INTRACELLULAR PH OF TISSUE-CULTURED BOVINE CORNEAL ENDOTHELIAL-CELLS

Intracellular pH (pH(i)) of bovine tissue-cultured corneal endothelial cells has been measured under several experimental conditions. Determinations were made on individual cells using video-imaging techniques that allowed assessment of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence at 440 and 490 nm. Each experiment had a calibration performed on a cell monolayer: this was performed using a high K+-nigericin solution. Resting pH(i) was 7.25 +/- 0.03 (n = 18) in bicarbonate solutIon at pH 7.4. Amiloride (1 mM) caused an acidification of approximately 0.2 U within 2 min: replacement with normal Ringer allowed a return to normal pH(i) after an alkali overshoot. Exposure to 20 mM NH4Cl caused alkalinization that became acidic upon washout of NH4Cl. In Na+-rich solution pH(i) returned to normal after acidification but pH(i) remained low in Na+-free solution until substituted by Na+-rich solution. Removal of HCO3- from the bathing solution caused a nonsignificant acidification of pH(i) by 0.1 U at 2 and 4 min, and 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS; 1 mM) acidified pH(i) by 0. 14 U at 2 min and 0.24 U at 4 min. Addition of DIDS (1 mM) in a HCO3--free solution had no effect on pH(i). Hydrogen peroxide acidified pH(i) by 0.3 U at 50 muM and 1 mM. These results indicate that a Na+:H+ antiport exists that regulates pH(i) even at normal ambient pH in the presence of bicarbonate: this process becomes highly activated after an acid load. There is a DIDS-sensitive HCO3- movement that is probably coupled to Na+ or Cl-.