Pharmacological modulation of ion transport across wild-type and DeltaF508 CFTR-expressing human bronchial epithelia |
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Authors: | Devor D C Bridges R J Pilewski J M |
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Institution: | Department of Cell Biology and Physiology, University of Pittsburgh, Pennsylvania 15261, USA. dd2+@pitt.edu |
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Abstract: | Forskolin,UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen(Methoxsalen; 8-MOP), and genistein were evaluated for theireffects on ion transport across primary cultures of human bronchialepithelium (HBE) expressing wild-type (wt HBE) and F508( F-HBE) cystic fibrosis transmembrane conductance regulator. In wtHBE, the baseline short-circuit current (Isc)averaged 27.0 ± 0.6 µA/cm2 (n = 350). Amiloride reduced this Isc by 13.5 ± 0.5 µA/cm2 (n = 317). In F-HBE,baseline Isc was 33.8 ± 1.2 µA/cm2 (n = 200), and amiloride reducedthis by 29.6 ± 1.5 µA/cm2 (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 Isc( Isc = 8.4 ± 0.8 µA/cm2; n = 119). Addition ofacetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid further reduced Isc, suggesting forskolin also stimulatesHCO3 secretion. This was confirmed by ionsubstitution studies. The forskolin-induced Iscwas inhibited by 293B, Ba2+, clofilium, and quinine,whereas charybdotoxin was without effect. In F-HBE the forskolinIsc response was reduced to 1.2 ± 0.3 µA/cm2 (n = 30). In wt HBE, mucosal UTPinduced a transient increase in Isc ( Isc = 15.5 ± 1.1 µA/cm2;n = 44) followed by a sustained plateau, whereas in F-HBE the increase in Isc was reduced to5.8 ± 0.7 µA/cm2 (n = 13). In wtHBE, 1-EBIO, NS004, 8-MOP, and genistein increased Isc by 11.6 ± 0.9 (n = 20), 10.8 ± 1.7 (n = 18), 10.0 ± 1.6 (n = 5), and 7.9 ± 0.8 µA/cm2(n = 17), respectively. In F-HBE, 1-EBIO, NS004, and8-MOP failed to stimulate Cl secretion. However, additionof NS004 subsequent to forskolin induced a sustained Cl secretory response (2.1 ± 0.3 µA/cm2,n = 21). In F-HBE, genistein alone stimulatedCl secretion (2.5 ± 0.5 µA/cm2,n = 11). After incubation of F-HBE at 26°C for24 h, the responses to 1-EBIO, NS004, and genistein were allpotentiated. 1-EBIO and genistein increased Na+ absorptionacross F-HBE, whereas NS004 and 8-MOP had no effect. Finally,Ca2+-, but not cAMP-mediated agonists, stimulatedK+ secretion across both wt HBE and F-HBE in aglibenclamide-dependent fashion. Our results demonstrate thatpharmacological agents directed at both basolateral K+ andapical Cl conductances directly modulate Cl secretion across HBE, indicating they may be useful in ameliorating theion transport defect associated with CF. |
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