ROLE OF PROTEIN-KINASE-C IN THE REGULATION OF GAP JUNCTIONAL COMMUNICATION

We have examined the effect of protein kinase (PKC) depletion in SV40- transformed Djungarian hamster fibroblasts (DM15 cells) on the level o f gap junction permeability, Cx43 electrophoretic mobility, and cell s ensitivity to different uncoupling stimuli. After 24 hr exposure to 12 -O-tetradecanoyl-phorbol-13-acetate (TPA), the total PKC activity in D M15 cells was reduced to 20-25% in comparison with intact cells. In PK C-depleted cells the level of dye coupling was 30-40% higher than in t he same untreated cultures. Western blot analysis revealed multiple fo rms of the gap junction protein connexin 43, which correspond to known phosphorylated and dephosphorylated forms of this protein. No decreas e in the level of connexin 43 phosphorylation after PKC depletion was observed. TPA(10(-7) g/ml), mezerein (10(-7) g/ml), teleocidin (10(-8) g/ml), Ca-ionophore A23I87 (10(-6) g/ml), insecticide 1,1,1-trichloro -2,2-bis-(p-chlorphenyl)-(DDT) (10(-4) g/ml), and nigericin (10(-5) M in hydrolysate lactalbumin solution, pH 6.3) induced a four-to six-fol d decrease in the number of recipient cells in the dye-coupling assay. PKC-depleted cells became almost completely resistant to the uncoupli ng effect of mezerein, teleocidin, and A23187, as well as to new expos ure to TPA, and became partially resistant to the effect of DDT. Niger icin inhibited intercellular communication between PKC-depleted cells to the same extent as between control cells. Thus, in the cell system studied, PKC plays a certain role in maintaining the basal level of ga p junction permeability and has an important significance as a mediato r of the uncoupling effects of such substances as TPA, mezerein, teleo cidin, and Ca2+. (C) 1994 Wiley-Liss, Inc.