Novel chloride-dependent acid loader in the guinea-pig ventricular myocyte: Part of a dual acid-loading mechanism

Abstract

1. The fall of intracellular pH (pH(i)) following the reduction of extracellular pH (pH(o)) was investigated in guinea-pig isolated ventricular myocytes using intracellular fluorescence measurements of carboxy-SNARF-1 (to monitor pH(i)). Cell superfusates were buffered either with a 5% CO2-HCO3- system or were nominally CO2-HCO3- free. 2. Reduction of pH(o) from 7.4 to 6.4 reversibly reduced pH(i) by about 0.4 pH units, independent of the buffer system used. 3. In HCO3--free conditions, acid loading in low pH(o) was not dependent on Na+-H+ exchange or on the presence of Na+. It was unaffected by high-K+ solution, by voltage-clamp depolarization, by various divalent cations (Zn2+, Cd2+, Ni2+ and Ba2+) and by the organic Ca2+ channel blocker diltiazem, thus ruling out proton influx through H+- or Ca2+-conductance channels or influx via a K+-H+ exchanger. The fall also persisted in the presence of glycolytic inhibitors, or the lactate transport inhibitor, α-cyano-4-hydroxy cinnamate. 4. In HCO3--free conditions, acid loading in low pH(o) was reversibly inhibited (by up to 85%) by Cl(o)- removal and was slowed by the stilbene drug DBDS (dibenzamidostilbene disulphonic acid). In contrast, the Cl--HCO3-exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) had no inhibitory effect. Acid loading is therefore mediated by a novel Cl--dependent, acid influx pathway. 5. After switching to CO2-HCO3--buffered conditions, acid loading was doubled. It was still not inhibited by Na+-free or high-K+ solutions but was once again inhibited (by 78%) in Cl--free solution. The HCO3--stimulated fraction of acid loading was inhibited by DIDS. We propose a model of acid loading in the cardiomyocyte which consists of two parallel carriers. One is Cl--HCO3- exchange, while we suggest the other to be a novel Cl--OH- exchanger (although we do not rule out the alternative configuration of H+-Cl- co-influx). The proposed dual acid-loading mechanism accounts for most of the sensitivity of pH(i) to a fall of pH(o).