Increased gap junction assembly between cultured cells upon cholesterol supplementation

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 μM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation. resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100μg ml^-1) and actinomycin D (10 μg ml^-1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed. Thus, cholesterol added in the presence of serum-supplemented medium resulted in a dramatic and rapid increase in the assembly of functional gap junctions between Novikoff cells.