Martin J Hug, Robert J Bridges
We have previously demonstrated that the airway serous cell line Calu-3 employs a number of pH regulatory mechanisms required for bicarbonate secretion by these cells [1]. The aim of the present study was to investigate the pH regulatory mechanisms of serous cells of freshly isolated submucosal glands (SMG). Porcine SMG were dissected out of pig tracheas obtained from a local slaughterhouse. Single glands were transferred into the chamber of an inverted microscope, immobilized by two holding pipettes and the serous cells loaded with the fluorescent pH probe 2’,7’-bis-(2- carboxyethyl)-5,6-carboxyfluorescein (BCECF). Fluorescence was monitored from small areas consisting of up to 20 cells. The fluorescence ratio of the emission after excitation at 488 nm and 436 nm respectively was used to estimate cytosolic pH (pHi). Resting pHi of SMG cells in the absence of HCO3- /CO2 was 7.1±0.16 (n=24). Addition of a solution buffered with HCO3- /CO2 to the bath transiently acidified the cells by 0.18±0.03 (n=18). pHi rapidly recovered to a slightly more alkaline value than baseline pHi. Removal of the HCO3- /CO2 buffer strongly alkalinized SMG cells by 0.2±0.03 (n=18). To challenge pH regulatory mechanisms we exposed the cells to 20 mmol/L NH4+ in the absence and presence of HCO3- /CO2. In both cases we observed a rapid increase in pHi followed by a slight recovery. Washout of NH4+ strongly acidified the cells. Realkalinization of pHi could only be observed in the presence of Na+ . This effect was inhibited by the addition of the specific Na+/H+ exchanger isoform 1 (NHE1) blocker 3- methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride (HOE 694, 10–100 µmol/L) with an halfmaximal inhibitory concentration (IC50) of approximately 20 µmol/L. Full recovery of pHi in the presence of HOE 694 was observed when the cells were bathed in HCO3- /CO2 solution. Addition of forskolin (5 µmol/L) in the presence of HCO3- /CO2 did not significantly alter pHi or change pHi recovery after acid loading. We conclude that SMG cells possess both HCO3- dependent and HCO3- independent pHi; regulatory mechanisms that require the presence of extracellular Na+ . Further studies are required to understand whether bicarbonate is only transported to regulate pHi or whether this transport determines the overall secretory capacity of SMG serous cells.
