Evidence for reduced Cl‐ and increased Na+ permeability in cystic fibrosis human primary cell cultures.

Abstract

SUMMARY1. Employing a primary cell culture system and intracellular microelectrodes, we quantitated and compared the Na+ and Cl-pathways in apical membranes of normal and cystic fibrosis (CF) human airway epithelia.2. Like the transepithelial difference (PD) in situ, the PD of CF epithelia in culture (-27+4 mV, mean+S.E.M.; n = 28) exceeded the PD of normal epithelia (-10 +1 mV; n = 22). The raised PD principally reflected an increase in the rate of active transport (equivalent short circuit, Ieq) for CF epithelia (61 + 9 ,tA cm-2) as compared with normal epithelia (23 + 3 ,uA cm-2). No significant differences in transepithelial resistance were detected. 3. As indicated by ion replacement studies (gluconate for Cl), the apical membrane of normal cells exhibits an apical membrane Cl-conductance (Gc1) that can be activated by isoprenaline. CF cells do not exhibit an apical membrane GC1, nor can a GC1 be activated by isoprenaline.4. CF cells exhibited a larger amiloride-sensitive Ieq and amiloride-sensitive apical membrane conductance (GNa) than normal cells. Further, the amiloride-sensitive Ieq was increased by isoprenaline in CF but not normal airway epithelia.5. Equivalent circuit analysis yielded evidence for a more positive electromotive force (EMF) across the apical membrane and a more negative EMF across the basolateral membrane of CF cells as compared with normal cells. Baseline resistances of the apical (Ra) and basolateral (Rb) membranes did not differ for normal and CF cells.6. Estimates of the resistance of the paracellular path to ion flow (R.) by equivalent circuit analysis or ion substitution detected no differences in R. between CF and normal cells.7. We conclude that abnormalities in both cellular Cl-permeability (reduced) and Na+ permeability (increased) are characteristic of the cultured CF respiratory epithelial cell. These data suggest that a defect in the regulation of apical membrane permeabilities is a central feature of this disease.

Interregulation of Proton-gated Na+ Channel 3 and Cystic Fibrosis Transmembrane Conductance Regulator

“…CFTR down-regulates amiloridesensitive ␣␤␥ ENaC activity in Xenopus oocytes (3)(4)(5)(6)(7)(8)(9)(10)(11). These results are in agreement with observations from normal and CF tissues (12)(13)(14)(15)(16)(17). On the other hand, ENaC up-regulates the CFTR current by up to 6-fold, and this process is not Na ϩ -dependent (7,8,18,19).…”

Mechanisms of Bicarbonate Secretion: Lessons from the Airways

“…These investigators observed that the intracellular pHs of normal and CF airway cells were equal (normal ¼ 7.15 + 0.02 vs. CF ¼ 7.11 + 0.05), but that the apical membrane potentials (V ap ) were different and of opposite polarity (normal ¼ 219 + 2 mV vs. CF ¼ 3 + 5 mV). This difference has been confirmed in several additional studies by Boucher and coworkers (Boucher et al 1988;Willumsen et al 1989a,b). Thus, at an extracellular HCO 3 2 concentration of 25 mM, an extracellular and intracellular pCO 2 of 40 mm Hg, and a pH of 7.15, there is an outwardly directed driving force for HCO 3 2 secretion of 6 mV across the apical membrane of normal cells.…”

“…Indeed, only after Na þ transport is down-regulated or inhibited with amiloride is it possible to observe Cl 2 secretion in normal cells. It is generally accepted that the difference in V ap between normal and CF cells is due to the loss of CFTR channels and the higher Na þ permeability in CF cells (Boucher et al 1986(Boucher et al , 1988Boucher 1994a,b). Besides Cl 2 , CFTR also conducts HCO 3 2 , as shown in the studies of Gray et al (1990) and Linsdell and coworkers (Linsdell et al 1997;Tang et al 2009;Li et al 2011) and more recently by Ishiguro et al (2009).…”

Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium

“…Confluent monolayers of primary human nasal epithelium were mounted in modified Ussing chambers (Knowles et al, 1983;Boucher et al, 1988). The studies were performed under short circuit current conditions at 37°C, and in some studies, amiloride was added to the apical bath (1O-4 M) to block transepithelial sodium transport.…”