NOVEL CHLORIDE-DEPENDENT ACID LOADER IN THE GUINEA-PIG VENTRICULAR MYOCYTE - PART OF A DUAL ACID-LOADING MECHANISM

1. The fall of intracellular pH (pH(i)) following the reduction of ext racellular pH (pH,) was investigated in guinea-pig isolated ventricula r myocytes using intracellular fluorescence measurements of carboxy-SN ARF-1 (to monitor pH(i)). Cell superfusates were buffered either with a 5% CO2-HCO3- system or were nominally CO2-HCO3- free. 2. Reduction o f 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 t he presence of Na+. It was unaffected by high-K+ solution, by voltage- clamp depolarization, by various divalent cations (Zn2+, Cd2+, Ni2+ an d 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 glyc olytic inhibitors, or the lactate transport inhibitor, alpha-cyano-4-h ydroxy 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'-diisothioc yanatostilbene-2,2'-disulphonic acid) had no inhibitory effect. Acid l oading is therefore mediated by a novel Cl--dependent, acid influx pat hway. 5. After switching to CO2-HCO3--buffered conditions, acid loadin g was doubled. It was still not inhibited by Na+-free or high-K+ solut ions but was once again inhibited (by 78%) in Cl--free solution. The H CO3--stimulated fraction of acid loading was inhibited by DIDS. 6. We propose a model of acid loading in the cardiomyocyte which consists of two parallel carriers. One is Cl--HCO3- exchange, while the suggest t he other to be a novel Cl--OH- exchanger (although rye 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).