US EPA

2262521 

Journal Article 

Abstract 

Alcohol decreases RhoA activity through a Nitric Oxide (NO)/Cyclic GMP (cGMP)/ Protein Kinase G (PKG) dependent pathway in the airway epithelium 

Bailey, K; Robinson, J; Gartin, C; Sisson, JH; Wyatt, TA 

2010 

Yes 

American Journal of Respiratory and Critical Care Medicine
ISSN: 1073-449X
EISSN: 1535-4970 

181 

A6377 

English 

WOS:000208771004736 

is part of a larger document 3452678 Proceedings of the American Thoracic Society 2010 International Conference, May 14-19, 2010, New Orleans

RATIONALE: Alcohol has been shown to have a number of harmful effects on the lung, including increasing the risk of pneumonia and bronchitis. How alcohol increases the risk of these diseases is poorly defined. We have previously shown that alcohol causes dysregulation of genes related to the innate immunity of the lung. Others have shown that changes in RhoA activity can act as a molecular switch to induce or suppress gene expression. It is not known how alcohol affects RhoA activity in the airway epithelium. We hypothesized that brief alcohol exposure modulates RhoA activity in the airway epithelium.

METHODS: Primary airway epithelial cells were obtained from donor lungs deemed unsuitable for transplant under an IRB approved protocol. Airway epithelial cells (Passage 1-5) were grown to 60% confluency and exposed to ethanol at various concentrations and times. The cell layers were harvested and RhoA activity was measured using the G-LISA RhoA activity kit (Cytoskeleton, Denver, CO).

RESULTS: We observed a 50% decrease in RhoA activity, compared to media controls in cells exposed to 25, 50 and 100mM alcohol after 1 and 4 hours of exposure. By 6 hours, there was an 80% decrease in RhoA activity at all concentrations. We were able to block this decrease in activity using Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME), a NOS inhibitor. Likewise, we were able to demonstrate the same decrease in RhoA activation using 0.1μM sodium nitroprusside (SNP), an NO donor. With SNP, we saw a 50% decrease in RhoA activity after only 1 hour. These findings suggest that NO is an important regulator in alcohol’s modulation of RhoA activity. To determine the role of cGMP/PKG, we pretreated the cells with a cGMP antagonist analogue, Rp-8Br-cGMP-S. This blocked the decrease in RhoA activity caused by alcohol, suggesting that alcohol exerts its effect on RhoA activity through cGMP/PKG.

CONCLUSIONS: Alcohol decreases RhoA activity through a NO/cGMP/PKG dependent pathway. RhoA activity controls many aspects of basic cellular functions, including cell morphology, tight junction formation, and cell cycle progression and gene regulation. Dysregulation of RhoA activity can therefore have several consequences, including dysregulation of inflammation. This may partially explain how alcohol increases the risk of pneumonia and bronchitis. 

American Thoracic Society 2010 International Conference 

New Orleans, LA 

May 14-19, 2010