Effects of aldosterone on the impedance properties of cultured renal amphibian epithelia.

The cultured renal amphibian cell line A6 has proven advantageous for studies of Na+ transport regulation. In the present study, the effects of aldosterone action on the transepithelial electrical properties of this epithelium were assessed. Specifically, the time course of aldosterone action was determined and the effects of chronic (10-18 day) aldosterone elevation were assessed using transepithelial equivalent circuit methods and impedance analysis techniques. Short-term (< 4 hr) exposure to aldosterone (0.1 microM) stimulated the amiloride-sensitive short-circuit current (Isc) by over twofold and increased the transepithelial conductance (GT) by approximately 12%. The increases in Isc and GT were maintained in epithelia subjected to chronic aldosterone exposure. In contrast to previous reports, paracellular resistance (Rj) was not altered by aldosterone. This difference may be related to the longer time of exposure or different basal Na+ transport rates in the present study. The apical membrane conductance was significantly increased for aldosterone-treated epithelia compared to aldosterone-depleted (i.e., serum-deprived) controls. Apical membrane area (capacitance) was not significantly affected. This finding is consistent with a higher density (number of channels per membrane area) of conducting Na+ channels in this membrane following aldosterone stimulation. Basolateral membrane properties were not significantly altered for aldosterone-treated tissues compared to serum-treated control tissues. In contrast, basolateral membrane-specific conductance (i.e., basolateral membrane conductance normalized to basolateral membrane capacitance) was significantly lower for serum-deprived epithelia than for serum-treated controls or aldosterone-treated tissues. The effects of chronic aldosterone exposure were also evaluated for the A6 subclonal cell line, 2F3. Similar to A6 epithelia, Isc was essentially doubled following aldosterone stimulation while Rj and cellular driving force (Ec) were not affected. Apical membrane conductances under control conditions for 2F3 epithelia were higher than those for A6, but were not significantly different from A6 following aldosterone exposure or serum deprivation. These findings suggest possible differences in the regulation of apical membrane Na+ channels for 2F3 and A6 epithelia.

Na+ transport and impedance properties of cultured renal (A6 and 2F3) epithelia.

Previous impedance analysis studies of intact epithelia have been complicated by the presence of connective tissue or smooth muscle. We now report the first application of this method to cultured epithelial monolayers. Impedance analysis was used as a nondestructive method for deducing quantitative morphometric parameters for epithelia grown from the renal cell line A6, and its subclonal cell line 2F3. The subclonal 2F3 cell line was chosen for comparison to A6 because of its inherently higher Na+ transport rate. In agreement with previous results, 2F3 epithelia showed significantly higher amiloride-sensitive short-circuit currents (Isc) than A6 epithelia (44 +/- 2 and 27 +/- 2 microA/cm2, respectively). However, transepithelial conductances (GT) were similar for the two epithelia (0.62 +/- 0.04 mS/cm2 for 2F3 and 0.57 +/- 0.04 mS/cm2 for A6) because of reciprocal differences in cellular (Gc) and paracellular (Gj) conductances. Significantly lower Gj and higher Gc values were observed for 2F3 epithelia than A6 (Gj = 0.23 +/- 0.02 and 0.33 +/- 0.04 mS/cm2 and Gc = 0.39 +/- 0.16 and 0.26 +/- 0.10 mS/cm2, respectively). Nonetheless, the cellular driving force for Na+ transport (Ec) and the amount of transcellular Na+ current under open-circuit conditions (Ic) were similar for the two epithelia. Three different morphologically-based equivalent circuit models were derived to assess epithelial impedance properties: a distributed model which takes into account the resistance of the lateral intercellular space and two models (the "dual-layer" and "access resistance" models), which corrected for impedance of small fluid-filled projections of the basal membrane into the underlying filter support. Although the data could be fitted by the distributed model, the estimated value for the ratio of apical to basolateral membrane resistances was unreasonably large. In contrast, the other models provided statistically superior fits and reasonable estimates of the membrane resistance ratio. The dual-layer model and access resistance models also provided similar estimates of apical and basolateral membrane conductances and capacitances. In addition, both models provided new information concerning the conductance and area of the basolateral protrusions. Estimates of the apical membrane conductance were significantly higher for 2F3 (0.79 +/- 0.23 mS/cm2) than A6 epithelia (0.37 +/- 0.07 mS/cm2), but no significant difference could be detected for apical membrane capacitances (1.4 +/- 0.04 and 1.2 +/- 0.1 microF/cm2 for 2F3 and A6, respectively) or basolateral membrane conductances (3.48 +/- 1.67 and 2.95 +/- 0.40 mS/cm2). The similar basolateral membrane properties for the two epithelia may be explained by their comparable transcellular Na+ currents under open-circuit conditions.