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

Abstract

The characteristics and mechanism of the inhibition of connexin-mediated gap junctional communication by the non-genotoxic rodent hepatocarcinogen, nafenopin, has been studied in rat hepatocytes. Nafenopin caused a time- and concentration-dependent inhibition of dye coupling in hepatocytes as assessed by transfer of microinjected lucifer yellow. A half-maximum inhibitory effect of nafenopin occurred at approximately 50 μM, which was not cytotoxic. The inhibitory effect was reversible since a significant recovery of communication was observed 3 h after removal of the chemical. The protein kinase inhibitor Go6976 prevented the inhibition of dye coupling, but a tyrosine kinase inhibitor (genistein) did not. Connexin 32 and 26 protein expression, as assessed by immunoblotting, was similar in nafenopin-treated hepatocytes compared to controls, with the exception that in a 10-h culture with nafenopin, the level of connexin 26 was elevated compared to controls. Immunohistochemistry indicated that the localization of plaques containing connexin 32 was not affected in hepatocytes by nafenopin. Immunoprecipitated connexin 32 was, however, detected by an anti-phosphoserine antibody following 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 connexin 32 function in rat hepatocytes by nafenopin.