Measurement of Multi Ion Transport through Human Bronchial Epithelial Cell Line Provides an Insight into the Mechanism of Defective Water Transport in Cystic Fibrosis

We measured concentration changes of sodium, potassium, chloride ions, pH and the transepithelial potential difference by means of ion-selective electrodes, which were placed on both sides of a human bronchial epithelial 16HBE14σ cell line grown on a porous support in the presence of ion channel blockers. We found that, in the isosmotic transepithelial concentration gradient of either sodium or chloride ions, there is an electroneutral transport of the isosmotic solution of sodium chloride in both directions across the cell monolayer. The transepithelial potential difference is below 3 mV. Potassium and pH change plays a minor role in ion transport. Based on our measurements, we hypothesize that in a healthy bronchial epithelium, there is a dynamic balance between water absorption and secretion. Water absorption is caused by the action of two exchangers, Na/H and Cl/HCO3, secreting weakly dissociated carbonic acid in exchange for well dissociated NaCl and water. The water secretion phase is triggered by an apical low volume-dependent factor opening the Cystic Fibrosis Transmembrane Regulator CFTR channel and secreting anions that are accompanied by paracellular sodium and water transport.

New ISE-Based Apparatus for Na+, K+, Cl−, pH and Transepithelial Potential Difference Real-Time Simultaneous Measurements of Ion Transport across Epithelial Cells Monolayer–Advantages and Pitfalls

Cystic Fibrosis (CF) is the most common fatal human genetic disease, which is caused by a defect in an anion channel protein (CFTR) that affects ion and water transport across the epithelium. We devised an apparatus to enable the measurement of concentration changes of sodium, potassium, chloride, pH, and transepithelial potential difference by means of ion-selective electrodes that were placed on both sides of a 16HBE14σ human bronchial epithelial cell line that was grown on a porous support. Using flat miniaturized ISE electrodes allows for reducing the medium volume adjacent to cells to approximately 20 μL and detecting changes in ion concentrations that are caused by transport through the cell layer. In contrast to classic electrochemical measurements, in our experiments neither the calibration of electrodes nor the interpretation of results is simple. The calibration solutions might affect cell physiology, the medium composition might change the direction of actions of the membrane channels and transporters, and water flow that might trigger or cut off the transport pathways accompanies the transport of ions. We found that there is an electroneutral transport of sodium chloride in both directions of the cell monolayer in the isosmotic transepithelial concentration gradient of sodium or chloride ions. The ions and water are transported as an isosmotic solution of 145 mM of NaCl.

Regulation of ion transport by pH and [HCO3−] in isolated gills of the crab Neohelice (Chasmagnathus) granulata

Posterior isolated gills of Neohelice ( Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3−] and pH. Elevating [HCO3−] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference ( Vte), a measure of ion transport. The elevation in [HCO3−] also induced a switch from acid secretion (−43.7 ± 22.5 μequiv·kg−1·h−1) in controls to base secretion (84.7 ± 14.4 μequiv·kg−1·h−1). The HCO3−-induced Vte increase was inhibited by basolateral acetazolamide (200 μmol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3− did not take place in Cl−-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 ± 18.4 μequiv kg−1 h−1. This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl−-free saline. Therefore, the cellular response to low pH is different from the HCO3−-stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3−-stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3−-dependent cotransporters mediate the response to low pH.