Regulation of connexin phosphorylation and cell-cell coupling in trabecular meshwork cells.
OBJECTIVE
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 gap junctions was assessed by observing the diffusion of fluorescent dye from 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 PKC activator phorbol 12-myristate 13-acetate (PMA; 10 nM, 1 hour) had no marked effect on the pattern of connexin43 immunofluorescence. Injection of a TM cell with lucifer yellow resulted in the spread of the dye into neighboring cells. Dye coupling was inhibited by PMA in a dose- and time-dependent 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 nonphosphorylated protein (43 kDa) and three phosphorylated forms (47, 48, and 49 kDa). Cells 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, and the communication is inhibited by PKC, probably, at least in part, through phosphorylation of connexin43.
Kimura
S
S
Department of Ophthalmology, Yamaguchi University School of Medicine, Ube City, Japan.
Suzuki
K
K
Sagara
T
T
Nishida
T
T
Yamamoto
T
T
Kitazawa
Y
Y
D000199:Actins / Q000378:metabolism; D000818:Animals; D000867:Anterior Chamber / Q000378:metabolism; D002417:Cattle; D002450:Cell Communication / Q000502:physiology*; D018031:Connexin 43 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000502:physiology; D007546:Isoquinolines / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D000199:Actins; D018031:Connexin 43; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007546:Isoquinolines; C017475:lucifer yellow; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate