pH regulation in horizontal cells of the skate retina

To examine the mechanisms by which horizontal cells regulate intracellular pH (pHi), measurements were recorded from isolated cells enzymatically dissociated from the skate retina utilizing the pH-sensitive dye BCECF. In a HCO3--containing Ringer solution, steady-state pHi was 7.32+/-0.13 (mean+/-S.D., n=70). Recovery from acidification was examined using the NH4+ prepulse technique. When NH4+ was removed from the extracellular solution, pHi dropped rapidly to approximately 0.3 pH units below the initial baseline, and then recovered at an initial rate of approximately 0.072 pH units/min. During recovery of pHi after the acid load, the removal of Na+ or the addition of amiloride from a HCO3--free extracellular solution reduced the rate of recovery by 79%+/-11% and 69%+/-14%, respectively. In the presence of DIDS, which inhibits primarily anion transport, or during the removal of Na+, the recovery from acidification was reduced by 83%+/-10% and 70%+/-11%, respectively, as compared to the control value in HCO3--containing solution. These results suggest that the skate horizontal cell possesses a Na/H exchanger as well as a Na+-and HCO3--dependent mechanism for removal of excess acid. Removal of HCO3- or Cl- from the extracellular solution had little effect on pHi, but removing external Na+ induced a marked decrease in pHi that fell at an initial rate of approximately 0.3 pH units min-1. This rate of acidification was decreased by 58%+/-19% in the presence of DIDS (500 micron) and reduced by 28%+/-13% with the addition of amiloride (2 mm). Thus, Na- and HCO3-dependent transport was about 2-fold more active than Na/H exchange during low Na+-induced acidification. The intrinsic pH-buffer capacity, determined from the pHi change induced by incremental reductions in the [NH4+] of the extracellular solution, was 24.2 mm/pH unit at the horizontal cell's resting pHi. Moreover, pHi was relatively insensitive to changes in membrane potential; in experiments under whole-cell voltage clamp (-70 mV), intracellular pH remained constant during depolarizing voltage swings to -30 mV or +30 mV, as well as during hyperpolarizing pulses to -90 or -110 mV.