Ca²⁺- and swelling-induced activation of ion conductances in bronchial epithelial cells

Abstract

The present study was performed to examine Ca²⁺-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (V _te) and resistance (R _te) were measured in confluent monolayers. Resting cells had a membrane voltage (V _m) of −36±1.1 mV (n=137) which was mainly caused by K⁺ and Cl⁻ conductances and to a lesser extent by a Na⁺ conductance. V _te was apical-side-negative after stimulation. Equivalent short-circuit current (I _sc = V _te/R _te) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1–10 μmol/l) and ATP (0.1–100 μmol/l). The histamine-induced I _sc was blocked by either basolateral diphenhydramine (0.1 mmol/l, n=4) or apical cimetidine (0.1 mmol/l, n=4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K⁺ conductance and hyperpolarized V _m. This effect was mimicked by the Ca²⁺ ionophore ionomycin (1 μmol/l, n=11). Inhibition of the bradykinin-induced I _sc by the blocker HOE140 (1 μmol/l, n=3) suggested the presence of a BK₂ receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP ≈ ATP > ITP > GTP ≈ CTP ≈ [β,γ-methylene] ATP ≈ 2-methylthio-ATP = 0 and was obtained in I _sc measurements and patch-clamp recordings. This suggests the presence of a P_2u receptor. Hypotonic cell swelling activated both Cl⁻ and K⁺ conductances. The Cl⁻ conductance was only slightly inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (0.5 mmol/ l, n=3). These data indicate that 16HBE140- bronchial epithelial cells, which are known to express high levels of cystic fibrosis transmembrane conductance regulator protein, form a secretory epithelium. While hypotonic cell swelling activates both K⁺ and Cl⁻ channels, the Ca²⁺-induced Cl⁻ secretion is due mainly to activation of basolateral K⁺ channels.