Phosphorylation of connexin43 and inhibition of gap junctional communication in 12-O-tetradecanoylphorbol-13-acetate-exposed R6 fibroblasts: Minor role of protein kinase CβI and μ

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits gap junctional communication in many cell culture systems, but TPA-induced phosphorylation of the gap junction protein connexin43 (Cx43) varies much between systems. We have here studied whether these responses and their sensitivities can be correlated with total protein kinase C (PKC) enzyme activity and if specific PKC isoenzymes are involved. Rat R6 fibroblasts transfected with the cDNA sequence encoding PKCβI (R6-PKC3) had a total PKC activity 7- to 16-fold higher than the corresponding control cells (R6-C1), depending on the selection pressure (G418 concentration). Still, R6-PKC3 cells were no more sensitive than R6-C1 cells to TPA-induced down-regulation of communication, except at the highest selection pressure (500 μg/ml G418). Thus, total PKC activity does not indicate absolute sensitivity of a cell system to TPA-induced suppression of communication, but within a certain cell system increasing PKC activity may enhance the sensitivity to TPA in this respect. The results also suggest that PKCβI is of minor importance for TPA-induced regulation of communication. Experiments with the Lilly compound 379196, a PKCβ-specific inhibitor, further supported this conclusion. Except for PKCβI in R6-PKC3 cells, both cell lines contained the TPA-responsive PKC isoenzymes α, δ, ε and μ. Long-term treatment with TPA caused strong down-regulation of PKCα, δ and ε, but little down-regulation of PKCμ. Concurrently, the cells became refractory to repeated exposure to TPA, indicating that PKCμ is of minor importance. Experiments with the general PKC inhibitor GF109203X and the PKCα (and β/γ) inhibitor Gö6976 suggested that both classical (α) and novel PKCs (δ and ε) might be involved in TPA-induced suppression of intercellular communication, while phosphorylation of Cx43 may mainly be mediated by PKCα in the present systems.