Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers

Abstract

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-β₁ (TGF-β₁) decreases overall monolayer resistance ( R_te) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-β₁ (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5–7 in culture, tight-junctional resistance ( R_tj, kΩcm²) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-β₁ or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/ R_te) and tight-junctional conductance (1/ R_tj) are 0.59 and 0.14 mS/cm² for control monolayers in MDSF, 1.59 and 0.38 mS/cm² for monolayers exposed to TGF-β₁, and 0.38 and 0.18 mS/cm² for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-β₁-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.