| Summary: | Regulation of intracellular pH (pH<sub>i</sub>) 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 pH<sub>i</sub> in single cells plated on coverslips and mounted in a temperature regulated superfusion chamber on a microscope stage. In both HCO<sub>3</sub><sup>-</sup>containing and HCO<sub>3</sub><sup>-</sup> -free conditions, pH<sub>i</sub> was ~ 7.20 and buffering capacity was found to be inversely related to pH<sub>i</sub>. Recovery from an acid load (induced by NH<sub>4</sub>Cl prepulse) was Na<sup>+</sup>-dependent. In HCO<sub>3</sub><sup>-</sup> -free conditions, inhibition of pH<sub>i</sub> recovery by hexamethylene amiloride (HMA) identified Na<sup>+</sup>/H<sup>+</sup> exchange as the principal mechanism of acid extrusion. In contrast, in HCO<sub>3</sub><sup>- </sup>- role of Na<sup>+</sup>/H<sup>+</sup> exchange in pH<sub>i</sub> regulation under more physiological conditions. However, dihydro- 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (H<sub>2</sub>DIDS) affected this Na<sup>+</sup> and HCO<sub>3</sub><sup>-</sup> -dependent pH<sub>i</sub> recovery and suggested that Na<sup>+</sup>-dependent recovery from intracellular acidification in HCO<sub>3</sub><sup>-</sup>- containing conditions was mediated primarily by an H<sub>2</sub>DIDS-sensitive component. Inhibition of pH<sub>i</sub> recovery by bafilomycin A<sub>1</sub> in HCO<sub>3</sub><sup>-</sup> -containing conditions also suggested that a proton pump was present in these cells. The failure of inwardly and outwardly - directed Cl<sup>-</sup> gradients to affect pH<sub>i</sub> recovery suggested that Cl<sup>-</sup> -dependent mechanisms were not present. The effects of HMA and H<sub>2</sub>DIDS on Na<sup>2+</sup> -dependent alkalinisation in cells preincubated in Na<sup>+ </sup>-free media were also investigated. Surprisingly, only HMA affected the rate of Na<sup>+</sup> -dependent alkalinisation. This directly contradicted the data reported above and implied that Na<sup>+</sup>/H<sup>+</sup> exchange was the primary pH<sub>i</sub> regulatory mechanism in HCO<sub>3</sub><sup>-</sup> - containing conditions in these cells. In conclusion, pH<sub>i </sub>in isolated sheep ciliated tracheal cells appears to be regulated by three acid-extruding mechanisms.
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