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Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.

Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units.