ACID EXTRUSION IN S3 SEGMENT OF RABBIT PROXIMAL TUBULE .1. EFFECT OF BILATERAL CO2/HCO3-

Monitoring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein, we studied intracellular pH (pH(i)) regulation in the isolated perfused S3 segment of rabbit proximal tubule. To explain a previous observation, that steady-state pH(i) is higher in the presence than in the absence of CO2/HCO3- (N. L. Nakhoul, L. K. Chen, and W. F. Boron. J. Gen. Physiol. 102: 1171-1205, 1993), we examined the effect of bilateral (i.e., luminal and basolateral) CO2/HCO3- on the acid extrusion processes responsible for recovery of pH(i) from acid loads. To compute fluxes from rates of pH(i) change, we determined the pH(i) dependence of intrinsic intracellular buffering power, which was similar to 50 mM/pH at pH(i) 6.5 and fell linearly to similar to 20 mM at pH(i) 7.4. In one series of experiments, we monitored the rate of pH(i) recovery from an acid load imposed by an NH4+/NH3 prepulse. Over a broad range of pH(i) values, total net acid extrusion was approximately four times higher in bilateral presence of CO2/HCO3- than in its absence. In a second group of experiments, which were designed to determine the effect of CO2/HCO3- on luminal Na+/H+ exchange, we monitored the rate of pH(i) recovery elicited by adding Na+ back to only the lumen, after first removing Na+ bilaterally. Initial rate of luminal Na+-dependent net acid extrusion in presence of CO2/HCO3- was similar to 229 mu M/s (pH(i) 6.92), similar to 1.8 times higher than the flux of similar to 127 mu M/s (P < 0.005) obtained in absence of CO2/HCO3- (pH(i) 6.66). CO2/HCO3- alkali-shifted the flux vs. pH(i) relationship by 0.3-0.4 pH units. In a final series of experiments, we examined the effect of CO2/HCO3- on the Na+-independent alkalinization that follows the rapid, initial acidification elicited by bilateral Na+ removal. In the presence of CO2/HCO3-, lag time for initiation of the Na+-independent alkalinization was only similar to 36 vs. similar to 211 s (P < 0.002) in absence of CO2/HCO3-. Also, Na+-independent net acid extrusion rate was approximately two to three times higher in presence than in absence of CO2/HCO3- at comparable pH(i). This Na+-independent acid extrusion was insensitive to N-ethylmaleimide (2 mM), but was inhibited similar to 94% by efforts to deplete intracellular ATP (i.e., removal of glucose and amino acids, plus addition of 2 mM cyanide and 10 mM iodoacetic acid). Stimulation of luminal Na+/H+ exchange and Na+-independent acid extrusion appears to be the major, if not the entire, explanation for the higher steady-state pH(i) caused by bilateral addition of CO2/HCO3-.