Evidence for the role of a Na+/HCO3- cotransporter in trout hepatocyte pHiregulation

The mechanisms of intracellular pH(pHi) regulation were examined in hepatoc ytes of the rainbow trout Oncorhynchus mykiss. pHi was monitored using the pH-sensitive fluorescent dye BCECF, and the effects of various media and ph armacological agents were examined for their influence on baseline pi-ii an d recovery rates from acid and base loading. Rates of Na+ uptake were measu red using Na-22, and changes in membrane potential were examined using the potentiometric fluorescent dye Oxonol VI, The rate of proton extrusion foll owing acid loading was diminished by the blockade of either Na+/H+ exchange (using amiloride) or anion transport (using DIDS), The removal of external HCO3- and the abolition of outward K+ diffusion by the channel blocker Ba2 + also decreased the rate of proton extrusion following acid load. Depolari zation of the cell membrane with 50 mmoll(-1) K+, however, did not affect p Hi, The rate of recovery front base loading was significantly diminished by the blockade of anion transport, removal of external HCO3- and, to a lesse r extent, by blocking Na+/H+ exchange. The blockade of K+ conductance had n o effect. The decrease in Na+ uptake rate observed in the presence of the a nion transport blocker DIDS and the DIDS-sensitive hyperpolarization of mem brane potential during recovery from acid loading suggest that a Na+-depend ent electrogenic transport system is involved in the restoration of pHi aft er intracellular acidification. The effects on baseline pHi indicate that t he different membrane exchangers are tonically active in the maintenance of steady-state pHi. This study confirms the roles of a Na+/H+ exchanger and a Cl-/HCO3- exchanger in the regulation of trout hepatocyte pHi and provide s new evidence that a Na+/HCO3- cotransporter contributes to pHi regulation .