Attenuation of gossypol cytotoxicity by cyclic AMP in a rat liver cell line

The effect of pretreatment of a rat liver cell line (Clone 9) with 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), an agent previously shown to increase gap junctional intercellular communication (GJIC), was used to determine if increased GJIC would attenuate the cytotoxic effects of gossypol acetic acid (GAA). Pretreatment with 8-Br-cAMP increased the inhibitory concentration 50% (IC50) for gossypol from 4.1 to 6.1 microM in Clone 9 cells. GJIC was suppressed by about 57% within 5 min when Clone 9 cells were treated with 1.0 microM GAA alone, whereas cells treated with 3.0 or 10 microM GAA were completely uncoupled within the same time frame. In contrast, GJIC was maintained near control levels in Clone 9 cells preincubated with 1.0 mM 8-Br-cAMP for 10 min prior to 1.0 microM GAA exposure. 8-Br-cAMP partially restored GJIC in cells treated with 3.0 microM GAA but was unable to protect cells exposed to 10 microM GAA. The effects of GAA and 8-Br-cAMP pretreatment on connexin43 (Cx43) protein expression was analyzed with Western blots. GAA treatment at concentrations of 1 and 3 microM caused a time- and dose-dependent increase in phosphorylation of Cx43 over a 20-min period, whereas 10 microM GAA caused a time-dependent degradation of Cx43 over the same interval. Pretreatment of cells for 10 min with 8-Br-cAMP completely reversed the effect of 1 microM GAA and partially blocked the effect of 3 microM GAA on Cx43 phosphorylation and suppressed Cx43 degradation at 10 microM. Additional fluorescence endpoints associated with cellular homeostasis mechanisms were also monitored to evaluate cytotoxicity and potential protective effects of 8-Br-cAMP pretreatment in cells exposed to GAA, including generation of reactive oxygen species (ROS), cytoplasmic acidification, glutathione (GSH) content, intracellular calcium levels ([Ca2+]i), and mitochondrial and plasma membrane potential. The adverse effects of GAA on the production of ROS, cytoplasmic acidification, GSH content, and [Ca2+]i were also attenuated. The primary protective effect of 8-Br-cAMP was observed at or below the IC50 of GAA, with greatest protective effects detected on early endpoints affected by GAA exposure. These studies suggest that the protective effect of 8-Br-cAMP in GAA-treated cells results from enhanced GJIC, which facilitates cellular homeostasis by providing cell-cell diffusion of essential metabolites, ions, and regulatory and informational molecules.