Pseudomonas aeruginosa is a gram-negative bacterium that causes chronic infection in cystic fibrosis patients. We reported recently that P. aeruginosa modulates epithelial Na(+) channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested whether LPS from P. aeruginosa alters ENaC expression and activity in alveolar epithelial cells. We found that LPS induces a approximately 60% decrease of ENaC apical current without significant changes in intracellular ENaC or surface protein expression. Because a growing body of evidence reports a key role for extracellular nucleotides in regulation of ion channels, we evaluated the possibility that modulation of ENaC activity by LPS involves extracellular ATP signaling. We found that alveolar epithelial cells release ATP upon LPS stimulation and that pretreatment with suramin, a P2Y(2) purinergic receptor antagonist, inhibited the effect of LPS on ENaC. Furthermore, ET-18-OCH3, a PLC inhibitor, and Go-6976, a PKC inhibitor, were able to partially prevent ENaC inhibition by LPS, suggesting that the actions of LPS on ENaC current were mediated, in part, by the PKC and PLC pathways. Together, these findings demonstrate an important role of extracellular ATP signaling in the response of epithelial cells to LPS.

Active Na(+) transport mediated by epithelial Na(+) channel (ENaC) is vital for fetal lung fluid reabsorption at birth and pulmonary edema resolution. Previously, we demonstrated that αENaC expression and activity are downregulated in alveolar epithelial cells by cycloheximide (Chx) and Pseudomonas aeruginosa. The regulatory mechanisms of αENaC mRNA expression by Chx and lipopolysaccharide (LPS) from P. aeruginosa were further studied in the present work. Both agents decreased αENaC mRNA expression to 50% of control values after 4 h. Chx repressed αENaC expression in a dose-dependent manner independently of protein synthesis. Although extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways were activated by the two treatments, their mechanisms of ENaC mRNA modulation were different. First, activation of the signaling pathways was sustained by Chx but only transiently by LPS. Second, ERK1/2 or p38 MAPK inhibition attenuated the effects of Chx on αENaC mRNA, whereas suppression of both signaling pathways was necessary to alleviate the outcome of LPS on αENaC mRNA. The molecular mechanisms involved in the decrease of αENaC expression were investigated in both conditions. LPS, but not Chx, significantly reduced αENaC promoter activity via the ERK1/2 and p38 MAPK pathways. These results suggest that LPS attenuates αENaC mRNA expression via diminution of transcription, whereas Chx could trigger some posttranscriptional mechanisms. Although LPS and Chx downregulate αENaC mRNA expression similarly and with similar signaling pathways, the mechanisms modulating ENaC expression are different depending on the nature of the cellular stress.

Pseudomonas aeruginosa is a gram negative bacteria that causes chronic infection in cystic fibrosis patients and is known to modulate the epithelial Na channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested if LPS from P. aeruginosa could modulate the expression and activity of ENaC in alveolar epithelial cells. Alveolar epithelial cells purified from rat lung were cultured on permeable filters for 4 days before the cells were treated on the apical side with 15 μg/ml of Pseudomonas LPS. The treatment decreased gradually the total transepithelial current with the lowest point reached at 4h with a 35% decline compared to untreated cells. The ENaC (amiloride‐sensitive) portion of this current was declined by 53% at 4h. The α and βENaC mRNA were decreased respectively to 55% and 54% of control by a 4h LPS treatment. Estimation of ENaC activity at the apical membrane following amphotericin B permeabilisation of the basolateral membrane in presence of a Na gradient shows a 52% decrease in 4h LPS treated cells. The decline in ENaC current elicited by LPS is therefore the consequence of a decreased activity of the channel. Ro‐31 8820 a PKC inhibitor abolished the downregulation of ENaC activity in the cells and also restored the α and γENaC mRNA level back to normal. These results show that LPS downregulates ENaC mRNA expression and activity in alveolar epithelial cells by a PKC‐sensitive pathway.Supported by CCFF and CIHR