Pharmacological modulation of ion transport across wild-type and ΔF508 CFTR-expressing human bronchial epithelia

Abstract

Forskolin, UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen (Methoxsalen; 8-MOP), and genistein were evaluated for their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and DeltaF508 (DeltaF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current (I(sc)) averaged 27.0 +/- 0.6 microA/cm(2) (n = 350). Amiloride reduced this I(sc) by 13.5 +/- 0.5 microA/cm(2) (n = 317). In DeltaF-HBE, baseline I(sc) was 33.8 +/- 1.2 microA/cm(2) (n = 200), and amiloride reduced this by 29.6 +/- 1.5 microA/cm(2) (n = 116), demonstrating the characteristic hyperabsorption of Na(+) associated with cystic fibrosis (CF). In wt HBE, subsequent to amiloride, forskolin induced a sustained, bumetanide-sensitive I(sc) (DeltaI(sc) = 8.4 +/- 0.8 microA/cm(2); n = 119). Addition of acetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4, 4'-dinitrostilben-2,2'-disulfonic acid further reduced I(sc), suggesting forskolin also stimulates HCO(3)(-) secretion. This was confirmed by ion substitution studies. The forskolin-induced I(sc) was inhibited by 293B, Ba(2+), clofilium, and quinine, whereas charybdotoxin was without effect. In DeltaF-HBE the forskolin I(sc) response was reduced to 1.2 +/- 0.3 microA/cm(2) (n = 30). In wt HBE, mucosal UTP induced a transient increase in I(sc) (Delta I(sc) = 15. 5 +/- 1.1 microA/cm(2); n = 44) followed by a sustained plateau, whereas in DeltaF-HBE the increase in I(sc) was reduced to 5.8 +/- 0. 7 microA/cm(2) (n = 13). In wt HBE, 1-EBIO, NS004, 8-MOP, and genistein increased I(sc) by 11.6 +/- 0.9 (n = 20), 10.8 +/- 1.7 (n = 18), 10.0 +/- 1.6 (n = 5), and 7.9 +/- 0.8 microA/cm(2) (n = 17), respectively. In DeltaF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl(-) secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl(-) secretory response (2.1 +/- 0.3 microA/cm(2), n = 21). In DeltaF-HBE, genistein alone stimulated Cl(-) secretion (2.5 +/- 0.5 microA/cm(2), n = 11). After incubation of DeltaF-HBE at 26 degrees C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na(+) absorption across DeltaF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca(2+)-, but not cAMP-mediated agonists, stimulated K(+) secretion across both wt HBE and DeltaF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K(+) and apical Cl(-) conductances directly modulate Cl(-) secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

In Situ Measurement of Airway Surface Liquid [K+] Using a Ratioable K+-sensitive Fluorescent Dye

“…The cell cultures expressed ENaC Na ϩ channels and CFTR Cl Ϫ channels, with electrophysiological responses similar to those reported previously (22,23). Fig.…”

The Small Conductance K+ Channel, KCNQ1

“…1-EBIO doubled the base-line I sc in our in vitro trachea preparation and thereby confirmed data gathered from experiments with primary cultured human and murine airway epithelial cells (7,44). Surprisingly, part of this 1-EBIO-induced rise of I sc was 293B-sensitive, which stands in clear contrast to the literature, where this current is predominantly attributed to mIK1 channels.…”

Organelle redox of CF and CFTR-corrected airway epithelia

“…Currents were larger when the apical solution contained no Cl − because the cell-to-apical electrochemical gradient for Cl − was increased so that Cl − left the cell through the activated CFTR. The need for a Cl − gradient as a driving force both to secrete Cl − across the apical membrane and to increase the sensitivity of the assay has been recognized previously (24,54,55,56).…”