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Abstract
This study provides the first information about pHi regulatory mechanisms in human retinal pigment epithelium (RPE). The experiments were carried out on fresh explant tissues from adult donor and fetal eyes, and pHi was measured using fluorescence microscopy techniques and the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In adult donor RPE, the resting pHi is 7.30 +/- 0.14 (mean +/- standard deviation; n = 6) in HCO3 Ringer's solution. In HCO3 Ringer's solution, apical Na removal caused rapid cell acidification with an initial rate of 0.40 +/- 0.10 pH U/min (n = 4). This Na-dependent acidification was partially inhibited by apical amiloride (n = 1) and DIDS (n = 1). In HCO3 Ringer's solution, pHi recovery from an acid load (NH4 prepulse) also was blocked by apical Na removal. In nominally HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells. These results suggest that the apical membrane of adult human RPE contains an Na/H exchanger and possibly a Na-dependent, DIDS-inhibitable pH regulatory mechanism, perhaps a NaHCO3 cotransporter. For the fetal RPE, the resting pHi was 7.16 +/- 0.10 (n = 9) and 7.19 +/- 0.10 (n = 20) in HCO3 and HCO3-free Ringer's solution, respectively. In HCO3 and HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells and the removal of apical Na caused cell acidification with an initial rate of 0.30 +/- 0.08 (n = 32) and 0.58 +/- 0.29 (n = 6) pH U/min, respectively. The pHi recovery from an acid load also was blocked by apical amiloride and apical Na removal. These results suggest that the apical membrane Na/H exchanger is the dominant acid extrusion mechanism in human fetal RPE.(ABSTRACT TRUNCATED AT 250 WORDS)