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(Circulation Research. 1996;79:698-704.)
© 1996 American Heart Association, Inc.

Articles

Role of an Electrogenic Na⁺-HCO₃^- Cotransport in Determining Myocardial pH_i After an Increase in Heart Rate

Maria C. Camilion de Hurtado, Bernardo V. Alvarez, Nestor G. Perez, Horacio E. Cingolani

Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Medicas, Universidad Nacional de La Plata (Argentina).

Correspondence to Dr Horacio E. Cingolani, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Medicas, Universidad Nacional de La Plata, Calle 60 y 120, 1900 La Plata, Argentina.

	Abstract

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The contribution of electrogenic Na⁺-HCO₃^- cotransport to pH_i regulation during changes in heart rate was explored in cat papillary muscles loaded with BCECF-AM in bicarbonate-free (HEPES) medium and in CO₂/HCO₃^--buffered medium. Stepwise increments in the frequency of contraction from 15 to 100 bpm induced a reversible increase in the pH_i from 7.13±0.03 to 7.36±0.03 (P<.05, n=5) in the presence of CO₂/HCO₃^- buffer. The same increase in the frequency of stimulation, however, decreased pH_i from 7.10±0.02 to 6.91±0.06 (P<.05, n=5), in the absence of bicarbonate. Moreover, in CO₂/HCO₃^--superfused muscles pretreated with SITS (0.1 mmol/L), this effect of increasing the contraction frequency was reversed, and a decrease of pH_i from 7.03±0.04 to 6.88±0.06 (P<.05, n=4) was observed when the pacing rate was increased stepwise from 15 to 100 bpm. High [K⁺]_o–induced depolarization of cell membrane alkalinized myocardial cells in the presence of HCO₃^- ions, whereas acidification was observed as a consequence of hyperpolarization induced by low external [K⁺]_o. Myocardial resting membrane potential became hyperpolarized upon exposure to HCO₃^--buffered media. This HCO₃^--induced hyperpolarization was not blocked by the inhibition of Na⁺,K⁺-ATPase activity by ouabain (0.5 µmol/L) but was prevented by SITS. The results suggested that membrane depolarization during cardiac action potential causes an increase in electrogenic Na⁺-HCO₃^- cotransport. Such depolarizations occurring as a consequence of increases in heart rate would thus, by means of elevated bicarbonate influxes, substantially increase the myocardial cell's ability to recover from an enhanced proton production.

Key Words: Na⁺-HCO₃^- cotransport • BCECF • pH_i • membrane potential • heart rate

	Introduction

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Myocardial pH_i is normally more alkaline than would be predicted by passive H⁺ distribution across the cell membrane, thus indicating the contribution of acid-extruding mechanisms to the maintenance of pH_i. Among them, the Na⁺-H⁺ exchange is the most widely known, and it is the only one active in the absence of bicarbonate.¹ In the presence of bicarbonate, two other ion transporter mechanisms have so far been implicated in pH_i homeostasis: a Na⁺-HCO₃^- symport^{2 3} and the extracellular Cl^-–intracellular HCO₃^- exchanger.⁴ The possible presence of a Na⁺-dependent intracellular Cl^-–extracellular HCO₃^- countertransport as found in cultured chick heart cells⁵ and a lactate-proton cotransport^{6 7 8} should be also mentioned. Na⁺-HCO₃^- cotransport has been described to be an electrogenic mechanism in many cell types, such as the mammalian proximal tubule,⁹ gastric oxyntic cells,¹⁰ retinal epithelium,¹¹ smooth muscle,¹² hepatocytes,¹³ human ciliary muscle,¹⁴ and glial cells.^{15 16 17} A stoichiometry ratio of 2 or 3 HCO₃^- ions to 1 Na⁺ ion was reported in these tissues. However, the electrogenicity of this symport in myocardium is still uncertain.^{2 3 18}

Depolarization of the cell membrane should increase the driving force of an electrogenic system transporting more HCO₃^- ions than Na⁺ ions into the cell. Therefore, an increase in the frequency of action potentials should favor HCO₃^- influx, inducing a rise in pH_i. However, previous studies of Purkinje fibers reported a decrease^{19 20 21 22} or no detectable changes²³ in myocardial pH_i after an increase in heart rate.

Experiments were carried out in order to test the following hypothesis: If the Na⁺-HCO₃^- symport is electrogenic, then an increase instead of a decrease in pH_i should be detected in the presence of bicarbonate after the increase in the frequency of depolarizations. Evidence showing that this is the case will be presented.

	Materials and Methods

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The experiments were performed on thin cat papillary muscles (mean cross-sectional area, 0.30±0.04 mm²) dissected from the right ventricle. The general procedure for isolating and mounting the muscles has been described previously.¹⁸ After mounting, the muscles were stretched progressively in order to reach the length at which maximal developed force was obtained (L max). The mean developed force exerted during the isometric contractions was 1.17±0.16 g/mm². Experiments were carried out at 30°C, and the muscles were superfused at a flow velocity of 2.7 mm/s with one of the following solutions: (1) a HEPES-buffered solution containing (mmol/L) NaCl 146.2, KCl 4.5, CaCl₂ 1.35, MgSO₄ 1.05, glucose 11, and HEPES 5 (pH was adjusted to 7.4 at 30°C with 3 mol/L NaOH; total Na⁺ amounted to 148.5 mmol/L; and the solution was gassed with 100% O₂); (2) a HCO₃^--buffered solution that contained (mmol/L) NaCl 128.3, KCl 4.5, CaCl₂ 1.35, NaHCO₃ 20.23, MgSO₄ 1.05, and glucose 11 (the solution was equilibrated with a CO₂/O₂ gas mixture to give a pH value of 7.39±0.01); (3) a HCO₃^--buffered solution in which [K⁺]_o was decreased to 1.0 mmol/L or alternatively increased either to 22.0 or 45.0 mmol/L, without osmotic compensation, in order to induce changes in resting membrane potential (E_m). During exposure to high [K⁺]_o, no significant change in resting tension was detected, but muscles became quiescent. To prevent adrenergic-receptor activation by possible catecholamine release from nerve endings, all the experiments were conducted in the presence of 1.0 µmol/L atenolol (Sigma Chemical Co) plus 1.0 µmol/L prazosin (Sigma Chemical Co). SITS (final concentration, 0.1 mmol/L; Sigma) or ouabain (final concentration, 0.5 µmol/L; Sigma) was added to the superfusing solution 30 minutes before the experiments. Muscles were paced with square-wave pulses of 10-millisecond duration, and a voltage intensity 10% over threshold was applied via two platinum wires running along the sides of the preparations. Two different protocols for increasing the frequency of stimulation were followed: in the first, the frequency of stimulation was increased stepwise from 15 to 30, from 30 to 60, from 60 to 90, and from 90 to 100 pulses·min^-1. Each step-up in rate induced an initial increase in developed force that subsequently decreased with time. On the average, in muscles bathed with CO₂/HCO₃^- buffer, the developed force initially increased by 17±5%, 37±12%, 44±13%, and 54±15%, respectively. By contrast, in HEPES buffer, this initial increase in developed force was 10±4%, 19±9%, 25±5%, and 54±16% at the same respective rates. Each rate increment was maintained for a 10-minute period to allow for stabilization of the response. In the second protocol, the frequency of stimulation was abruptly increased from 15 to 100 pulses·min^-1 and then returned to the control rate.

pH_i Measurements
pH_i determinations in the isolated muscles were made after loading the muscles with the acetoxymethyl ester form of the pH-sensitive dye BCECF (BCECF-AM, Molecular Probes) as previously described.¹⁸ BCECF fluorescence was excited at 450 and 495 nm, and the fluorescence emission was monitored at 535±5 nm. To limit photobleaching, a neutral density filter (1% transmittance) was placed in the excitation light path and a manual shutter was used to select sampling intervals (3 seconds every 15 seconds). At the end of each experiment, fluorescence emission was calibrated by the high-K⁺ nigericin technique.²⁴ The calibration solution contained (mmol/L) KCl 140.0, MgCl₂ 1.0, CaCl₂ 1.0, HEPES 5.0, nigericin 0.01, sodium cyanide 4.0, and 2,3-butanedionemonoxime 20.0 (the latter to prevent muscle contracture).²⁵ The pH was adjusted with KOH to four different values ranging from 7.5 to 6.5. Such a calibration gave a linear relation (r=.98±.002, n=28) between pH and the fluorescence ratio (F₄₉₅/F₄₅₀), with the latter calculated as follows: ratio=(fluorescence at 495 nm-autofluorescence at 495 nm)/(fluorescence at 450 nm-autofluorescence at 450 nm).

E_m Determinations
Ultraflexible glass microelectrodes were pulled as described by Fedida et al²⁶ and filled with 3 mol/L KCl in order to measure transmembrane potential (resistance, 25±4 M). Impalements were performed in quiescent preparations. After stable impalements were obtained, the pacing of the muscle was resumed. The microelectrodes were coupled to a high-input–impedance electrometer amplifier (Neuroprobe amplifier, A-M System). Ground was established by means of a Ag-AgCl/agar-KCl bridge. This type of ultraflexible electrode allows continuous recording of E_m in contracting muscles for long periods. The criteria for satisfactory impalement were (1) an abrupt potential drop within a few microns of advance of the microelectrode tip, (2) upon withdrawal of the electrode, an electrode resistance within 4 M of its original value, and the return of the electrode potential to the zero level ±2 mV, and (3) under control conditions, a maximum resting potential more negative than -70 mV. On the basis of these criteria, 15% of the impalements were rejected. Switching from bicarbonate-free to bicarbonate-buffered medium might have an effect on junction potential at the interface between the reference electrode and the superfusate. To examine whether such changes could be influencing the determination of E_m values, the junction potential of microelectrodes in both HEPES- and HCO₃^--buffered media was measured. The mean variation in 40 determinations (involving 10 different microelectrodes) was not different from zero (-0.23±1.07 mV, P>.05). For this reason, the difference was not considered for the analysis of the data.

Calculation of Mean E_m Depolarization Induced by an Increased Frequency of Stimulation or by High [K⁺]_o
When the frequency of action potentials is increased during a given period, the fraction of time within which the cell membrane is depolarized also increases. The mean degree of membrane depolarization induced by any given frequency of stimulation was calculated as the time integral of the E_m values above the basal resting potential divided by the 10-minute duration of step-up in rate. Likewise, the mean extent of membrane depolarization effected by high [K⁺]_o was calculated as the time integral of E_m values above basal resting potential divided by the 10-minute duration of exposure to each [K⁺]_o.

Statistics
Data are expressed as mean±SEM and were compared with Student's t test or the nonparametric Kruskal-Wallis H test. A value of P<.05 was considered statistically significant.

	Results

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Changes in pH_i Induced by Increasing the Contraction Frequency
One-Step Increase in the Contraction Frequency
Fig 1A shows a representative experiment in which the beating frequency of a papillary muscle bathed in CO₂/HCO₃^--buffered medium was abruptly increased from 15 to 100 bpm. Similar results were obtained in four other papillary muscles. An elevation of the beating rate induced a gradual rise in pH_i that within 6 to 7 minutes reached a new steady state value at close to 7.40. On the average, an increase in the beating rate from 15 to 100 bpm increased the pH_i by 0.28±0.05, with a time constant for this increment in pH_i of 6.2±1.4 minutes (n=5). These data suggest that 10 minutes after the beginning of the increase in rate, myocardial pH_i reaches a new steady state value at 0.3 pH unit above the control level. However, we cannot rule out the possibility that a slow bicarbonate (or acid equivalent) extrusion might have returned the pH_i to control levels if the high pacing rate had instead been maintained for a longer period. The effect of increasing the stimulation frequency was fully reversible. When the stimulation frequency was returned to the control rate (15 pulses·min^-1), the pH_i became restored to basal value with a time constant of 1.2±0.3 minutes (n=5).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of increasing the frequency of stimulation on myocardial pHi in CO2/HCO3--buffered medium. A, A representative experiment in which the rate of stimulation was abruptly increased from 15 to 100 bpm. B and C, The effect of intermediate increases in the frequency (from 15 to 30 and from 30 to 60 bpm, respectively). Arrows denote the beginning of high pacing rate. In every instance, the increase in pHi reached a steady state value within 10 minutes.

Multistep Increase in the Contraction Frequency
Increments in beating rate of intermediate magnitude elicited intermediate increases in pH_i, each of which reached a new steady state value well within the 10-minute stabilization period allowed. This is illustrated in Fig 1B and 1C, in which data are shown from representative experiments in which the frequency of stimulation was stepwise increased. When the stimulation rate was increased to 30 bpm, pH_i reached a new steady state value in 5 minutes (Fig 1B). When the stimulation rate was increased to 60 bpm, a new steady state pH_i value was attained in 4 minutes (Fig 1C). On average, the time constant of the rise in pH_i was 4.2±1.2 minutes at 30 bpm, 3.8±0.7 minutes at 60 bpm, 2.9±0.9 minutes at 90 bpm, and 2.4±1.4 minutes at 100 bpm (n=5).

A summary of the results of pHi changes induced by such stepwise increments in the frequency of contraction of papillary muscles superfused with CO₂/HCO₃^--buffered media is shown in Fig 2A. Myocardial pH_i was 7.13±0.03 (n=5) at a rate of 15 bpm and increased with each stepwise increment in the frequency up to a value of 7.36±0.03 at 100 bpm. However, when the stimulation frequency was suddenly returned to control rates, myocardial pH_i became restored to a value not different from baseline, with a time constant of 1.7±0.4 minutes.

View larger version (19K): [in this window] [in a new window]   Figure 2. Changes in myocardial pHi induced by stepwise increments in the frequency of contractions of papillary muscles bathed in either bicarbonate-buffered solution (A), bicarbonate-free medium (B), or bicarbonate plus SITS (C). Increases in frequency induced a progressive elevation in pHi in papillary muscles bathed in a CO2/bicarbonate medium (A, n=5). In contrast, the same increments in frequency evoked a decrease in pHi in muscles either bathed in nominally bicarbonate-free medium (B, n=5) or in CO2/HCO3--buffered solution containing 0.1 mmol/L SITS (C, n=5). *P<.05 compared with pHi values at basal contraction frequency (15 bpm) in each group (paired t test). #P<.05 compared with either the HEPES or the CO2/HCO3-+SITS groups (ANOVA).

The same protocol of stepwise increments in the frequency of contraction was applied to muscles bathed in bicarbonate-free medium. The effect obtained in muscles superfused with HEPES buffer bubbled with oxygen, a condition in which there is virtually no bicarbonate in the medium, is shown in Fig 2B. Under this condition, the increase in beating rate produced a progressive decrease in pH_i from 7.10±0.02 to 6.92±0.06. The rapid return to the control rate induced a return of pH_i as well: pH_i returned to 7.02±0.05 with a time constant of 3.5±1.7 minutes. These findings are in agreement with previous reports describing that a decrease in pH_i was detected when the pacing rate was increased in sheep cardiac Purkinje fibers bathed in bicarbonate-free medium.^{19 20 21 22} The acidification probably reflected an increase in proton production and/or an imbalance between myocardial oxygen requirement and supply.^{22 27}

To assess if the increases in myocardial pH_i elicited by increasing the rate of beating in the presence of bicarbonate might be mediated by a bicarbonate-dependent membrane-transport mechanism, the effect of increasing the frequency of stimulation on pH_i was tested in muscles bathed in CO₂/bicarbonate-buffered medium after the addition of 0.1 mmol/L of the disulfonic stilbene derivative SITS (Fig 2C). Baseline pH_i, at 15 bpm, was somewhat lower (7.04±0.05) in the presence of SITS than without SITS (7.13±0.03). Even though this difference did not reach statistical significance (P>.05), such a trend is consistent with the role of Na⁺-HCO₃^- cotransport in the regulation of myocardial pH_i. Pretreatment with SITS reversed the effect of increasing the frequency of contractions in the presence of bicarbonate, for here a gradual decrease of pH_i was observed when the frequency of contractions was increased stepwise from 15 to 100 bpm. Thus, an elevation in the frequency of contractions in the presence of SITS evoked effects on pH_i qualitatively and quantitatively similar to those observed in the nominal bicarbonate-free medium (compare Fig 2B with 2C). The decrement in pH_i induced by the high pacing rate was 0.18±0.04 (n=5) in HEPES buffer and 0.15±0.04 (n=5, P=NS) in CO₂/bicarbonate buffer plus SITS. Once the stimulation rate was returned to 15 pulses·min^-1, the myocardial pH_i of SITS-pretreated muscles returned to control values with a time constant of 2.4±0.2 minutes.

Taken together, the above results suggest that a bicarbonate-dependent alkalinizing mechanism is activated by increasing the frequency of contractions. The possibility that this activation could occur during the course of single contractions was explored by comparing the values of myocardial pH_i determined at the peak of the twitch with those measured during the diastole. No appreciable difference, however, was detected over a 10-minute period. On the average, myocardial pH_i was found to be 7.11±0.001 at systole and 7.11±0.001 at diastole in a group of seven muscles paced at a rate of 15 pulses·min^-1. In view of these data and the fact that the time constant values of the rise in pH_i in response to an increased number of depolarizations are in the order of minutes, either long-lasting depolarizations of a certain magnitude or many successive depolarizations would appear to be necessary in order to elicit a significant increase in pH_i.

Changes in Myocardial pH_i in Response to Changes in [K⁺]_o
In order to explore the effect of sustained changes of E_m on pH_i, papillary muscles bathed in CO₂/HCO₃ were exposed to four different [K⁺]_o levels (1.0, 4.5, 22.0, or 45.0 mmol/L), and the changes in pH_i induced by changes in [K⁺]_o were plotted as a function of the mean E_m values determined at each [K⁺]_o (Fig 3). Under control conditions ([K⁺]_o, 4.5 mmol/L), the mean pH_i value was 7.16±0.08 (n=4). The depolarization of E_m by increasing [K⁺]_o resulted in myocardial alkalinization, and this is illustrated in Fig 3, inset A. By contrast, when cell membrane was hyperpolarized by decreasing [K⁺]_o to 1.0 mmol/L (Fig 3, inset B), the opposite effect, ie, intracellular acidification, was observed. The linearity of the relationship (pH_i versus E_m) is consistent with the expected presence of an electrogenic Na⁺-HCO₃^- symport driven by K⁺-induced changes in E_m, and the data can thus be further used to estimate the cotransport stoichiometry. At equilibrium, and assuming that [K⁺]_i and [Na⁺]_i remained about the same during the changes in [K⁺]_o, the pH_i induced by a given change in [K⁺]_o from [K⁺]_o1 to [K⁺]_o2 obeys an equation of the following form:

where n is the number of HCO₃^- ions transported per Na⁺ via the cotransporter. The deviation of E_m from the Nernst equation (48-mV change in E_m rather than 58 mV for a 10-fold change in [K⁺]_o) was compensated for by multiplying log [K⁺]_o2/[K⁺]_o1 by a factor of 0.827 (ie, 48/58). The estimate gave an n value of 1.63±0.15, a figure close to the one previously reported for astrocytes.¹⁷

View larger version (19K): [in this window] [in a new window]   Figure 3. Linear relationship between the K+-induced changes in myocardial pHi and resting Em. Muscles were exposed to media with different [K+]o levels to induce either hyperpolarization or depolarization of Em. At control, the [K+]o (4.5 mmol/L) pHi was 7.16±0.08 (n=4). Changes in pHi were calculated from the difference between the pHi value at the control [K+]o and that measured at a given [K+]o. Inset A shows data from one experiment illustrating the increase in pHi induced by raising [K+]o. Inset B depicts data from the same experiment in panel A but showing the decrease in pHi induced by low [K+]o.

Effect of Bicarbonate on E_m
We have previously reported that a hyperpolarization of cardiac cell membranes occurred when the myocardium was suddenly exposed to bicarbonate-containing medium,¹⁸ a finding consistent with an excess of negative charges accompanying Na⁺-HCO₃^- symport–mediated HCO₃^- influx. It is well known that in myocardium, as in most cells, exposure to CO₂/HCO₃^- produces a rapid intracellular acidification as a result of permeation by CO₂. The initial decrease in pH_i is followed by a gradual recovery owing to the activation of the acid-extrusion mechanisms. During the phase of pH_i recovery, the myocardial [Na⁺]_i rises,^{28 29} and this increase in [Na⁺]_i might, in turn, increase Na⁺,K⁺-ATPase activity, thereby producing the hyperpolarization of the cell membrane. To test this possibility, experiments were carried out in which the effect of changing from HEPES to bicarbonate on E_m was tested in the presence of ouabain (Fig 4A) or in the absence of the drug (Fig 4B). The inhibition of Na⁺,K⁺-ATPase by ouabain did not block the hyperpolarizing effect of bicarbonate: In the presence of ouabain, the E_m value changed from -73.0±0.4 mV to -82.5±0.9 mV (n=4) after muscles were exposed to CO₂/HCO₃^- buffer. The magnitude of this hyperpolarizing effect was very similar to the one obtained in the absence of ouabain (Fig 4B). The hyperpolarizing effect of HCO₃^- was clearly evident despite the fact that intracellular acidosis per se decreases the conductance of the inward rectifier K⁺ channel³⁰ and causes membrane depolarization. These data, therefore, would suggest that the addition of bicarbonate elicited an outward current compatible with the existence of an electrogenic Na⁺-HCO₃^- cotransport with a coupling ratio of Na⁺:HCO₃^-<1. The blockade of HCO₃^--induced hyperpolarization by SITS was previously reported by us in cat papillary muscles.¹⁸

View larger version (22K): [in this window] [in a new window]   Figure 4. Hyperpolarization of Em in response to introduction of bicarbonate into the extracellular space. A, Mean±SEM values of the resting Em in muscles pretreated with 0.5 µmol/L ouabain in order to inhibit Na+,K+-ATPase activity (n=4). B, For comparison, the HCO3--induced changes in Em obtained in the absence of ouabain (n=10). An immediate hyperpolarizing response of Em was obtained when the HEPES-buffered superfusate was switched to CO2/HCO3--buffered solution, either in the absence or in the presence of ouabain. The hyperpolarizing effect persisted as long as HCO3- ions were present.

If the driving force for HCO₃^- efflux is augmented either by a sustained-high-[K⁺]_o depolarization or by phasic depolarizations induced by an increased heart rate, then the changes in myocardial pH_i should be a function of the mean membrane depolarization. This possibility was examined by plotting the pH_i data as a function of the mean membrane depolarization induced either by any given step-up in beating rate or by an alteration in the [K⁺]_o value (see "Materials and Methods"). The results are shown in Fig 5. The correlation coefficient between pH_i values and mean membrane depolarization was .91. Note that the regression line intercepts the y axis at a value of 7.12. This figure, representing the myocardial pH_i of muscles with no depolarization above resting potential, is close to the pH_i value of 7.14±0.06 recently reported for quiescent cat papillary muscles.³¹

View larger version (19K): [in this window] [in a new window]   Figure 5. Relationship between myocardial pHi values and the mean membrane depolarization. The mean membrane depolarization induced by a given frequency of stimulation or [K+]o was calculated (see "Materials and Methods") as the time integral of the values of Em above resting potential divided by the 10-minute duration of each rate step-up or each [K+]o. The least squares regression line for the combined data is shown along with the 95% confidence limits. The correlation coefficient between pHi values and mean membrane depolarization was .91.

	Discussion

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Several authors have previously explored the effect of increasing heart rate on myocardial pH_i. Most of those studies were conducted on sheep Purkinje fibers. In this preparation, a rate-dependent decrease in pH_i was observed in the absence of bicarbonate.^{19 20 21 22} This fall in pH_i is probably the result of increased anaerobic glycolysis.²² In the presence of CO₂/bicarbonate, the frequency-induced intracellular acidification of Purkinje fibers was markedly reduced.^{21 23} The response of contractile myocardium to the same intervention, however, is different. The frequency-induced decrease in pH_i normally seen in papillary muscles bathed in bicarbonate-free medium was reversed to an alkalinization in the presence of bicarbonate. A similar response was reported in whole-heart experiments; ie, the beating rate–dependent decrease in pH_i was reversed to a small increase in isolated perfused ferret hearts when anaerobic glycolysis was prevented.²⁷ A transitory alkaline shift in myocardial pH_i was also observed when the pacing rate of perfused rabbit hearts was doubled.³² The different response of ventricular myocardium and that of the fast conduction system to increases in heart rate might reflect a different functional role of Na⁺-HCO₃^- cotransport in the two cell types. It has been shown that this symport activity accounts for 40% to 50% of total acid extrusion in ventricular myocardium^{3 18} but for only 20% in Purkinje fibers.² However, different perfusion conditions in the experimental chambers might also account for these discrepant observations. The "unstirred layer" covering the surface of the cells in isolated preparations might contribute to intracellular acidification.³³ The transitory alkaline shift in myocardial pH_i recently described by Eijgelshoven et al³² in rabbit hearts was attributed by those authors to a decrease in phosphocreatine (PCr) levels. If the changes in pH_i reported here had been the result of changes in PCr levels, then the decrease in PCr induced by increasing the stimulation frequency would have also occurred in HEPES buffer and would not have been blocked by SITS. Nevertheless, a contribution to the alkalinizing effect by PCr breakdown still cannot be entirely ruled out.

The effect of increasing the rate of contractions was reversible, and myocardial pH_i returned to control value after the pacing rate was decreased to control value. However, the rate of pH_i recovery was slower in HEPES medium compared with bicarbonate, although the difference did not reach statistical significance. In the presence of bicarbonate, the possible more rapid return of myocardial pH_i probably reflects two simultaneous events: the reduction in the force driving the activity of the Na⁺-HCO₃^- cotransport and the contribution of the Na⁺-independent Cl^--HCO₃^- exchanger, which might be operating at a higher rate because of the more alkaline pH_i.³⁴ In HEPES medium, in contrast, the restoration of pH_i would only be due to the activity of the Na⁺-H⁺ antiporter.

The rapid membrane hyperpolarization detected after the addition of bicarbonate is an indication that the symport carries an excess of HCO₃^- ions into the cell during the diastole. Data showing the hyperpolarizing effect of bicarbonate have already been reported,¹⁸ and the possibility of an indirect effect mediated by Na⁺,K⁺-ATPase activation can now be excluded as well. The hyperpolarizing effect of added bicarbonate was not generally observed by Dart and Vaughan-Jones² in Purkinje fibers. Nevertheless, in some experiments a hyperpolarization did in fact occur but was not blocked by DIDS. The differences between our results and theirs could be attributable to species and/or tissue differences or to the use of SITS instead of DIDS.

Even though we are aware that under our experimental conditions, changes in extracellular osmolarity and/or in [Na⁺]_i and [Ca²⁺]_i may have partially contributed to the [K⁺]_o-induced changes in pH_i, an estimate of the cotransport stoichiometry was obtained from the slope of the linear relationship between changes in pH_i and [K⁺]_o-induced changes in E_m. The estimation of the minimum number of HCO₃^- ions transported with each Na⁺ ion yielded an n value of 1.63. One interpretation of our estimate of the HCO₃^-:Na⁺ stoichiometry ratio is that two HCO₃^- ions are carried into the cell along with each Na⁺ but that the exact value of n was underestimated because of the failure to reach thermodynamic equilibrium within the course of the experiment.³⁵ Alternatively, this n value could reflect a heterogeneous population of cardiac cells expressing Na⁺-HCO₃^- cotransporter molecules of different stoichiometries. This latter possibility would explain the observation of Lagadic-Gossmann et al,³ who reported that in some, but not all, guinea pig ventricular myocytes an increased recovery from acidosis was observed after cell mem brane depolarization. Different stoichiometries for the Na⁺-HCO₃^- cotransporter have been reported for several tissues.^{9 11 16 17 36 37}

We do not propose that the steady state myocardial pH_i is a function of heart rate. In spite of species differences, the values for myocardial pH_i reported in the literature cited vary between 7.05 and 7.26. Even in hearts beating at a rate significantly higher (200 bpm) than the ones used in the present study, the myocardial pH_i does not reach values as high³⁸ as those we are reporting here as a consequence of increased depolarization frequency. Considering the previous studies and our own data, we conclude that after a certain time following an increase in heart rate, the mechanisms regulating myocardial pH_i eventually bring [H⁺]_i back to control levels. We made no attempt to study either the time required for such a return of the pH_i to basal levels or the mechanisms involved in that recovery. However, two possibilities for the latter are (1) a Na⁺-independent Cl^--HCO₃^- exchange, an acidifying mechanism that is activated when pH_i increases above 7.30 to 7.40,⁴ and (2) a passive H⁺ influx. In the latter instance, the actual H⁺ ion influx would be small as a result of the low H⁺ ion concentration in biological solutions. If we assume that E_m=-80 mV, pH_i=7.36, pH_o=7.40, and intracellular buffer capacity (ß_i)=30 mmol/L,¹⁸ such a passive H⁺ ion influx would result in a cytoplasmic acidification of 0.033 pH unit/h.

At all events, the decrease in myocardial pH_i following an increase in heart rate, as previously reported and likewise observed by us, appears to be a drop in pH_i that occurs when the most important physiological buffer, bicarbonate, is omitted. By contrast, the availability of bicarbonate during an increase in heart rate would provide the myocardium with a mechanism that, by means of an elevated HCO₃^- influx, substantially increases the cell's ability to recover from an enhanced proton production. This mechanism, however, might not be necessary for Purkinje fibers.

	Acknowledgments

 
Dr Perez is the recipient of a postdoctoral fellowship from Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Argentina; Dr Alvarez is the recipient of a predoctoral fellowship from Comision de Investigaciones Cientificas, Buenos Aires, Argentina; and Drs de Hurtado and Cingolani are Established Investigators of CONICET, Argentina. The authors thank Karen Mesa Noack for her excellent secretarial assistance during the preparation of the manuscript.

Received February 8, 1996; accepted June 27, 1996.

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