1. The perforated whole-cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+-HCO3- cotransport in isolated rat ventricular myocytes. 2. Switching from Hepes buffer to HCO3- buffer at constant extracellular pH (pH(o)) hyperpolarized the resting membrane potential (RMP) by 2.9 ± 0.4 mV (n = 9, P < 0.05). In the presence of HCO3-, the anion blocker SITS depolarized RMP by 2.6 ± 0.5 mV (n = 5, P < 0.05). No HCO3--induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50% of repolarization time (APD50) was 29.2 ± 6.1% shorter in the presence of HCO3- than in its absence (n = 6, P < 0.05). 3. Quasi-steady-state currents were evoked by voltage-clamped ramps ranging from -130 to +30 mV, during 8 s. The development of a novel component of Na+-dependent and Cl--independent steady-state outward current was observed in the presence of HCO3-. The reversal potential (E(rev)) of the Na+-HCO3- cotransport current (I(Na.Bic)) was measured at four different levels of extracellular Na+. A HCO3-:Na+ ratio compatible with a stoichiometry of 2:1 was detected. I(Na,Bic) was also studied in isolation in standard whole-cell experiments. Under these conditions, I(Na,Bic) reversed at -96.4 ± 1.9 mV (n = 5), being consistent with the influx of 2 HCO3- ions per Na+ ion through the Na+-HCO3- cotransporter. 4. In the presence of external HCO3-, after 10 min of depolarizing the membrane potential (E(m)) with 45 mm extracellular K+, a significant intracellular alkalinization was detected (0.09 ± 0.03 pH units; n = 5, P < 0.05). No changes in pH(i) were observed when the myocytes were pre-treated with the anion blocker DIDS (0.001 ± 0.024 pH units; n = 5, n.s.), or when exposed to Na+-free solutions (0.003 ± 0.037 pH units; n = 6, n.s.). 5. The above results allow us to conclude that the cardiac Na+-HCO3- cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.
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