Mechanism of oleic acid-induced gap junctional disassembly in rat cardiomyocytes.
This study investigated the mechanism of oleic acid (OA) on gap junctions and identified the protein kinase C (PKC) isoforms involved in OA-mediated gap junction disassembly in cardiomyocytes. Control cardiomyocytes showed continuous staining of the plasma membrane at cell-cell contact areas using antibodies reacting with connexin 43 (Cx43). The spontaneous contraction rate of cultured cardiomyocytes was reduced in a time-dependent manner by OA. In addition, Cx43 expression at cell-cell junction decreased, suggesting the disassembly of gap junction. Staining for PKC and PKCalpha, which were shown to colocalize with Cx43, also decreased with increased duration of OA treatment. The effects of OA on these distributional changes at cell junctions were reversed by 24 h incubation in fresh culture medium devoid of OA. Immunoprecipitation assays confirmed the biochemical binding between Cx43 and PKC/PKCalpha, and this protein interaction was not affected by OA. This may provide the basis for simultaneous detachment of Cx and PKC/PKCalpha from the cell-cell junction to the cytosol upon OA stimulation. Western blot analysis showed that OA-induced Cx43 Ser368 phosphorylation, and that this effect could be blocked by cotreatment with the general PKC inhibitor, calphostin C, the PKC inhibitor, eV1-2, or the Src kinase inhibitor, PP1, but not by the PKCalpha inhibitor, Gö6976. eV1-2 also prevented the OA-induced disassembly of gap junctions. Taken together, these data suggest that OA-induced Cx43 Ser368 phosphorylation is mediated by activation of PKC and Src kinase and might be responsible for OA-induced gap junctional disassembly.
Huang
Yuahn-Sieh
YS
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, 1-1 Jen-Ai Road, Taipei, Taiwan 100, ROC.
Tseng
Yung-Zu
YZ
Wu
Jiahn-Chun
JC
Wang
Seu-Mei
SM
D000818:Animals; D002450:Cell Communication / Q000187:drug effects; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D019301:Oleic Acid / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects
D018031:Connexin 43; D019301:Oleic Acid; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon