Na+-H+ exchange in isolated myocytes from adult rat heart.

The intracellular pH (pHi) in ventricular myocytes isolated from adult rat heart was measured in suspension using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Steady-state pHi in bicarbonate-free media [extracellular pH (pHo) = 7.4] was 7.16 +/- 0.11 at 37 degrees C. With the use of the ammonium chloride prepulse technique, pHi was acidified, and the rate of return to resting pHi was determined. Initial rate analysis of the recovery was used to characterize the kinetics of proton net efflux via the Na+-H+ exchanger. At pHo = 7.4, proton extrusion was stimulated by extracellular sodium with a K1/2 = 58 +/- 16 mM and a maximal rate of recovery of 55 +/- 7 mmol/(1 cell H2O.min). Amiloride, which inhibited greater than 90% of the observed proton movements, was a competitive inhibitor with respect to Nao, with an inhibition constant of 3.5 microM. Proton net efflux was also inhibited by extracellular protons, with a maximal flux occurring above pHo = 8 and no net efflux occurring below pHo = 6.0. Efflux was stimulated by intracellular protons over a much narrower range (pHi = 6.6-7.1). This steep dependence indicates the involvement of at least one additional proton binding site besides the intracellular transport site, in accordance with the kinetic behavior observed in other cell systems.