Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking

Given the rapid turnover of connexin proteins, gap junction (GS) assembly r epresents an important means of regulating the extent of GJ communication b etween cells, This report describes an increase in the level of GJ assembly ,within one hour following treatment with cAMP-elevating reagents or low de nsity lipoprotein (LDL), Dye transfer methods and freeze-fracture,vith elec tron microscopy were used to assay junctional permeability and structure, r espectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells, Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye tra nsfer rates between cells and the extent of GJ formation 2- to 3-fold. Thes e data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly, The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level o f connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hour s. However, three agents (brefeldin A, monensin and nocodazole), that inhib it intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or expo sure to LDL, Related studies, which employed trafficking inhibitors at diff erent stages in GJ assembly, suggested that Cx43 trafficking during enhance d assembly is regulated, in part, by cell contact, Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic m embrane systems. We conclude that an increase in GJ assembly: (i) occurs ra pidly in the presence of elevated cAMP or LDL, (ii) does not require an inc rease in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.