Nafenopin causes protein kinase c-mediated serine phosphorylation and lossof function of connexin 32 protein in rat hepatocytes without aberrant expression or localization

The characteristics and mechanism of the inhibition of connexin-mediated ga p junctional communication by the non-genotoxic rodent hepatocarcinogen, na fenopin, has been studied in rat hepacocytes. Nafenopin caused a time- and concentration-dependent inhibition of dye coupling in hepatocytes as assess ed by transfer of microinjected lucifer yellow. A half-maximum inhibitory e ffect of nafenopin occurred at approximately 50 mu M, which was not cytotox ic. The inhibitory effect was reversible since a significant recovery of co mmunication was observed 3 h after removal of the chemical, The protein kin ase inhibitor Go6976 prevented the inhibition of dye coupling, but a tyrosi ne kinase inhibitor (genistein) did not. Connexin 32 and 26 protein express ion, as assessed by immunoblotting, was similar in nafenopin-treated hepato cytes compared to controls, with the exception that in a 10-h culture with nafenopin, the level of connexin 26 was elevated compared to controls, Immu nohistochemistry indicated that the localization of plaques containing conn exin 32 was not affected in hepatocytes by nafenopin, Immunoprecipitated co nnexin 32 was, however, detected by an anti-phosphoserine antibody followin g nafenopin treatment, but not in controls. This serine phosphorylation was prevented in the presence of Go6976. The results give further support for a role of protein kinase C in the post-translational inactivation of connex in 32 function in rat hepatocytes by nafenopin.