Carbenoxolone inhibits junctional transfer and upregulates Connexin43 expression by a protein kinase A-dependent pathway

We have been investigating the function and gene expression of connexins by vascular wall cells, especially Connexin43 (Cx43) in bovine aortic endothelial cells (BAEC). In this study, we tested the effects of carbenoxolone (CBN), a gap junction communication (GJC) blocker on the junctional transfer of Lucifer yellow in BAEC. CBN is a water-soluble derivative of the liquorice-root extract 18-alpha-glycyrrhetinic acid. CBN rapidly abolished dye-transfer in the scrape-load transfer assay (a measure of GJC) in a reversible and dose-dependent fashion. We then asked whether the BAEC might somehow compensate for the loss of junctional communication by altering the expression of connexins. Thus, we treated BAEC with 100 mu M CBN in serum free medium and determined the total Cx43 cellular distribution (immunostaining) and protein content (immunoblotting). Besides changes in distribution, by 6 h, Cx43 content levels increased to 166% +/- 22% (P < 0.0001) of controls. RNA blot data showed two-three fold increases in Cx43 message in BAEC after 6 h of CBN treatment, suggesting transcriptional control. Since CBN has structural similarities to corticosteroids, we tested both aldosterone and prednisolone but neither drug increased Cx43 levels, suggesting that the CBN response was not due to a generalized steroid effect. Staurosporine inhibited the CBN-induced increase in Cx43 content, suggesting a role for kinases in the signaling pathway. Further studies with inhibitors indicated that PKA but not PKC was implicated. In summary, CBN blocks junctional communication and modulates Cx43 expression in BAEC. These results suggest a feedback mechanism for control of connexin expression based on junctional patency.