Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO−3 by a Cl −-independent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO−3 secretion and caused the secretion of Cl − by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO−3 secretion to Cl − secretion because the uptake of HCO−3 across the basolateral membrane is mediated by a 4,4 ′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive Na+:HCO−3 cotransporter. Since the stoichiometry reported for Na +:HCO−3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO−3 entry and favor the secretion of Cl −. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO−3 and Cl − secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO−3 and a Cl − channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO−3 secretion in the human airways warrants greater attention.
Bicarbonate and Chloride Secretion in Calu-3 Human Airway Epithelial Cells
1Abbreviations used in this paper: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CTX, charybdotoxin; DNDS, 4,4′-dinitrostilben-2,2′disulfonic acid; 1-EBIO, 1-ethyl-2-benzimidazolinone; NBC, Na+:HCO−3 cotransporter; R T, transepithelial resistance.
White (1989) has reported a complete inhibition by 1 mM DNDS of a Na+:HCO−3 cotransporter in the basolateral membrane of salamander intestine. Newman (1991) has reported a 73% inhibition by 2 mM DNDS of a Na+:HCO−3 cotransporter in retinal glial cells of the salamander. Although perhaps not directly comparable, Boron and Knakal (1989) reported a DNDS Ki of 300 μM of a Na+- and Cl−- dependent HCO−3 cotransporter in the squid axon.
The net secretion of HCO−3 can be calculated from the equation J (mol · cm−2 · h−1) = buffer capacity (βCO2) · ΔpH · h−1 · volume · area−1, where βCO2 = 2.3 (25 mM HCO−3 ), final volume = 100 μl, and area = 1.1 cm2. Thus, JHCO3 = 57.5 · 0.33 ΔpH · h−1 · 0.1 × 10−3 liters · 1.1 cm−2 = 1.7 μeq · cm−2 · h−1. Although the final volume was not measured, it was consistently greater in the forskolin-stimulated monolayers compared with the control monolayers. Therefore, the actual net flux of HCO−3 would be proportionally higher and be in even closer agreement with the forskolin-stimulated increase in Isc.
Together with the measured net secretion of HCO−3 of ∼60 μA · cm−2, one can use the values for EHCO3 (−13 mV) and ECl (−35 mV) to obtain an estimate of the apical membrane HCO−3 conductance (gHCO3), where gHCO3 = (ECl − EHCO3)/IHCO3 = 2.7 mS · cm−2. This estimation assumes the apical membrane is at ECl. Results from impedance analysis on Calu-3 cells indicate forskolin increases the apical membrane conductance (gapical) to ∼20 mS · cm−2 (Bridges, R.J., unpublished observations). This remarkably high conductance would ensure the apical membrane potential is at or near ECl, but also yields a HCO−3 to Cl− conductance ratio of ∼0.15 (where gCl = gapical − gHCO3 = 20 − 2.7 = 17.3 mS · cm−2 so that gHCO3/gCl = 2.7/17.3 = 0.15), a value in good agreement with the patch clamp estimates of 0.15–0.25 for CFTR. Moreover, an apical membrane gCl of 17.3 mS · cm−2 means a driving force of only 3.5 mV is required to achieve a net Cl− secretion of 60 μA · cm−2.
Daniel C. Devor, Ashvani K. Singh, Linda C. Lambert, Arthur DeLuca, Raymond A. Frizzell, Robert J. Bridges; Bicarbonate and Chloride Secretion in Calu-3 Human Airway Epithelial Cells . J Gen Physiol 1 May 1999; 113 (5): 743–760. doi: https://doi.org/10.1085/jgp.113.5.743
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