1. Intracellular pH (pH(i)) was recorded ratiometrically in isolated guinea-pig ventricular myocytes using the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphthorhodafluor). 2. Following an intracellular acid load (10 mm NH4 Cl removal), pH(i) recovery in HEPES-buffered Tyrode solution was inhibited by 1-5 mm amiloride (Na+-H+ antiport blocker). In the presence of amiloride, switching from HEPES buffer to HCO3-/CO2 (pH(o) of both solutions = 7-4) stimulated a pH(i) recovery towards more alkaline levels. 3. Amiloride-resistant, HCO3-dependent pH(i) recovery was inhibited by removal of external Na+ (replaced by N-methyl-D-glucamine), whereas removal of external Cl- (replaced by glucuronate, leading to depletion of internal Cl-), removal of external K+, or decreasing external Ca2+ by almost-equal-to tenfold had no inhibitory effect. These results suggest that the amiloride-resistant recovery is due to a Na+-HCO3- cotransport into the cell. 4. The stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 500 mum) slowed Na+-HCO3--dependent pH(i) recovery. 5. Intracellular pH increased in Cl--free solution and this increase still occurred in Na+-free solution indicating that it is not caused via Na+-HCO3- symport and is more likely to be due to Cl- efflux in exchange for HCO3- influx on a sarcolemmal Cl--HCO3- exchanger. The lack of any significant pH(i) recovery from intracellular acidosis in Na+-free solution suggests that this exchanger does not contribute to acid-equivalent extrusion. 6. Possible voltage sensitivity and electrogenicity of the co-transport were examined by using the whole-cell patch clamp technique in combination with SNARF- 1 recordings of pH(i). Stepping the holding potential from - 110 to - 40 mV did not affect amiloride-resistant pH(i) recovery from acidosis. Moreover, following an intracellular acid load, the activation of Na+-HCO3- co-influx (by switching from HEPES to HCO3-/CO2 buffer) produced no detectable outward current (outward current would be expected if the coupling of HCO3- with Na+ were > 1.0). 7. Intracellular intrinsic buffering power (beta(i)) was assessed as a function of pH(i) (beta(i) computed from the decrease of pH(i) following reduction of extracellular NH4Cl in amiloride-containing solution). Beta(i) in the ventricular myocyte increases roughly linearly with a decrease in pH(i) according the following equation: beta(i) = -28(pH(i))+222.6. 8. Comparison of acid-equivalent efflux via Na+-HCO3- symport and Na+-H+ antiport showed that, following an intracellular acidosis, the symport accounts for about 40 % of total acid efflux, the other 60 % being carried by the antiport. Over the pH(i) range 7.1-6.9, the activity of both acid extrusion systems increases similarly as pH(i) falls below 7.1. 9. Application of 100 mum acetazolamide (a carbonic anhydrase inhibitor) slowed and attenuated intracellular acid loads imposed by introduction of CO2-buffered Tyrode solution and slowed subsequent pH(i) recovery. This suggests that carbonic anhydrase is present in mammalian myocardium. 10. In conclusion, acid-equivalent extrusion from the guinea-pig ventricular myocyte in a HCO3-/CO2-buffered medium is achieved by both Na+-H+ antiport and a voltage-insensitive Na+-HCO3- symport. The efficiency of the latter system is dependent upon carbonic anhydrase activity.
