ANALYZING PHORBOL ESTER EFFECTS ON GAP JUNCTIONAL COMMUNICATION - A DRAMATIC INHIBITION OF ASSEMBLY

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junctio n assembly between Novikoff hepatoma cells was examined. Cells were di ssociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the c ell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron m icroscopy of reaggregated control cells showed interfaces containing a n average of more than 600 aggregated intramembranous gap junction par ticles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions wa s unaffected by 60 min of TPA treatment. Therefore, TPA dramatically i nhibited gap junction assembly but did not alter channel gating nor en hance disassembly of preexisting gap junction structures. Short term T PA treatment (<30 min) increased phosphorylation of the gap junction p rotein molecular weight of 43,000 (Cx43), but did not change the cellu lar level of Cx43. Cell surface biotinylation experiments suggested th at TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C pro bably exerts an effect on assembly of gap junctions at the plasma memb rane level.