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+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity.

Solution osmolarity is known to affect Na+ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na+ currents (I(sc)). I(sc) for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25 +/- 2, 9 +/- 2, 3 +/- 0.4, and 0.6 +/- 0.5 microA/cm2, respectively. Similar results were obtained following changes in the serosal tonicity: mucosal changes did not significantly affect I(sc). The changes in I(sc) were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na+ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na+ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na+ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na+ transport rates for A6 epithelia reported in the literature.

Amiloride-sensitive Na+ transport across cultured renal (A6) epithelium: evidence for large currents and high Na:K selectivity.

Electrical techniques were used to determine the Na:K selectivity of the amiloride-sensitive pathway and to characterize cellular and paracellular properties of A6 epithelium. Under control conditions, the mean transepithelial voltage (VT) was -57 +/- 5 mV, the short-circuit current (Isc) averaged 23 +/- 2 microA/cm2 and the transepithelial resistance (RT) was 2.8 +/- 0.3 k omega cm2 (n = 13). VT and Isc were larger than reported in previous studies and were increased by aldosterone. The conductance of the amiloride-sensitive pathway (Gamil) was assessed before and after replacement of Na+ in the mucosal bath by K+, using two independent measurements: (1) the slope conductance (GT), determined from current-voltage (I-V) relationships for control and amiloride-treated tissues and (2) the maximum amiloride-sensitive conductance (Gmax) calculated from the amiloride dose-response relationship. The ratio of Gamil in mucosal Na+ solutions to Gamil for mucosal K+ solutions was 22 +/- 6 for GT measurements and 15 +/- 2 for Gmax data. Serosal ion replacements in tissues treated with mucosal nystatin indicated a potassium conductance in the basolateral membrane. Equivalent circuit analyses of nystatin and amiloride data were used to resolve the cellular (Ec) and paracellular (Rj) resistances (approximately 5 k omega cm2 and 8-9 k omega cm2, respectively). Analysis of I-V relationships for tissues depolarized with serosal K+ solutions revealed that the amiloride-sensitive pathway could be described as a Na+ conductance with a permeability coefficient (PNa) = 1.5 +/- 0.2 x 10(-6) cm/s and the intracellular Na+ concentration (Nai) = 5 +/- 1 mM (n = 5), similar to values from other tight epithelia. We conclude that A6 epithelia are capable of expressing large amiloride-sensitive currents which are highly Na+ selective.