Abstract: | Using thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),we examined the effect of hyperosmolar solutions, which presumablycaused cell shrinkage, on intracellular pH(pHi) regulation in mesangialcells (single cells or populations) cultured from the rat kidney. Thecalibration of BCECF is identical in shrunken and unshrunken mesangialcells if the extracellular K+concentration (K+])is adjusted to match the predicted intracellularK+]. ForpHi values between ~6.7 and~7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (~300mosmol/kgH2O). In the nominalabsence ofCO2/HCO 3,exposing cell populations to a HEPES-buffered solution supplementedwith ~300 mM mannitol (600 mosmol/kgH2O) causes steady-statepHi to increase by ~0.4. The pHi increase is due to activationofNa+/H+exchange because, in single cells, it is blocked in the absence ofexternal Na+ or in the presence of50 µM ethylisopropylamiloride (EIPA). Preincubating cells in aCl -free solution for atleast 14 min inhibits the shrinkage-induced pHi increase by 80%. Wecalculated the pHi dependence oftheNa+/H+exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) thepHi dependence of the totalacid-extrusion rate and 2) thepHi dependence of theEIPA-insensitive acid-loading rate. Shrinkage alkali shifts thepHi dependence ofNa+/H+exchange by ~0.7 pH units. |