Transport properties of cell culture models of the alveolar epithelium

Abstract

Alveolar epithelial cells are a main target of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which are characterized by fluid accumulation and impaired ion transport in the lungs. This thesis characterizes cell culture models of the alveolar epithelium and studies ion transport under normal and ALI conditions. The human distal pulmonary epithelial cell line NCIH441 was found to express high levels of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase. Consistent with this phenotype the cell line formed a functional barrier with active ion transport as assessed by measurements of transepithelial electrical resistance (TEER), paracellular permeability and transepithelial potential difference (TEPD). Phenotypic and functional properties of NCI-H441 cells were optimized by varying cell seeding density and concentrations of dexamethasone and insulin-transferrin-selenium supplements to achieve in vivo-like polarized monolayers with high levels of electrical resistance, potential difference and expression of claudin-3 and α1-Na+-K+-ATPase. Treatment with inhibitors of sodium and chloride channels and the cyclic adenosine monophosphate (cAMP) agonist forskolin showed that sodium absorption through ENaC is the primary transport pathway for sodium under baseline and stimulated conditions. Transcellular chloride transport could not be detected at baseline while it was secreted through apical channels under forskolin stimulation. Under an oleic acid-induced ALI model, the tight epithelium became leaky as assessed by the deterioration and disappearance of the restrictive component of paracellular permeability and the absorptive sodium flux was impaired. These changes were associated with altered expression of tight junctions (decreased claudin-3 but elevated ZO-1) and ion transport proteins (downregulated α1-Na+-K+-ATPase and β-ENaC subunit). The extracellular signal regulated kinases (ERK) pathway was found to play a complex role in the regulation of TEER and TEPD. Under baseline conditions inhibition of ERK phosphorylation reduced the expression of α1-Na+-K+-ATPase and consequently TEPD, but had no effect on tight junction proteins and TEER. Oleic acid was found to activate the ERK pathway in a dose dependent manner. But inhibition of the pathway did not block the effects of oleic acid on the TEER and TEPD, suggesting that oleic acid mediated injury proceeds via an ERK-independent pathway.