ISSN:
1432-2013
Keywords:
Key wordsαhENaC
;
Airway epithelial cells
;
Amiloride
;
CFTR
;
Patch clamp
;
RT-PCR
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract According to previous studies, amiloride-sensitive (Amil+) Na+ channels are present in apical membranes of airway epithelial cells. When isolated from intact tissue and grown in primary culture or as immortalized cell lines, these cells tend to lose these Amil+ Na+ channels. The present study examines this issue in immortalized human bronchial epithelial cells (16HBE14o-cell line). The mRNA of one subunit of the Na+ channel (αhENaC) was semi-quantified by polymerase chain reaction of reverse transcribed RNA. Transcripts were significantly increased when cells were exposed to aldosterone and dexamethasone irrespective of whether grown on permeable supports or plastic. When grown on plastic dishes 16HBE14o-cells showed cAMP-dependent Cl− currents in whole-cell (WC) patch-clamp experiments, corresponding to expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Na+ currents could not be detected although cells expressed significant amounts of αhENaC as demonstrated by Northern blot analysis. In contrast, when cells were grown on permeable supports or cultured in the presence of butyrate (5 mmol/l, plastic or permeable support) or aldo-sterone and dexamethasone (both 1 μmol/l, plastic or permeable support), amiloride (10 μmol/l) hyperpolarized the membrane voltage (ΔV m) by 2–9 mV, paralleled by small reductions of WC conductances (ΔG m) of 0.4–4.0 nS. The effects of amiloride on ΔVm were generally more pronounced (up to 12 mV) when cells were grown on permeable supports. The amiloride effect (ΔV m) was concentration dependent with an inhibitory constant, K i, of about 0.1 μmol/l. We further examined whether the induction of an Amil+ Na+ conductance was paralleled by additional changes in membrane conductance. In fact, the cAMP-activated Cl− conductance was significantly attenuated by approximately 80% (n = 35) in cells responding to amiloride, whilst the ATP-activated K+ conductance remained unaffected. The present data suggest that cellular mechanisms determining differentiation control the functional expression of Na+ and Cl− conductances in human airway epithelial cells.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s004240050038
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