Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions

Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single-channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+ channels that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+ selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (6.6 ± 3.4 pS, n = 94), highly Na+-selective channel (HSC) with a P Na/ P K >80 that is inhibited by submicromolar concentrations of amiloride ( K 0.5 = 37 nM) and is similar in biophysical properties to ENaC channels described in other epithelia. To establish the relationship of this HSC channel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (α, β, and γ) and used patch-clamp techniques to determine the density of this channel in apical membrane patches of ATII cells. Overnight treatment of cells with antisense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of HSC channels in the apical membrane cell-attached patches. Taken together, these results show that when grown on permeable supports in the presence of steroids and air interface, the predominant channels expressed in ATII cells have single-channel characteristics resembling channels that are associated with the coexpression of the three cloned ENaC subunits α-, β-, and γ-ENaC.

Detection of Cl- flux in the apical microenvironment of cultured foetal distal lung epithelial cells

A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (J(Cl)) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmol × cm(−2) × s(−1)). A Cl- effusion gradient was used to confirm that empirical measurements of J(Cl) using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical J(Cl) was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50 mmol × l(−1), an apical-to-basolateral (inward) flux, maximal at 400 pmol × cm(−2) × s(−1), was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol × cm(−2) × s(−1) at [Cl]e>100 mmol × l(−1). At [Cl-]e>100 mmol × l(−1), isoproterenol (basolaterally applied, 10 micromol × l(−1)) activated a Cl- influx of 561 pmol × cm(−2) × s(−1), whereas UTP (apically applied, 100 micromol × l(−1)) stimulated a Cl- efflux of 300 pmol × cm(−2) × s(−1). In all cases, 50–70 % of J(Cl) was abolished by Cl- channel blockade using 10 micromol × l(−1) diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl-]e and in response to Cl- channel blockade and to beta2 adrenoreceptor and P2Y receptor agonists.