1. Intracellular pH (pH(i)) was recorded in isolated sheep cardiac Purkinje fibres using liquid sensor ion-selective microelectrodes in conjunction with conventional (3 M-KCl) microelectrodes (to record membrane potential). 2. In HEPES-buffered solution (pH(o) 7.4), pH(i) recovery from an intracellular acid load (20 mm-NH4Cl removal) was blocked by 1 mM-amiloride, consistent with the inhibition of Na+-H+ exchange. Replacement of the HEPES buffer with CO2-HCO3- caused a transient acidosis followed by an amiloride-resistant recovery of pH(i) to more alkaline levels (n = 43). This implies the presence of a HCO3--dependent pH(i) regulatory mechanism. 3. Comparison of the membrane potential with the equilibrium potential for HCO3- ions (EHCO3) estimated during amiloride-resistant pH(i) recovery, showed that for polarized fibres (membrane potential E(m) approximately -80 mV), there was a net outward electrochemical driving force for HCO3- ions. Hence the amiloride-resistant pH(i) recovery cannot be explained in terms of passive HCO3- influx through membrane channels. 4. Removal of external Na+ (Na+ replaced by N-methyl-D-glucamine) inhibited HCO3--dependent pH(i) recovery, whereas removal of external Cl- (leading to depletion of internal Cl-; Cl0- replaced by glucuronate) or short-term removal of extracellular K+ had no inhibitory effect. We suggest that a Na+-HCO3- co-influx causes the recovery. Replacement of external Na+ with Li+ greatly reduced HCO3--dependent pH(i) recovery indicating that Li0+ cannot readily substitute for Na0+ on the co-transport. 5. The stilbene drug DIDS (4,4-diisothiocyano-stilbene-disulphonic acid, 500-mu-m) slowed HCO3--dependent pH(i) recovery. 6. Depolarization of the membrane potential in high K0+ (44-5 mm) solution or with 5 mm-BaCl2 had no effect upon the rate of HCO3--sensitive pH(i) recovery. This observation, when coupled with the fact that activation of HCO3--dependent pH(i) recovery was associated with no consistent change of membrane potential, suggests that the Na+-HCO3- co-influx is electroneutral and voltage insensitive. 7. HCO3--dependent pH(i) recovery was unaffected by the Na+-K+-2Cl- co-transport inhibitor, bumetanide (150-mu-m). 8. The contribution of Na+-H+ exchange and Na+-HCO3 co-transport to net acid efflux was assessed. At a pH(i) of 6-6, we estimate that the co-transport should account for 20 % of total acid equivalent efflux. Because of the different slopes for the pH(i) dependence of the two transporters, this contribution should increase as pH(i) increases so that. at a pH(i) of 7.0, Na+-HCO3 symport should account for approximately 30% of total acid-equivalent efflux. 9. lt is concluded that in CO2-HCO3--buffered media, acid equivalent extrusion is mediated not only by -Na+-H+ exchange but also by an electroneutral Na+-HCO3-co-influx mechanism.
