Effect of Basolateral COz/HCO on Intracellular pH Regulation in the Rabbit $3 Proximal Tubule

A B S TRACT We used the absorbance spectrum of the pH-sensitive dye dimethylcarboxyfluorescein to moni tor intracellular pH (pHi) in the isolated perfused $3 segment of the rabbit proximal tubule, and examined the effect on pHi of switching from a HEPES to a CO2/HCOf buffer in the lumen and /o r the bath (i.e., basolateral solution). Solutions were ti trated to p H 7.40 at 37°C. With 10 mM acetate present bilaterally (lumen and bath), this causing steady-state pHi to be ra ther high ( ~ 7.45), bilaterally switching the buffer from 32 mM HEPES to 5% CO2/25 mM HCO~ caused a sustained fall in pHi of ~ 0.26. However, with acetate absent bilaterally, this causing steady-state p h i to be substantially lower (~6.9) , bilaterally switching to CO2/HCO s caused a transient pHi fall (due to the influx of CO2), followed by a sustained rise to a level ~ 0.18 higher than the initial one. The remainder of the experiments was devoted to examining this alkalinization in the absence of acetate. Switching to CO~/HCO~ only in the lumen caused a sustained p h i fall of ~0.15, whereas switching to CO~/HCO~ only in the bath caused a transient fall followed by a sustained pHi increase to ~ 0.26 above the initial value. This basolateral CO2/HCO~-induced alkalinization was not inhibited by 50 I~M DIDS appl ied shortly after CO2/HCO~ washout, but was slowed ~73% by DIDS appl ied more than 30 min after CO2/HCO~ washout. The rate was unaffected by 100 ~M bilateral acetazolamide, although this drug greatly reduced COs-induced p h i transients. The alkalinization was not blocked by bilateral removal of Na + per se, but was abolished at pHi values below ~6.5. The alkalinization was also unaffected by short-term bilateral removal of CIor SO i . Basolateral CO2/HCO~ elicited the usual pHi increase even when all solutes were replaced, short or long-term (> 45 min), by N-methyl-D-glucammonium/glucuronate (NMDG+/GIr-) . Luminal CO2/HCO~ did not elicit a pHi increase in NMDG+/GIr . Although the