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Out-of-equilibrium pH transients in the guinea-pig ventricular myocyte

Chae-Hun Leem and Richard D. Vaughan-Jones

1. Following an intracellular alkali load (imposed by acetate prepulsing in CO₂/HCO₃^- buffer), intracellular pH (pH_i) of the guinea-pig ventricular myocyte (recorded from intracellular SNARF fluorescence) recovers to control levels. Recovery has two phases. An initial rapid phase (lasting up to 2 min) is followed by a later slow phase (several minutes). Inhibition of sarcolemmal acid-loading carriers (by removal of extracellular Cl^-) inhibits the later, slow phase but the initial rapid recovery phase persists. It also persists in the absence of extracellular Na⁺ and in the presence of the HCO₃^- transport inhibitor DIDS (4,4-di-isothiocyanatostilbene-2,2-disulphonic acid).

2. The rapid recovery phase is not evident if the alkali load has been induced by reducing P_CO2 (from 10 to 5 %), and it is inhibited in the absence of CO₂/HCO₃^- buffer (i.e. Hepes buffer). It is also slowed by the carbonic anhydrase (CA) inhibitor acetazolamide (ATZ). We conclude that it is caused by buffering of the alkali load through the hydration of intracellular CO₂ (CO₂-dependent buffering).

3. The time course of rapid recovery is consistent with an intracellular CO₂ hydration rate constant (k₁) of 0·36 s^-1 in the presence of CA activity, and 0·14 s^-1 in the absence of CA activity. This latter k₁ value matches the literature value for uncatalysed CO₂ hydration in free solution. Natural CO₂ hydration is accelerated 2·6-fold in the ventricular myocyte by endogenous CA.

4. The rapid recovery phase represents a period when the intracellular CO₂/HCO₃^- buffer is out of equilibrium (OOE). Modelling of the recovery phase using our k₁ value, indicates that OOE conditions will normally extend for at least 2 min following a step rise in pH_i (at constant P_CO2). If CA is inactive, this period can be as long as 5 min. During normal pH_i regulation, the recovery rate during these periods cannot be used as a measure of sarcolemmal acid loading since it is a mixture of slow CO₂-dependent buffering and transmembrane acid loading. The implication of this finding for quantification of pH_i regulation during alkalosis is discussed.

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