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

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 Delta F508 (Delta F-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current (I-sc) averaged 27.0 +/- 0.6 mu A/cm(2) (n = 350). Amiloride reduced this I-sc by 13.5 +/- 0.5 mu A/cm(2) (n = 317). In Delta F-HBE, baseline I-sc was 33.8 +/- 1.2 mu A/cm(2) (n = 200), and amiloride reduced this by 29.6 +/- 1.5 mu A/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 (Delta I-sc = 8.4 +/- 0.8 mu A/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 HCO3- secretion. This was confirmed by ion substitution studies. The forskolin-induced I-sc was inhibited by 293B, Ba2+, clofilium, and quinine, whereas charybdotoxin was without effect. In Delta F-HBE the forskolin I-sc response was reduced to 1.2 +/- 0.3 mu A/cm(2) (n = 30). In wt HBE, mucosal UTP induced a transient increase in I-sc (Delta I-sc = 15.5 +/- 1.1 mu A/cm(2); n = 44) followed by a sustained plateau, whereas in Delta F-HBE the increase in I-sc was reduced to 5.8 +/- 0.7 mu A/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 mu A/cm(2) (n = 17), respectively. In Delta F-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 mu A/cm(2), n = 21). In Delta F-HBE, genistein alone stimulated Cl- secretion (2.5 +/- 0.5 mu A/cm(2), n = 11). After incubation of Delta F-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 Delta F-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca2+-, but not cAMP-mediated agonists, stimulated K+ secretion across both wt HBE and Delta F-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.