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 (pH(i)) 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 pH(i) values between similar to 6.7 and similar to 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/ kgH(2)O) is threefold larger than in unshrunken cells (similar to 300 mosmol/kgH(2)O). In the nominal absence of CO2/HCO3-, exposing cell populations to a HEPES-buffered solution supplemented with similar to 300 mM mannitol (600 mosmol/kgH(2)O) causes steady-state pH(i) to increase by similar to 0.4. The pH(i) 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 mu M ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pH(i) increase by 80%. We calculated the pH(i) dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pH(i) dependence of the total acid-extrusion rate and 2) the pH(i) dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pH(i) dependence of Na+/H+ exchange by similar to 0.7 pH units.