Mechanisms of intracellular pH regulation in ciliated tracheal epithelial cells
Regulation of intracellular pH (pHi) was studied in ciliated epithelial cells isolated from sheep tracheae. Microspectrofluorimetry of the pH -sensitive dye 2'; 7' - biscarboxyethyl -5(6) -carboxyfluorescein (BCECF) was used to measure pHi in single cells plated on coverslips and mounted in a temperature regulated superfusion chamber on a microscope stage. In both HCO3-containing and HCO3- -free conditions, pHi was ~ 7.20 and buffering capacity was found to be inversely related to pHi. Recovery from an acid load (induced by NH4Cl prepulse) was Na+-dependent. In HCO3- -free conditions, inhibition of pHi recovery by hexamethylene amiloride (HMA) identified Na+/H+ exchange as the principal mechanism of acid extrusion. In contrast, in HCO3- - role of Na+/H+ exchange in pHi regulation under more physiological conditions. However, dihydro- 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (H2DIDS) affected this Na+ and HCO3- -dependent pHi recovery and suggested that Na+-dependent recovery from intracellular acidification in HCO3-- containing conditions was mediated primarily by an H2DIDS-sensitive component. Inhibition of pHi recovery by bafilomycin A1 in HCO3- -containing conditions also suggested that a proton pump was present in these cells. The failure of inwardly and outwardly - directed Cl- gradients to affect pHi recovery suggested that Cl- -dependent mechanisms were not present. The effects of HMA and H2DIDS on Na2+ -dependent alkalinisation in cells preincubated in Na+ -free media were also investigated. Surprisingly, only HMA affected the rate of Na+ -dependent alkalinisation. This directly contradicted the data reported above and implied that Na+/H+ exchange was the primary pHi regulatory mechanism in HCO3- - containing conditions in these cells. In conclusion, pHi in isolated sheep ciliated tracheal cells appears to be regulated by three acid-extruding mechanisms.