Regulation of connexin phosphorylation and cell-cell coupling in trabecular meshwork cells

PURPOSE. TO investigate the expression of functional gap junctions and the effect of protein kinase C (PKC) on such junctions in confluent cultures of bovine trabecular meshwork (TM) cells. METHODS. Expression of the gap junction protein connexin43 in TM cells was examined by immunofluorescence microscopy. Intercellular communication by g ap junctions was assessed by observing the diffusion of fluorescent dye fro m an individual cell, injected with lucifer yellow. The phosphorylation of connexin43 was evaluated by immunoblot analysis with a monoclonal antibody to this protein. RESULTS. Immunofluorescence staining revealed that connexin43 was localized to sites of contact between adjacent TM cells. Exposure of cells to the PK C activator phorbol 12-myristate 13-acetate (PMA; 10 mu M, 1 hour) had no m arked effect on the pattern of connexin43 immunofluorescence. Injection of a TPA cell with lucifer yellow resulted in the spread of the dye into neigh boring cells. Dye coupling was inhibited by PMA in a dose- and time-depende nt manner, and this inhibition was prevented by pretreatment of cells with the PKC inhibitor bisindolylmaleimide I. Immunoblot analysis of control TM cell lysates yielded connexin43 bands corresponding to the nonphosphorylate d protein (43 kDa) and three phosphorylated forms (47, 48, and 49 kDa). Cel ls exposed to PMA (10 nM, 1 hour) yielded an additional band corresponding to a 44-kDa form of phosphorylated connexin43 and showed a decrease in the intensity of the band corresponding to the nonphosphorylated protein and an increase in the intensity of the 47-kDa band. CONCLUSIONS. TM cells communicate with each other through gap junctions, an d the communication is inhibited by PKC, probably, at least in part, throug h phosphorylation of connexin43.