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Inhibition of Cx43‐induced gap junctional communication on growth of cultured endothelial monolayers
Author(s) -
Houser Sandra Dean,
Quadri Sadiqa K.,
Bhattacharya Jahar
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.932.3
Subject(s) - mutant , connexin , fluorescence recovery after photobleaching , gap junction , intracellular , microbiology and biotechnology , biology , bullfrog , chemistry , biochemistry , endocrinology , gene , membrane
Deletion of the gap junctional protein, connexin 43 (Cx43) blocks endothelial (EC) proliferation and vasculogenesis. However, since Cx43 supports intercellular gap junctional communication (GJC), the growth inhibition is potentially attributable to lack of Cx43 expression, or blockade of GJC. To distinguish between these possibilities, in rat lung microvascular EC (RLMEC) we expressed full length GFP‐Cx43 cDNA containing a mutation on threonine 154 (Cx43T154). In HeLa cells, this mutant blocks Cx43‐dependent GJC, but not Cx43 expression (JBC, 2006. 281:7994‐8009). Both wild type (WT; n=3) and Cx43T154‐expressing (n=3) RLMEC monolayers grew to confluence, indicating that the mutant did not inhibit EC proliferation. To assay GJC, we loaded RLMEC with the intracellular cytosolic fluorescent marker, calcein‐AM. Then in single cells viewed by confocal microscopy, we bleached fluorescence to 50% of initial fluorescence (Io), then determined the fluorescence recovery after photobleaching (FRAP). In WT monolayers, FRAP was 22 ± 6% of Io in 3 min (mean ± SD; n=3). In Cx43T154‐expressing monolayers, FRAP was markedly less at 4.2 ± 3 % of Io (n=3; P<0.05). Hence, expression of the mutant Cx43 markedly impaired GJC. We conclude that inhibition of GJC does not inhibit EC proliferation to form stable monolayers (HL36024, HL57556, HL64896).