PH REGULATION IN HORIZONTAL CELLS OF THE SKATE RETINA

To examine the mechanisms by which horizontal cells regulate intracell ular pH (pH(i)), measurements were recorded from isolated cells enzyma tically dissociated from the skate retina utilizing the pH-sensitive d ye BCECF, In a HCO3--containing Ringer solution, steady-state pH(i) wa s 7.32+/-0.13 (mean+/-S.D., n = 70). Recovery from acidification was e xamined using the NH4+ prepulse technique. When NH4+ was removed from the extracellular solution, pH(i) dropped rapidly to similar to 0.3 pH units below the initial baseline, and then recovered at an initial ra te of similar to 0.072 pH units/min, During recovery of pH(i) after th e acid load, the removal of Na+ or the addition of amiloride from a HC O3--free extracellular solution reduced the rate of recovery by 79%+/- 11% and 69%+/-14%, respectively. In the presence of DIDS, which inhibi ts primarily anion transport, or during the removal of Na+, the recove ry from acidification was reduced by 83%+/-10% and 70%+/-11%, respecti vely, as compared to the control value in HCO3--containing solution. T hese results suggest that the skate horizontal cell possesses a Na/H e xchanger as well as a Na+- and HCO3--dependent mechanism for removal o f excess acid. Removal of HCO3- or Cl- from the extracellular solution had little effect on pH(i), but removing external Na+ induced a marke d decrease in pH(i) that fell at an initial rate of similar to 0.3 pH units min(-1). This rate of acidification was decreased by 58%+/-19% i n the presence of DIDS (500 mu M) and reduced by 28%+/-13% with the ad dition of amiloride (2 mM). Thus, Na- and HCO3-dependent transport was about 2-fold more active than Na/H exchange during low Na+-induced ac idification. The intrinsic pH-buffer capacity, determined from the pH( i) change induced by incremental reductions in the [NH4+] of the extra cellular solution, was 24.2 mM/pH unit at the horizontal cell's restin g pH(i). Moreover, pH(i) was relatively insensitive to changes in memb rane potential: in experiments under whole-cell voltage clamp (-70 mV) , intracellular pH remained constant during depolarizing voltage swing s to -30 mV or +30 mV, as well as during hyperpolarizing pulses to -90 or -110 mV. (C) 1998 Academic Press Limited.