PGE(2) activation of apical membrane Cl(-) channels in A6 epithelia: impedance analysis.

Measurements of transepithelial electrical impedance of continuously short-circuited A6 epithelia were made at audio frequencies (0.244 Hz to 10.45 kHz) to investigate the time course and extent to which prostaglandin E(2) (PGE(2)) modulates Cl(-) transport and apical membrane capacitance in this cell-cultured model epithelium. Apical and basolateral membrane resistances were determined by nonlinear curve-fitting of the impedance vectors at relatively low frequencies (<50 Hz) to equations (Paunescu, T. G., and S. I. Helman. 2001. Biophys. J. 81:838--851) where depressed Nyquist impedance semicircles were characteristic of the membrane impedances under control Na(+)-transporting and amiloride-inhibited conditions. In all tissues (control, amiloride-blocked, and amiloride-blocked and furosemide-pretreated), PGE(2) caused relatively small (< approximately 3 microA/cm(2)) and rapid (<60 s) maximal increase of chloride current due to activation of a rather large increase of apical membrane conductance that preceded significant activation of Na(+) transport through amiloride-sensitive epithelial Na(+) channels (ENaCs). Apical membrane capacitance was frequency-dependent with a Cole-Cole dielectric dispersion whose relaxation frequency was near 150 Hz. Analysis of the time-dependent changes of the complex frequency-dependent equivalent capacitance of the cells at frequencies >1.5 kHz revealed that the mean 9.8% increase of capacitance caused by PGE(2) was not correlated in time with activation of chloride conductance, but rather correlated with activation of apical membrane Na(+) transport.

cAmp activation of apical membrane Cl(-) channels: theoretical considerations for impedance analysis.

Transepithelial electrical impedance analysis provides a sensitive method to evaluate the conductances and capacitances of apical and basolateral plasma membranes of epithelial cells. Impedance analysis is complicated, due not only to the anatomical arrangement of the cells and their paracellular shunt pathways, but also in particular to the existence of audio frequency-dependent capacitances or dispersions. In this paper we explore implications and consequences of anatomically related Maxwell-Wagner and Cole-Cole dielectric dispersions that impose limitations, approximations, and pitfalls of impedance analysis when tissues are studied under widely ranging spontaneous rates of transport, and in particular when apical membrane sodium and chloride channels are activated by adenosine 3',5'-cyclic monophosphate (cAMP) in A6 epithelia. We develop the thesis that capacitive relaxation processes of any origin lead not only to dependence on frequency of the impedance locus, but also to the appearance of depressed semicircles in Nyquist transepithelial impedance plots, regardless of the tightness or leakiness of the paracellular shunt pathways. Frequency dependence of capacitance precludes analysis of data in traditional ways, where capacitance is assumed constant, and is especially important when apical and/or basolateral membranes exhibit one or more dielectric dispersions.

LY-294002-inhibitable PI 3-kinase and regulation of baseline rates of Na(+) transport in A6 epithelia.

Blocker-induced noise analysis of epithelial Na(+) channels (ENaCs) was used to investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na(+) transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na(+) transport rates (I(Na)) of 4.0 +/- 0.1 (unstimulated) and 9.1 +/- 0.9 microA/cm(2) (aldosterone), 10 microM LY-294002 caused, following a relatively small initial increase of transport, a completely reversible inhibition of transport within 90 min to 33 +/- 6% and 38 +/- 2% of respective baseline values. Initial increases of transport could be attributed to increases of channel open probability (P(o)) within 5 min to 143 +/- 17% (unstimulated) and 142 +/- 10% of control (aldosterone) from baseline P(o) averaging near 0.5. Inhibition of transport was due to much slower decreases of functional channel densities (N(T)) to 28 +/- 4% (unstimulated) and 35 +/- 3% (aldosterone) of control at 90 min. LY-294002 (50 microM) caused larger but completely reversible increases of P(o) (215 +/- 38% of control at 5 min) and more rapid but only slightly larger decreases of N(T). Basolateral exposure to LY-294002 induced no detectable effect on transport, P(o) or N(T). We conclude that an LY-294002-sensitive PI 3-kinase plays an important role in regulation of transport by modulating N(T) and P(o) of ENaCs, but only when presented to apical surfaces of the cells.

Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption.

Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+ reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.