Mechanisms of intracellular pH regulation in ciliated tracheal epithelial cells

• Main Author: Maddox, Angela D. M.
• Published: University of Aberdeen 1999
• Subjects: 636.089; Veterinary sciences & veterinary medicine
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287601

Regulation of intracellular pH (pH_i) 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_i 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, pH_i was ~ 7.20 and buffering capacity was found to be inversely related to pH_i. Recovery from an acid load (induced by NH₄Cl prepulse) was Na⁺-dependent. In HCO₃^- -free conditions, inhibition of pH_i 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 pH_i regulation under more physiological conditions. However, dihydro- 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (H₂DIDS) affected this Na⁺ and HCO₃^- -dependent pH_i recovery and suggested that Na⁺-dependent recovery from intracellular acidification in HCO₃^-- containing conditions was mediated primarily by an H₂DIDS-sensitive component. Inhibition of pH_i 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 pH_i 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 pH_i regulatory mechanism in HCO₃^- - containing conditions in these cells. In conclusion, pH_iin isolated sheep ciliated tracheal cells appears to be regulated by three acid-extruding mechanisms.