦-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the ␦-opioid receptor activation. A ␦-opioid receptor agonist, [D-Ala 2 ,D-Leu 5 ]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-ε inhibitor [PKC-ε translocation inhibitory peptide (PKC-ε-TIP)] but was not affected by a PKC-␦ inhibitor (rottlerin). After DADLE infusion, PKC-ε, but not PKC-␦, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKCdependent site (Ser 368 ) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-ε-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin ϩ DADLE, which allows PKC-ε activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-ε plays a crucial role in ␦-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic ␦-opioid receptor activation. infarct size; preconditioning PRECONDITIONING (PC) with brief ischemia substantially salvages the myocardium from necrosis after sustained ischemia and reperfusion (22,35). This cardioprotective effect of PC has been demonstrated not only in intact hearts, but also in isolated cardiomyocytes subjected to simulated ischemia, indicating that PC can directly protect each cardiomyocyte under ischemia-reperfusion. However, the extent of protection by PC is generally less in isolated cell preparations than in whole hearts (1,19,22,35). Furthermore, there is also evidence to suggest that a part of myocardial protection by PC in whole hearts is achieved by chemical uncoupling of cardiomyocytes during ischemia-reperfusion, resulting in suppressed extension of injury within the area at risk (8,20,23,28). In our recent studies (20, 23), both ischemic PC and an opener of the mitochondrial ATP-sensitive K ϩ (mK ATP ) channel, which is involved in the PC mechanism, significantly reduced gap junction permeability to a chemical tracer in the ischemic rabbit myocardium.

Ischemia Enhances Translocation of Connexin43 and Gap Junction Intercellular Communication, Thereby Propagating Contraction Band Necrosis After Reperfusion

“…7 On the other hand, an excess of [Ca 2+ ]i causes GJ channel closure. 37 Consistent with a previous report in perfused rat heart, 38 in the present study GJs were functional in 30 min of ischemia as demonstrated by dye transfer assay. We conclude that brief ischemia enhances translocation of Cx43 to GJs, leading to GJIC enhancement and CBN spread in the risk area, whereas the non-risk area has low GJIC activity and no spread of CBN after reperfusion.…”

Functional suppression of E-cadherin by protein kinase Cδ

“…Unlike PKCδ, the function of PKCα at cell-cell junctions is thought to modulate desmosomal adhesion of epithelial cells in response to wounding (Wallis et al, 2000) and to increase endothelial permeability by the disruption of VE-cadherin junctions (Sandoval et al, 2001). As for PKCε, this has been shown to reduce gap junction permeability (Doble et al, 2000;Miura et al, 2007), weaken tight junctions (D'Souza et al, 2007) and suppress adherens junctions (Imamdi et al, 2004). PKCγ was recently reported to have a role in the regulation of gap junctions (Akoyev and Takemoto, 2007).…”

Exercise Preconditioning-Induced Early and Late Phase of Cardioprotection Is Associated With Protein Kinase C Epsilon Translocation

“…27 Phosphorylation of Cx43 by PKCε plays a crucial role in δ-opioid-induced suppression of GJ permeability in ischemic myocardium. 28 During the late phase of EP, PKCε did not translocate to intercalated disks, but myocardial injury was still significantly attenuated, as it was during the early phase of EP. The cause of this might be related to PKCε phosphorylation.…”