Chronic ethanol ingestion increases expression of the angiotensin II type 2 (AT₂) receptor and enhances tumor necrosis factor-α- and angiotensin II-induced cytotoxicity via AT₂ signaling in rat alveolar epithelial cells

Background: Alcohol abuse increases the risk of acute lung injury in critically ill patients. We have shown that alveolar epithelial cell (AEC) apoptosis in response to inflammatory mediators, including tumor necrosis factor-α (TNF-α), parallels endotoxin-mediated acute lung injury in ethanol-fed rats. Although angiotensin II mediates TNF-α-induced apoptosis of AECs in vitro, its role in ethanol-mediated susceptibility to AEC apoptosis is unknown. Methods: Adult male rats were fed the Lieber-DeCarli diet for 6 weeks. AECs were isolated, and TNF-α- and angiotensin II-induced cytotoxicity (by terminal transferase-mediated dUTP nick end labeling staining) was determined with or without the addition of the angiotensin-converting enzyme inhibitor (lisinopril) or a selective blocker of the angiotensin II type 1 receptor (AT₁) or type 2 receptor (AT₂). Finally, the relative expression of the AT₁ and AT₂ receptors in AECs was determined by Western blot analysis. Results: TNF-α-induced cytotoxicity, but not angiotensin II-induced cytotoxicity, was prevented by lisinopril, indicating that de novo angiotensin II synthesis is required for TNF-α-induced apoptosis in these cells. Both TNF-α- and angiotensin II-induced cytotoxicity in AECs from control-fed and ethanol-fed rats were inhibited by the selective AT₂ blocker, PD123319, but not by the selective AT₁ blocker, losartan. In parallel, ethanol ingestion doubled AT₂ expression in AECs (by Western blot) but had no significant effect on AT₁ receptor expression. Conclusions: Chronic ethanol ingestion increases AT₂ expression in the alveolar epithelium and enhances TNF-α- and angiotensin II-induced cytotoxicity, both of which act via AT₂. Together, these findings suggest that selective AT₂ receptor inhibition could limit the development of acute lung injury in alcoholic patients.