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

Tetsuji Miura,¹ Toshiyuki Yano,¹ Kazuyuki Naitoh,¹ Masahiro Nishihara,¹ Takayuki Miki,¹ Masaya Tanno,^1,2 , and Kazuaki Shimamoto¹

¹Second Department of Internal Medicine and ²Department of Pharmacology, Sapporo Medical University School of Medicine, Sapporo, Japan

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-Ala²,D-Leu⁵]-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- inhibitor [PKC- translocation inhibitory peptide (PKC--TIP)] but was not affected by a PKC- inhibitor (rottlerin). After DADLE infusion, PKC-, but not PKC-, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser³⁶⁸) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC--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 + DADLE, which allows PKC- activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC- 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.

Key Words: infarct size • preconditioning

Address for reprint requests and other correspondence: T. Miura, Second Dept. of Internal Medicine, Sapporo Medical Univ. School of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan (e-mail: miura{at}sapmed.ac.jp)

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