TPA-induced inhibition of gap junctional intercellular communication is not mediated through free radicals

The present investigation was designed to determine whether the inhibition of gap junction-mediated intercellular communication (GJIC) induced by TPA (12-O-tetradecanoylphorbol-13-acetate) in rat liver epithelial (WB-F344) cells in vitro is mediated through free radical production. As assessed by fluorescence redistribution after photobleaching (FRAP) analysis, GJIC was significantly inhibited in cells treated for 1 hr with either 10 ng/ml TPA or 500 microM hydrogen peroxide (H2O2). Addition of 1000 U/ml catalase or 25 microM N',N'-diphenyl-p-phenylenediamine (DPPD) to TPA-treated cells did not alleviate the TPA-induced inhibition of GJIC. However, the concurrent addition of 1000 U/ml catalase to the culture medium prevented the H2O2 inhibition of GJIC. 2'-7'-dichlorofluorescein-mediated fluorescence, a measure of free radical production utilizing the Meridian ACAS 470 interactive laser cytometer, was not significantly increased in WB-F344 cells treated with 10 and 100 ng/ml TPA when compared to control cells. However, polymorphonuclear leukocytes (PMNs) treated for 10 min with 100 ng/ml TPA showed a substantial oxidative burst, as did WB-F344 cells treated for 1 hr with 500 microM H2O2. The concurrent addition of 1000 U/ml catalase to the culture medium attenuated H2O2-mediated free radical production in both PMNs and WB-F344 cells. Data from this study do not support a role for free radicals in the TPA-induced inhibition of GJIC in WB-F344 cells.