Proceedings of The Physiological Society
Kings College London (2005) J Physiol 565P, PC170
Use of simultaneous short circuit current and intracellular pH monitoring to investigate ion transport in a human airway cell line
Finlay, Lisa ; Wilson, Stuart ; Olver, Richard ;
1. Division of Maternal and Child Health Sciences, Tayside Institute of Child Health, Dundee, United Kingdom.
The transepithelial transport of anions (Cl-and HCO3-) is dependent upon the coordinated activity of anion channels, anion co-transporters, and K+ channels. Since the activity of at least some of these is pH-sensitive, we hypothesised that this transport may be influenced by changes in the intracellular pH (pHi). Moreover, since HCO3- is itself a physiologically relevant buffer, changes to the rate at which this anion is transported may influence pHi. In the present study we therefore explore the possibility of simultaneously measuring short circuit current (Isc), a measure of active ion transport, and intracellular pH (pHi) (see technique used by Ko et al, 1999) in polarised human lung epithelial cells (Calu-3s). Cells grown to confluence on Snapwell filters (3-4 days) were loaded with the pH-sensitive fluorescent dye, BCECF (2 hours). Filters were then inserted into miniature Ussing chambers and placed on the stage of an inverted microscope. The apical and basolateral sides of the cells were perfused independently with buffered physiological salt solution. The short circuit current (Isc) was recorded using the standard Ussing chamber technique. Simultaneously, cells were excited alternately at 440 and 490 nm and the BCECF fluorescence emitted was detected at 540 nm using a photomultiplier tube. At the end of each experiment, an in situ calibration was carried out using the high K+/nigericin technique. Preliminary experiments reveal that Calu-3 cells bathed in Hepes-buffered solution typically respond to alkalinisation brought about by a NH4Cl pulse with an increase in Isc (8.7 ± 2.43 μA cm-2 above basal Isc, n=9), that is followed by a decrease in Isc during subsequent intracellular acidification (3.97 ± 1.23 μA cm-2 below basal Isc). This is typically followed by recovery of both pHi and Isc back to near baseline values (0.99 ± 0.62 μA cm-2 below basal Isc). These early results indicate that changes in intracellular pH do influence electrogenic ion transport in these cells and this technique will enable us to examine the role of pH in Cl- and HCO3- transport and to study the mechanisms by which airway epithelial cells regulate changes in pHi.
Where applicable, experiments conform with Society ethical requirements