Cystic fibrosis and non-cystic-fibrosis human nasal epithelium show analogous Na + absorption and reversible block by phenamil

Abstract

Transepithelial short-circuit current (ISC), potential (VT) and resistance (RT) of confluent monolayers of human nasal epithelium cultured from patients with and without cystic fibrosis (CF) were measured. In our Ussing chamber experiments with monolayers derived from non-CF and CF patients neither ISC (non-CF: 14.1 +/- 1.0 microA/cm2, n = 77; CF: 16.7 +/- 1.5 microA/cm2, n = 42), nor RT (non-CF: 288 +/- 15 Omega . cm2; CF: 325 +/- 20 Omega . cm2) showed any significant differences, only VT showed moderate but significant different values (non-CF: -3.6 +/- 0.4 mV; CF: -5.6 +/- 0.7 mV, respectively). Total ISC in CF cells was nearly completely inhibited by amiloride (92 +/- 9.6%), while in non-CF tissue amiloride-insensitive conductances mediated a considerable amount of the ISC (36.3 +/- 6.1%), indicating a lower activity of amiloride-sensitive Na+ conductances in non-CF cells. In both tissues the amiloride-sensitive ISC could also be blocked by the amiloride analogues benzamil, phenamil and 5-(N-ethyl-N-isopropyl)2', 4'-amiloride (EIPA) with different affinities. However, amiloride had a significant lower affinity in CF tissue (half-maximal blocker concentration, K1/2 = 586 +/- 59 nM) compared with non-CF tissue (K1/2 = 294 +/- 22 nM). Astonishingly, phenamil, a blocker which irreversibly blocks all epithelial Na+ channels hitherto described, inhibited the Na+ conductances of human nasal epithelium in a completely reversible way, but nevertheless with high affinity (non-CF: K1/2 = 12.5 +/- 1.2 nM; CF: K1/2 = 17.1 +/- 1.1 nM). Even in high doses none of these blockers had any effect on intracellular Ca2+ concentration as measured with Fura-2. From these findings, we conclude that the epithelial Na+ conductances of human CF nasal epithelium show modified regulation or are functionally different from those of other tissues.

Evaluation of Second Generation Amiloride Analogs as Therapy for Cystic Fibrosis Lung Disease

“…With respect to potency on ENaC, our finding that benzamil is more potent than phenamil is not in agreement with early studies performing similar structure-activity relationships with similar tissues (Stutts et al, 1995) or different tissue models (Kleyman and Cragoe, 1988). One study (Blank et al, 1997), however, reported that phenamil was not as potent as benzamil at blocking ENaC in human airway epithelia. The differences in rank order likely reflect variations in raw data collection.…”

“…Two second generation amiloride analogs, benzamil and phenamil, have been considered as alternatives to amiloride for CF pharmacotherapy (Blank et al, 1997;Hofmann et al, 1998;Rodgers and Knox, 1999). Benzamil and phenamil have substituted benzyl and phenyl groups, respectively, on the terminal nitrogen atom of the guanidino moiety of amiloride ( Fig.…”

Minor Role of Cl^– Secretion in Non-Cystic Fibrosis and Cystic Fibrosis Human Nasal Epithelium

“…Volume, viscosity and composition of airway surface liquid (ASL) are regulated by ion transport processes which were generally believed to follow a composite model combining several transport processes. Na + , passively followed by water, is absorbed by amiloride-sensitive Na + channels as well as by amiloride-insensitive Na + conductances [8] in the apical membrane and is extruded via a basolateral Na + /K + -ATPase, the rate limiting step for this Na + absorption being the entry of Na + through the apical Na + channels. Cl -enters the cell via a basolateral Na + /K + /2Cl -cotransporter and is thought to contribute to apical Cl -secretion through at least 3 different classes of Cl -channels: (I) a PKA-and PKC-activated Cl -channel which is sensitive to DPC termed CFTR; (II) a DIDS-sensitive outwardly rectifying Clchannel which is activated by intracellular Ca 2+ (ORCC), and (III) a Cl -channel activated by extracellular ATP.…”

“…Using the technique we showed in some recent papers that there is no net balance between Na + absorption and Cl -secretion in primary cultures of human respiratory epithelium and the major part of transepithelial ion transport processes is mediated by Na + in CF as well as in non-CF epithelia [7,8]. In this paper we confirm and extend our previous observations that in human nasal epithelium of CF tissue and, even more surprisingly, non-CF tissue Cl -secretion via CFTR or other Clchannels plays only a minor role in the generation of the total transepithelial short-circuit current.…”

Optimization of CFTR-mRNA transfection in human nasal epithelial cells

“…As expected, the control cells had the highest amiloride-sensitive current. It is well known that primary HNE cells have an amiloride-sensitive current [38,39]. In comparison, in cells transfected with different doses of wtCFTR-mRNA the transepithelial ENaC current decreased stepwise, possibly showing a trend in the down-regulation of ENaC by CFTR (Fig.…”