The collecting duct of the inner stripe outer medulla (OMCDi) is a major site of distal nephron acidification. Using the pH sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5,6,-carboxyfluorescein (BCECF) and quantitative spectrofluorometry to measure intracellular pH in isolated perfused OMCDi, we have characterized basolateral transport processes responsible for regulation of intracellular pH. Experiments suggesting the existence of basolateral Cl-/base exchange were performed. In HCO3- containing buffers, bath Cl- replacement resulted in reversible alkalinization of the OMCDi from 7.22 +/- 0.05 to 7.57 +/- 0.12. Similarly 0.1 mM bath 4',4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) alkalinized the OMCDi from 7.14 +/- 0.09 to 7.34 +/- 0.09 and blocked further alkalinization by bath Cl- removal (delta = + 0.02 pH units). The concentration dependence kinetics of Cl-/base exchange revealed a K1/2 of 10 mM for external Cl- with a Vmax of 0.50 pH U/min. Experiments suggesting the existence of basolateral Na+/H+ exchange were also performed. Replacement of bath Na+ by tetramethylammonium resulted in reversible cell acidification (7.14 +/- 0.09 to 6.85 +/- 0.1). Tubules that were acidified by a brief exposure to NH4Cl displayed recovery of cell pH back to baseline at a rate that was highly dependent on bath Na+ concentration. Half maximal recovery rate was achieved at 7 mM bath Na+ and Vmax was 0.605 pH U/min. The Na+-dependent rate of cell pH recovery after acidification was blocked by 0.2 mM bath amiloride. These results suggest that intracellular pH in the OMCDi is regulated by parallel basolateral Na+/H+ exchange and Cl-/base exchange.
M D Breyer, H R Jacobson
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