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Shinji Kimura, Katsuyoshi Suzuki, Takeshi Sagara, Teruo Nishida, Tetsuya Yamamoto, Yoshiaki Kitazawa; Regulation of Connexin Phosphorylation and Cell–Cell Coupling in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2000;41(8):2222-2228.
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© ARVO (1962-2015); The Authors (2016-present)
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 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.
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