Impedance analysis of EGTA‐treated primary rat alveolar epithelial cell monolayers (RAECM)

Abstract

We recently reported, using an apical permeabilization technique, that RAECM paracellular ion conductance (Gj)/total ion conductance (Gdc) is ~0.25. Using impedance analysis, we estimated in this study the effects of a calcium chelator, EGTA (2 mM in both bathing fluids for 0.5h), on tight junctional resistance (Rj, kΩ) and individual resistances (Ra and Rb, kΩ) and capacitances (Ca and Cb, μF) of both apical (a) and basolateral (b) cell membranes of RAECM. Epithelial impedance (Z) was obtained as a ratio between multisinusoid input voltages and resultant current responses in the frequency domain. When Z data obtained prior to EGTA exposure were fit to a lumped equivalent circuit model for epithelial barriers, Ra ~2.4, Rb ~0.7, Ca ~1.2, Cb ~1.6 and Rj ~8.2 were estimated, with overall monolayer resistance (Rdc, kΩ) ~2.2 and Gj/Gdc ~0.28. EGTA decreased Rdc, Ra, Rb and Rj without appreciable changes in Ca and Cb, while Gj/Gdc increased to ~0.48. These data indicate that RAECM transcellular (Gc) and paracellular (Gj) ion conductances are both increased by calcium chelation (with the increase in Gj greater than that in Gc), and suggest that non‐invasive impedance analysis represents an improved approach to estimation of ion transport parameters for paracellular (i.e., tight junctional) and transcellular pathways of alveolar epithelium. (Support: NIH and Hastings Foundation.)