Nonselective Cation Channels Maintain Alveolar Fluid Clearance After Exposure to Toxins from Respiratory Pathogens.

Normal gas exchange in the alveoli is facilitated by a thin fluid layer. This layer is carefully regulated by two ion channels: highly selective channels (HSC or ENaC) and nonselective cation channels (NSC). Previous research found that both channels contribute about equally to alveolar fluid clearance (AFC). Since the molecular composition of NSC is a recent discovery, not much is known about NSC's response to various respiratory pathogens. NSC are composed of an alpha-ENaC subunit and at least one acid-sensing ion channel 1a (ASIC1a) subunit. Our study aims are to understand how NSC contribute to alveolar epithelial permeability and how the lungs respond to toxins from respiratory pathogens. We hypothesize that AFC will be lower in the absence of ASIC1a (ASIC1 KO) when exposed to toxins from respiratory pathogens: Lipopolysaccharide (LPS) from E. coli and Pneumolysin (PLY) from P. pneumoniae. Additionally, we hypothesize that ASIC1 KO animals will have increased baseline alveolar epithelium permeability. All animal experiments were approved by Emory's IACUC. We introduced pathogen toxins via intratracheal instillation to ASIC1 KO (KO) and wildtype (WT) mouse models (both on a C57Bl6 background). After a four-hour incubation period, we euthanized the mice by isoflurane overdose. We determined AFC by instilling their lungs with 0.7ml of a 2.0% Evans Blue (EB) or Fluorescein isothiocyanate-dextran (FITC) solution via the trachea. We took samples at t=0 and t=30 minutes after instillation. We determined the EB/ FITC concentration of both samples using spectrophotometry and calculated AFC from the change in dye concentration. Our preliminary findings suggest that LPS and PLY inhibit all AFC associated with HSC leaving only AFC associated with NSC. The AFC of LPS exposed KO animals is low compared to AFC for LPS exposed WT animals (6.04 ± 3.02, n= 15 vs 15.8 ± 5.61, n= 11, p< 0.001). Similarly, AFC of PLY exposed KO mice is lower than AFC for PLY exposed WT mice (2.78 ± 0.96, n= 10 vs 12.4 ± 4.36, n= 4, p< 0.001). In the toxin exposed KO groups, neither HSC nor NSC are fully functioning or contributing to AFC. We are still collecting data on the permeability of the alveolar epithelium. There is evidence to suggest that the regulation of HSC and NSC are drastically different. Given the critical nature of the alveoli fluid layer, it would be logical that NSC channels serve as a compensatory mechanism when HSC are inhibited by pathogens. These findings can help guide treatment for alveolar flooding caused by toxins from different respiratory pathogens.