Nicotinic Acetylcholine Receptors are Sensors for Ethanol in Lung Fibroblasts

Background Chronic ethanol ( E t OH ) 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 E t OH affects lung cells remain incompletely elucidated. In earlier work, we reported that E t OH increased the expression in lung fibroblasts of fibronectin, a matrix glycoprotein implicated in lung injury and repair. This effect was blocked by α‐bungarotoxin, a neurotoxin that binds certain nicotinic acetylcholine receptors (n AC h R s) thereby implicating n AC h R s in this process. Here, we examine the identity of these receptors. Methods Mouse lung fibroblasts were stimulated with E t OH (60 mM) or acetylcholine (100 to 500 μM) and evaluated for the expression of fibronectin and n AC h R s. Inhibitors to n AC h R s or the antioxidant N ‐acetyl cysteine (NAC) were used to assess changes in fibronectin expression. Animals exposed to E t OH for up to 6 weeks were used to evaluate the expression of n AC h R s in vivo. Results First, in E t OH ‐treated fibroblasts, we observed increased expression of α4 and α9 n AC h R subunits. Second, we found that acetylcholine, a natural ligand for n AC h R s, mimicked the effects of E t OH . Dihydro‐β‐erythroidin hydrobromide, a competitive inhibitor of α4 n AC h R , blocked the increase in fibronectin expression and cell proliferation. Furthermore, E t OH ‐induced fibronectin expression was inhibited in cells silenced for α4 n AC h R . However, E t OH ‐treated cells showed increased α‐bungarotoxin binding suggesting that α4 n AC h R mediates the effects of E t OH via a ligand‐independent pathway. Knowing there are several important cysteine residues near the ligand‐binding site of α4 n AC h R s, we tested the antioxidant NAC and found that it too blocked the induction of fibronectin expression by E t OH . Also, fibroblasts exposed to oxidant stress showed increased fibronectin expression that was blocked with α‐bungarotoxin. Finally, we showed increased expression of α4 n AC h R s in the lung tissue of mice and rats exposed to E t OH suggesting a role for these receptors in vivo. Conclusions Altogether, our observations suggest that α4 n AC h R s serve as sensors for E t OH ‐induced oxidant stress in lung fibroblasts, thereby revealing a new mechanism by which E t OH may affect lung cells and tissue remodeling and pointing to n AC h R s as potential targets for intervention.