Intracellular pH regulation in fresh and cultured bovine corneal endothelium. II. Na+:HCO3- cotransport and Cl-/HCO3- exchange.

The comparison of intracellular pH (pHi) regulation between fresh (FBCE) and cultured (CBCE) bovine corneal endothelium was extended to HCO3- transport mechanisms. Upon introduction of CO2/HCO3- Ringer's solution, there was a small, sharp acidification followed by an alkalinization .09 and .18 pH U above the HCO3- free resting pHi for FBCE and CBCE, respectively. This increase in pHi totally depended upon the presence of Na+, independent of Cl- and blocked by the anion transport inhibitor, H2DIDS (0.5 mmol/l). Recovery from an (NH4)2SO4-induced acid load also was blocked by Na+ removal and inhibited 62% and 84% by 0.5 mmol/l H2DIDS for FBCE and CBCE, respectively. These results indicate the presence of a Na+ dependent HCO3- transporter. Additions of 22 mmol/l K+ or 5 mmol/l Ba2+ led to substantial H2DIDS-inhibitable base influx in HCO3- Ringer's, which was significantly reduced in the absence of HCO3- for FBCE and CBCE, consistent with the presence of electrogenic Na+:nHCO3- cotransport. Using the Na+ sensitive intracellular dye, SBFI, we confirmed that the addition of HCO3- resulted in a H2DIDS-sensitive Na+ influx. The mean steady-state [Na+i] = 14 +/- 3 mmol/l in bicarbonate-free Ringer's and 31.5 +/- 2 mmol/l in CO2/HCO3-. HCO3- induced Na+ influx was reduced 74% by 0.5 mmol/l H2DIDS. Removal of Cl- from bicarbonate Ringer's alkalinized FBCE and CBCE by .12 +/- .01 and .21 +/- .03, respectively. The increased pHi was completely blocked by 0.5 mmol/l H2DIDS. Similar alkalinizations were seen when Cl- was removed from air equilibrated bicarbonate-free Ringer's. However, because of the lowered buffering capacity in the absence of HCO3-, the flux was reduced by 80%. Cl- free alkalinizations were eliminated by equilibrating the bicarbonate-free Ringer's with 100% nitrogen gas, indicating that residual CO2 can act as a substrate. Bicarbonate efflux on readdition of Cl- was Na+ independent and pHi sensitive (inactivated by low pHi), and showed simple saturating kinetics with respect to bath Cl, K1/2 = 22 and 16 mmol/l for FBCE and CBCE, respectively. These data are consistent with the presence of Cl-/HCO3- exchange in FBCE and CBCE. Application of 0.5 mmol/l H2DIDS to resting cells in HCO3- Ringer's significantly reduced pHi by .23 +/- .03 and .18 +/- .02 in FBCE and CBCE, respectively, indicating net HCO3- influx in resting cells and suggesting that the stoichiometry of the Na+:nHCO3- cotransporter favors Na+ and HCO3- uptake, ie, n < or = 2.