Culture-dependent expression of Na+ conductances in airway epithelial cells

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 alphahENaC) 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 alphahENaC 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 aldosterone and dexamethasone (both 1 micromol/l, plastic or permeable support), amiloride (10 micromol/l) hyperpolarized the membrane voltage (deltaVm) by 2-9 mV, paralleled by small reductions of WC conductances (deltaGm) of 0.4-4.0 nS. The effects of amiloride on deltaVm were gnerally more pronounced (up to 12 mV) when cells were grown on permeable supports. The amiloride effect (deltaVm) was concentration dependent with an inhibitory constant, Ki, of about 0.1 micromol/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 function expression of Na+ and Cl- conductances in human airway epithelial cells.