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Am J Physiol Cell Physiol 278: C1218-C1229, 2000;
0363-6143/00 $5.00
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Vol. 278, Issue 6, C1218-C1229, June 2000

Extracellular Clminus modulates shrinkage-induced activation of Na+/H+ exchanger in rat mesangial cells

Yukio Miyata, Shigeaki Muto, Satoru Yanagiba, and Yasushi Asano

Department of Nephrology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi 329-0498, Japan

To examine the effect of hyperosmolality on Na+/H+ exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pHi) in a single MC from rat glomeruli. All the experiments were performed in CO2/HCO-3-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH2O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 µM ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular Na+. The hyperosmolality shifted the Na+-dependent acid extrusion rate vs. pHi by 0.15-0.3 pH units in the alkaline direction. Removal of extracellular Cl- by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the Na+-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on Na+-dependent pHi recovery rate or Na+-dependent acid extrusion rate. The Cl--dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS, and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of pHi, 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular Cl-, and 4) the Cl--dependent NHE activation under hyperosmotic conditions partly occurs via Cl- channel and microtubule-dependent processes.

intracellular pH; chloride ion; chloride channel; microtubule


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