Phosphorylation of connexin43 and inhibition of gap junctional communication in 12-O-tetradecanoylphorbol-13-acetate-exposed R6 fibroblasts: minor role of protein kinase C beta I and mu

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits gap junctional communic ation 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 correl ated with total protein kinase C (PKC) enzyme activity and if specific PKC isoenzymes are involved, Rat R6 fibroblasts transfected with the cDNA seque nce encoding PKC betaI (R6-PKC3) had a total PKC activity 7- to 16-fold hig her than the corresponding control cells (R6-C1), depending on the selectio n pressure (G418 concentration), Still, R6-PKC3 cells were no more sensitiv e than R6-C1 cells to TPA-induced down-regulation of communication, except at the highest selection pressure (500 mug/ml G418), Thus, total PKC activi ty does not indicate absolute sensitivity of a cell system to TPA-induced s uppression of communication, but within a certain cell system increasing PK C activity may enhance the sensitivity to TPA in this respect. The results also suggest that PKC betaI is of minor importance for TPA-induced regulati on of communication, Experiments with the Lilly compound 379196, a PKC beta -specific inhibitor, further supported this conclusion Except for PKC beta I in R6-PKC3 cells, both cell lines contained the TPA-responsive PKC isoenz ymes alpha, delta, epsilon and mu, Long-term treatment with TPA caused stro ng down-regulation of PKC alpha, delta and epsilon, but little down-regulat ion of PKC mu, Concurrently, the cells became refractory to repeated exposu re to TPA, indicating that PKC mu is of minor importance. Experiments with the general PKC inhibitor GF109203X and the PKC alpha (and beta/gamma) inhi bitor Go6976 suggested that both classical (alpha) and novel PKCs (delta an d epsilon) might be involved in TPA-induced suppression of intercellular co mmunication, while phosphorylation of Cx43 may mainly be mediated by PKC al pha in the present systems.