PKC isoenzymes in the chicken lens and TPA-induced effects on intercellular communication

PURPOSE. Because lens connexins are phosphoproteins and intercellular commu nication between lens cells may be modulated by connexin phosphorylation, e xperiments were designed to characterize the expression of protein kinase C (PKC) isoenzymes in the chicken lens and in lentoid-containing cultures an d to study the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatm ent on the distribution of PKC isoenzymes and intercellular communication. METHODS. The presence and distribution of PKC isoenzymes were studied by im munoblot analysis and immunofluorescence in chicken lens sections and in ce ll cultures under control conditions and after treatment with TPA. Intercel lular communication was assessed by transfer of microinjected Lucifer yello w. RESULTS. PKC alpha, gamma, iota, epsilon, and mu were detected in lens homo genates by immunoblot analysis. The levels of PKC alpha, gamma, iota, and m u decreased between the 7th and the 18th embryonic days. Levels of PKC epsi lon remained relatively constant during the period of study. Similarly, len s cells in culture expressed isoenzymes alpha, gamma, epsilon, iota, and mu . PKC beta was not detected in lens or culture homogenates. In lens section s, all PKC isoenzymes analyzed were present in epithelial cells, in the ann ular pad region, and in the posterior aspect of fiber cells. The anti-PKC g amma antibody also stained fiber cell membranes. Analysis of lentoid cultur es by immunofluorescence revealed that PKC gamma, epsilon, and iota and min imal amounts of PKC alpha were present in lentoid cells. Treatment with 200 nM TPA for 15 to 30 minutes induced translocation of PKC gamma to the plas ma membrane of lentoid cells and significantly reduced the transfer of micr oinjected Lucifer yellow. CONCLUSIONS. Several PKC isoenzymes are expressed by lens cells in situ and in culture. The gamma isoenzyme, present in lens fibers, was activated in lentoid cells by TPA, a known activator of PKC. We have previously demonstr ated TPA-induced phosphorylation of the gap junction protein connexin56 (Cx 56). The new data presented in the current study demonstrate that TPA treat ment also decreased intercellular communication. Taken together, the result s suggest that differential phosphorylation of Cx56 by PKC gamma may induce a conformational change in the protein which, in turn, might lead to chann el closure.