Intracellular pH was recorded fluorimetrically by using carboxy‐SNARF‐1, AM‐loaded into superfused ventricular myocytes isolated from guinea‐pig heart. Intracellular acid and base loads were induced experimentally and the changes of pHi used to estimate intracellular buffering power (β). The rate of pHi recovery from acid or base loads was used, in conjunction with the measurements of β, to estimate sarcolemmal transporter fluxes of acid equivalents. A combination of ion substitution and pharmacological inhibitors was used to dissect acid effluxes carried on Na+‐H+ exchange (NHE) and Na+‐HCO3− cotransport (NBC), and acid influxes carried on Cl−‐HCO3− exchange (AE) and Cl−‐OH− exchange (CHE). The intracellular intrinsic buffering power (βi), estimated under CO2/HCO3−‐free conditions, varied inversely with pHi in a manner consistent with two principal intracellular buffers of differing concentration and pK. In CO2/HCO3−‐buffered conditions, intracellular buffering was roughly doubled. The size of the CO2‐dependent component (βCO2) was consistent with buffering in a cell fully open to CO2. Because the full value of βCO2 develops slowly (2·5 min), it had to be measured under equilibrium conditions. The value of βCO2 increased monotonically with pHi. In 5 % CO2/HCO3−‐buffered conditions (pHo 7·40), acid extrusion on NHE and NBC increased as pHi was reduced, with the greater increase occurring through NHE at pHi < 6·90. Acid influx on AE and CHE increased as pHi was raised, with the greater increase occurring through AE at pHi > 7·15. At resting pHi (7·04‐7·07), all four carriers were activated equally, albeit at a low rate (about 0·15 mM min−1). The pHi dependence of flux through the transporters, in combination with the pHi and time dependence of intracellular buffering (βi+βCO2), was used to predict mathematically the recovery of pHi following an intracellular acid or base load. Under several conditions the mathematical predictions compared well with experimental recordings, suggesting that the model of dual acid influx and acid efflux transporters is sufficient to account for pHi regulation in the cardiac cell. Key properties of the pHi control system are discussed.

Following an intracellular alkali load (imposed by acetate prepulsing in CO2/HCO3− buffer), intracellular pH (pHi) 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). The rapid recovery phase is not evident if the alkali load has been induced by reducing PCO2 (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). 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. 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 pHi (at constant PCO2). If CA is inactive, this period can be as long as 5 min. During normal pHi 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 pHi regulation during alkalosis is discussed.

1. The fall of intracellular pH (pHi) following the reduction of extracellular pH (pH.) was investigated in guinea-pig isolated ventricular myocytes using intracellular fluorescence measurements of carboxy-SNARF-1 (to monitor pH,). Cell superfusates were buffered either with a 5% CO2-HCO3 system or were nominally C02-HC03-free.2. Reduction of pHo from 7A4 to 6A4 reversibly reduced pHi by about 0 4 pH units, independent of the buffer system used.3. In HC03--free conditions, acid loading in low pHo was not dependent on Na`-H+ exchange or on the presence of Na+. It was unaffected by high-K+ solution, by voltage-clamp depolarization, by various divalent cations (Zn2+, Cd2+, Ni2+ and Ba2P) and by the organic Ca2+ channel blocker diltiazem, thus ruling out proton influx through H+-or Ca2+-conductance channels or influx via a K+-H+ exchanger. The fall also persisted in the presence of glycolytic inhibitors, or the lactate transport inhibitor, a-cyano-4-hydroxy cinnamate.4. In HC03--free conditions, acid loading in low pH0 was reversibly inhibited (by up to 85 %) by Cl-removal and was slowed by the stilbene drug DBDS (dibenzamidostilbene disulphonic acid). In contrast, the Cl--HCO3 exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) had no inhibitory effect. Acid loading is therefore mediated by a novel CF-dependent, acid influx pathway. 5. After switching to C02-HC03--buffered conditions, acid loading was doubled. It was still not inhibited by Nae-free or high-K+ solutions but was once again inhibited (by 78 %) in Clfree solution. The HC03--stimulated fraction of acid loading was inhibited by DIDS. 6. We propose a model of acid loading in the cardiomyocyte which consists of two parallel carriers. One is Cl--HCO3-exchange, while we suggest the other to be a novel ClF-OHexchanger (although we do not rule out the alternative configuration of H+-Cl-co-influx). The proposed dual acid-loading mechanism accounts for most of the sensitivity of pHi to a fall of pHo.Intracellular pH is an important physiological modulator of cardiac contraction (see Orchard & Kentish, 1990 for review). This is largely because reducing pH, reduces Ca2+ binding to troponin-C, thereby attenuating the force of contraction. Of the many conditions that can reduce pHi in cardiac cells, a common one is a reduction of extracellular pH. Over the physiological range of pH, intracellular pH is related approximately linearly to pH., with a transfer function (slope of pH1 vs. pHO) of 0 3-0'4 (Ellis & Thomas, 1976;Vaughan-Jones, 1986). Thus, because extracellular pH is an important modulator of pHi in cardiac cells, it becomes an important modulator of contractility in the heart. The mechaWism, however, whereby pHi is linked to pH0 remains unknown (Sun & Vaughan-Jones, 1994). This is the case not only for cardiac cells, but also for many other cell types. In the present work, we have investigated the mechanism linking pHi to pH0 in single enzymically isolated cardiomyocytes, while measuring pH1 using the pH-sensitive fluorophore, ...

It is often assumed that pH(i) is spatially uniform within cells. A double-barreled microperfusion system was used to apply solutions of weak acid (acetic acid, CO(2)) or base (ammonia) to localized regions of an isolated ventricular myocyte (guinea pig). A stable, longitudinal pH(i) gradient (up to 1 pH(i) unit) was observed (using confocal imaging of SNARF-1 fluorescence). Changing the fractional exposure of the cell to weak acid/base altered the gradient, as did changing the concentration and type of weak acid/base applied. A diffusion-reaction computational model accurately simulated this behavior of pH(i). The model assumes that H(i)(+) movement occurs via diffusive shuttling on mobile buffers, with little free H(+) diffusion. The average diffusion constant for mobile buffer was estimated as 33 x 10(-7) cm(2)/s, consistent with an apparent H(i)(+) diffusion coefficient, D(H)(app), of 14.4 x 10(-7) cm(2)/s (at pH(i) 7.07), a value two orders of magnitude lower than for H(+) ions in water but similar to that estimated recently from local acid injection via a cell-attached glass micropipette. We conclude that, because H(i)(+) mobility is so low, an extracellular concentration gradient of permeant weak acid readily induces pH(i) nonuniformity. Similar concentration gradients for weak acid (e.g., CO(2)) occur across border zones during regional myocardial ischemia, raising the possibility of steep pH(i) gradients within the heart under some pathophysiological conditions.