Nicotinic Acetylcholine Receptors are Sensors for Ethanol in Lung Fibroblasts

Background Chronic ethanol (EtOH) abuse in humans is known to independently increase the incidence of and mortality due to acute lung injury in at-risk individuals. However, the mechanisms by which EtOH affects lung cells remain incompletely elucidated. In earlier work, we reported that EtOH increased the expression in lung fibroblasts of fibronectin, a matrix glycoprotein implicated in lung injury and repair. This effect was blocked by -bungarotoxin, a neurotoxin that binds certain nicotinic acetylcholine receptors (nAChRs) thereby implicating nAChRs in this process. Here, we examine the identity of these receptors. Methods Mouse lung fibroblasts were stimulated with EtOH (60mM) or acetylcholine (100 to 500M) and evaluated for the expression of fibronectin and nAChRs. Inhibitors to nAChRs or the antioxidant N-acetyl cysteine (NAC) were used to assess changes in fibronectin expression. Animals exposed to EtOH for up to 6weeks were used to evaluate the expression of nAChRs in vivo. Results First, in EtOH-treated fibroblasts, we observed increased expression of 4 and 9 nAChR subunits. Second, we found that acetylcholine, a natural ligand for nAChRs, mimicked the effects of EtOH. Dihydro--erythroidin hydrobromide, a competitive inhibitor of 4 nAChR, blocked the increase in fibronectin expression and cell proliferation. Furthermore, EtOH-induced fibronectin expression was inhibited in cells silenced for 4 nAChR. However, EtOH-treated cells showed increased -bungarotoxin binding suggesting that 4 nAChR mediates the effects of EtOH via a ligand-independent pathway. Knowing there are several important cysteine residues near the ligand-binding site of 4 nAChRs, we tested the antioxidant NAC and found that it too blocked the induction of fibronectin expression by EtOH. Also, fibroblasts exposed to oxidant stress showed increased fibronectin expression that was blocked with -bungarotoxin. Finally, we showed increased expression of 4 nAChRs in the lung tissue of mice and rats exposed to EtOH suggesting a role for these receptors in vivo. Conclusions Altogether, our observations suggest that 4 nAChRs serve as sensors for EtOH-induced oxidant stress in lung fibroblasts, thereby revealing a new mechanism by which EtOH may affect lung cells and tissue remodeling and pointing to nAChRs as potential targets for intervention.