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(Circulation Research. 1995;77:1192-1200.)
© 1995 American Heart Association, Inc.

Articles

pH_i Regulation in Myocardium of the Spontaneously Hypertensive Rat

Compensated Enhanced Activity of the Na⁺-H⁺ Exchanger

Néstor G. Pérez, Bernardo V. Alvarez, María C. Camilión de Hurtado, Horacio E. Cingolani

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

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

	Abstract

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Abstract To elucidate the mechanisms controlling pH_i in myocardium of the spontaneously hypertensive rat (SHR), experiments were performed in papillary muscles (isometrically contracting at 0.2 Hz) from SHR and age-matched normotensive Wistar-Kyoto (WKY) rats loaded with the pH-sensitive fluorescent probe BCECF-AM. An enhanced activity of the Na⁺-H⁺ exchanger was detected in the hypertrophic myocardium of SHR. This conclusion was based on the following: (1) The myocardial pH_i was more alkaline in SHR (7.23±0.03) than in WKY rats (7.10±0.03) (P<.05) in HEPES buffer. (2) SITS (0.1 mmol/L in HEPES buffer) did not alter pH_i in the SHR (pH_i 7.26±0.03 and 7.28±0.03 before and after SITS, respectively). (3) The fall in pH_i observed after 20 minutes of Na⁺-H⁺ exchanger inhibition [5 µmol/L 5-(N-ethyl-N-isopropyl)amiloride (EIPA)] was greater in SHR (-0.16±0.01) than in WKY rats (-0.09±0.02, P<.05). (4) The velocity of pH_i recovery from an intracellular acid load was faster in SHR than in WKY rats (0.068±0.02 versus 0.014±0.002 pH units/min at pH_i 6.99, P<.05). (5) After EIPA inhibition, the rate of pH_i recovery from the same acid load decreased to a similar value in both rat strains (0.0032±0.002 pH units/min in SHR and 0.0032±0.002 pH units/min in WKY rats). Under the more physiological HCO₃^--CO₂ buffer, no significant difference in steady state myocardial pH_i was detected between rat strains (7.15±0.03 in SHR and 7.11±0.05 in WKY rats). This finding suggested that an acidifying bicarbonate-dependent mechanism was fully compensating for the hyperactivity of the Na⁺-H⁺ exchanger in SHR. The following pieces of evidence support an enhanced activity of the Na⁺-independent Cl^--HCO₃^- exchanger as the mechanism accounting for the compensation: (1) SITS (0.1 mmol/L) increased steady state pH_i in the presence of HCO₃^--CO₂ buffer in SHR (+0.08±0.02, P<.05) but not in WKY rats (+0.04±0.04). (2) The rate of pH_i recovery from an alkaline load was faster in SHR than in WKY rats (0.075±0.028 versus 0.027±0.016 pH units per minute, respectively; P<.05). (3) The enhanced recovery from an alkaline load in the SHR was Na⁺ independent. (4) No difference in the rate of pH_i recovery was detected between SHR and WKY rats when the alkaline load was performed after SITS blockade. Comparison of net HCO₃^- efflux at a given pH_i suggests that an increased pH_i is not the cause of the hyperactivity of the anion exchanger. Since this anion exchanger is not driving Na⁺, the offset of the increase in pH_i induced by the antiport would not prevent an increase in intracellular Na⁺ mediated by the Na⁺-H⁺ exchanger.

Key Words: myocardial pH_i • Na⁺-H⁺ exchange • Na⁺-independent Cl^-/HCO₃^- exchange • spontaneously hypertensive rats • BCECF

	Introduction

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Left ventricular hypertrophy, a major independent risk factor for morbidity and mortality from cardiovascular disease, can be a consequence of mechanical overload. The degree of hypertrophy, however, is not uniform in patients with arterial hypertension. Recent studies have shown that many subjects with left ventricular hypertrophy have normal blood pressure and also that hypertrophy may precede the elevation of blood pressure.^{1 2} This apparent discordance between blood pressure values and left ventricular mass suggests that factors other than blood pressure elevation promote the development of left ventricular hypertrophy.^{3 4} In a recent study by de la Sierra et al,⁵ an increased activity of Na⁺-H⁺ exchanger in erythrocytes was associated with the presence of left ventricular hypertrophy in hypertensive patients.

The hyperactivity of the Na⁺-H⁺ exchanger could result in a rise of both pH_i and [Na⁺]_i. The increase in [Na⁺]_i would lead to a secondary increase in [Ca²⁺]_i via the Na⁺-Ca²⁺ exchanger. The activity of Na⁺-H⁺ exchanger has been widely studied in different cell types in experimental and human hypertension with a variety of results.^{6 7 8 9 10 11 12 13} However, measurements of pH_i in hypertrophied myocardium are scanty.^{14 15}

The present study reports a hyperactivity of the Na⁺-H⁺ exchanger in the myocardium of the SHR and elucidates the mechanism by which the alteration is offset under physiological conditions.

	Materials and Methods

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Male SHR (6 months old, n=20) and age-matched normotensive WKY (n=19) rats were used. The SHR and WKY rats were originally derived from Charles River Breeding Farms, Wilmington, Mass. All animals were housed under identical conditions and had free access to standard dry meal and water. Systolic blood pressure was measured weekly with the indirect tail-cuff technique.¹⁶ The preparations and general protocol were similar to those previously reported.¹⁷ Briefly, isolated papillary muscles were horizontally mounted between a force transducer and a fixed hook in a flow chamber (constant flow rate, 4 mL/min) on the stage of an Olympus CK 2 inverted microscope. After they were mounted, the muscles were progressively stretched until they reached the length of maximal force development. Muscles were paced at 0.2 Hz with square wave pulses of 10-millisecond duration and a voltage intensity 10% over threshold. Temperature was kept constant at 30°C throughout the experiments.

Solutions
The muscles were superfused with one of the following solutions: (1) HEPES-buffered solution containing (mmol/L) NaCl 133.8, KCl 4.5, CaCl₂ 1.35, MgSO₄ 1.05, glucose 11, and HEPES 25 (the pH of the buffer solution [pH_o] was adjusted to 7.38±0.03 at 30°C with 3N NaOH [total Na⁺ amounted to 148.8 mmol/L], and the solution was gassed with 100% O₂); (2) HCO₃^-/CO₂-buffered solution, which contained (mmol/L) NaCl 128.3, KCl 4.5, CaCl₂ 1.35, NaHCO₃ 20.23, NaH₂PO₄ 0.35, MgSO₄ 1.05, and glucose 11 (the solution was equilibrated with CO₂/O₂ gas mixture to ensure a PCO₂ value of 35 mm Hg at the chamber level, with the pH_o of HCO₃^-/CO₂-buffered solutions being 7.37±0.01 at 30°C); (3) Na⁺-free HCO₃^-/CO₂-buffered solution in which 20.23 mmol/L NMG was substituted for NaHCO₃ and in which NaCl and NaH₂PO₄ were replaced with an equimolar amount of choline chloride (saturation of the solution with 5% CO₂/95% O₂ gave a pH of 7.38±0.01). The Na⁺-H⁺ exchanger was inhibited with 5 µmol/L EIPA (RBI). Anion exchanger mechanisms were inhibited by SITS (Sigma Chemical Co) added to the superfusate (final concentration, 0.1 mmol/L).

pH_i Measurements
Muscles were loaded with the ester form of the pH-sensitive fluorescent dye BCECF (Molecular Probes). The dye was solubilized in dimethyl sulfoxide (Sigma Chemical Co) containing pluronic acid F-127 (20% [wt/vol]) and diluted to a final concentration of 5 µmol/L in HCO₃^-/CO₂-buffered solution. After autofluorescence levels were recorded, the muscles were incubated for 3 hours in the BCECF-AM–containing solution. At the end of the loading period, fluorescence was uniformly distributed throughout the muscle, and it amounted to about four to five times the autofluorescence level. In BCECF-AM–loaded cells, it has been reported that most of the fluorescence arises from cytoplasmic-located BCECF, therefore making the potential error in pH_i measurements due to compartmentation relatively small.¹⁸ Since our experiments lasted several hours, loading of intracellular organelles was assessed in four papillary muscles at the end of the experimental protocol. The cell membrane was permeabilized with 15 µmol/L digitonin to induce the release of cytosolic BCECF. Afterwards, 1% Triton X-100 was applied. The data obtained from the decrease in fluorescence at 450-nm excitation wavelength showed that 60% to 64% of the fluorescent probe was in the cytosol, in agreement with previous reports in cardiac tissue.^{19 20} During the loading process, contractility decreased by 40% to 50%, but it gradually recovered during the washout. Washout of the extracellular space with dye-free solution was carried out for 90 minutes before any pH_i determination was done.

To measure fluorescence emission from BCECF, excitation light from a 75-W Xenon lamp was band-pass–filtered alternatively at 450 and 495 nm and was then transmitted to the muscles under study by a dichroic mirror (reflecting wavelengths, <510 nm) located beneath the microscope. Fluorescence emission was collected by the microscope objective (x10) and transmitted through a band-pass filter at 535±5 nm to a photomultiplier (model R2693, Hamamatsu). The output of the photomultiplier, together with the force transducer signals, was collected via an A/D converter (model 2801 A, Data Translation) and stored in a personal computer for later analysis. 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 (2 seconds every 10 seconds) during the protocol. At the end of each experiment, fluorescence emission was calibrated by exposing the muscles to a high-KCl solution containing 10 µmol/L nigericin, a H⁺-K⁺ exchanger that equals [H⁺]_o to [H⁺]_i when extracellular and intracellular K⁺ are the same. The calibration solution contained (mmol/L) KCl 140.0, MgCl₂ 1.0, CaCl₂ 1.35, HEPES 5.0, sodium cyanide 4, and BDM 20.0 to prevent muscle contracture.¹⁸ Buffer pH was adjusted with KOH or HCl to four different values ranging from 7.5 to 6.5. Such a calibration revealed a linear relation (r=.99±.001, n=39) between pH and the fluorescence ratio (F₄₉₅/F₄₅₀) calculated as follows: ratio=(fluorescence at 495 nm-autofluorescence at 495 nm)/(fluorescence at 450 nm-autofluorescence at 450 nm). Within this range of pH values, the "in vivo" calibrations could be superimposed with calibrations of BCECF-free acid solutions. This makes unlikely the possibility that the fluorescence signals could have been distorted by incomplete hydrolysis of the sequestered dye within intracellular organelles (see above). The fluorescence ratio was not altered when either EIPA (5 µmol/L) or SITS (0.1 mmol/L) was added to the bath during the calibration.

Calculation of Intracellular Buffering Power and Net H⁺ or HCO₃^- Fluxes
J_H+ and J_HCO3^- were estimated from the rate of pH_i recovery (dpH_i/dt) after an intracellular acid or alkali load, respectively. Alkali load was imposed by rapidly exposing the muscles to TMA hydrochloride.²¹ Ten-minute pulses of different TMA concentrations (10, 20, or 30 mmol/L) were applied without osmotic compensation, and the values of dpH_i/dt were determined as the change in pH_i observed during the first minute after the peak alkalosis. Intracellular acidification was induced by switching from HEPES-buffered superfusate to the CO₂/HCO₃^--buffered one. The values of pH_i during the recovery phase from the acid load values were fitted to an exponential curve of the form pH_it=pH_i(1-e^-kt), where pH_it and pH_i are the changes in pH_i from the initial value at time t and after steady state has been reached, respectively, and k is the rate coefficient. From this curve fit, values of dpH_i/dt (change in pH_i per minute) for each experiment were calculated. The following equation was used for calculation of net J_H⁺ or J_HCO3^- (mmol/L/min): J_H⁺/_HCO3^-=ß_totxdpH_i/dt, where ß_tot was calculated as the sum of intrinsic buffer capacity (ß_i) plus the buffering power due to intracellular CO₂/HCO₃^- (ß_CO2). ß_CO2 was considered to be 2.3 times [HCO₃^-]_i, assuming an open system for CO₂ and that its solubility and pK value are the same at either side of the cell membrane. [HCO₃^-]_i at any given pH_i was calculated from the Henderson-Hasselbalch equation to be [HCO₃^-]_i=[HCO₃^-]_ox10^(pHi-pHo). ß_i was calculated from the change in [HCO₃^-]_i produced by exposing the preparations to CO₂ as ß_i=[HCO₃^-]_i/pH_i observed when HEPES-buffer superfusate was switched to CO₂/HCO₃^- buffer.²² [HCO₃^-]_i was considered to equal the value of [HCO₃^-]_i during the exposure to CO₂, since in the absence of external CO₂, the value of [HCO₃^-]_i is very low (50 µmol/L²³ ). The main problem for estimating ß_i is that acid extrusion during the loading period may blunt the acidosis, thus leading to overestimation of ß_i value (see Reference 22²² for details). To reduce errors due to acid extrusion, we (1) blocked the Na⁺-H⁺ exchanger with 5 µmol/L EIPA 20 minutes before the acid load and (2) extrapolated the pH_i recovery back to a point where it intersected the line defining the maximum initial rate of acid loading. Both methods (ie, blockade and backextrapolation) have been used in previous works,^{24 25 26 27 28} and they are an attempt to correct the errors in the estimation of ß_i due to Na⁺-H⁺ exchange or any other H⁺ extruder mechanism. In the absence of Na⁺-H⁺ exchange blockade, using only the backextrapolation method, the estimated values of ß_i were 57.78±4.62 mmol/L at a mean pH_i of 6.99±0.01 in SHR (n=7) and 46.33±6.50 mmol/L in WKY rats (pH_i 6.89±0.05, n=5) (P=NS). After EIPA blockade and also by use of the backextrapolation method, ß_i values of 57.18±6.99 at pH_i 6.89±0.03 in SHR (n=5) and 45.98±5.53 in WKY rats (pH_i 6.85±0.04, n=4) (P=NS) were estimated. The fact that the values of ß_i estimated by the backextrapolation method were not significantly decreased after Na⁺-H⁺ exchange blockade suggested that the backextrapolation alone was enough to reduce errors. However, since it was suggested that the backextrapolation method does not always make an adequate correction,²⁸ the values estimated in the presence of EIPA blockade plus backextrapolation were used in the present study. No statistically significant difference in ß_i was detected between the myocardium of both strains. Actually, higher values of ß_i were determined in the myocardium of SHR. If this were a real difference, we would be underestimating ion fluxes in the myocardium of SHR.

Statistics
Data are presented as mean±SEM. Statistical analysis was performed by using either a paired t test or Student's t test, as appropriate. A value of P<.05 was considered significant.

	Results

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Table 1 shows values of systolic blood pressure and the heart weight–to–body weight index ratio of SHR and WKY rats. Hearts from SHR exhibited myocardial hypertrophy, as shown by the heart weight–to–body weight ratio. None of the animals studied showed clinical evidence of cardiac failure. The mechanical performance of the muscles from both experimental groups is shown in Table 2. No significant differences were detected in any of the contractile parameters studied.

View this table: [in this window] [in a new window]   Table 1. General Characteristics of Normotensive and Hypertensive Rats

View this table: [in this window] [in a new window]   Table 2. Characteristics of the Papillary Muscles

When the myocardium was exposed to HEPES buffer bubbled with oxygen (nominally bicarbonate free), pH_i was higher in SHR than in WKY rats, as shown in Fig 1. In HEPES buffer, myocardial pH_i in the normotensive rats was 7.10±0.04, a value in agreement with previous reports in this species.²⁹ In the SHR, however, mean pH_i was significantly higher (7.23±0.03, P<.05). Since the estimation of cellular buffer capacity showed no significant difference between the myocardium of both rat strains (see "Materials and Methods") and proton production is unlikely decreased in the SHR, the only mechanism that could account for the increased myocardial pH_i of the SHR was an enhanced H⁺ extrusion (or acid equivalent). Under nominally bicarbonate-free conditions, the main mechanism extruding H⁺ from the cell is presumably the Na⁺-H⁺ exchanger.³⁰ Even though the existence of a lactate/proton carrier in the heart cell membrane has been demonstrated, its contribution to the maintenance of steady state pH_i seems to be negligible, since specific inhibition of the transporter does not alter the steady state value of pH_i.^{20 31} Therefore, the simple finding of the higher value of pH_i in HEPES buffer strongly suggested a hyperactivity of the exchanger in the hypertrophic myocardium of the SHR. This was confirmed in additional experiments in which inhibition of bicarbonate-dependent regulatory pH_i mechanisms by SITS (0.1 mmol/L) did not change myocardial pH_i in SHR (7.26±0.03 and 7.28±0.03 before and 20 minutes after SITS administration, respectively; n=3). This is important, since a small contribution of bicarbonate-dependent mechanisms even in the absence of bicarbonate in the perfusate was recently described.³²

View larger version (13K): [in this window] [in a new window]   Figure 1. Myocardial pHi of SHR alkalinizes in the absence of HCO3-/CO2 buffer. Mean±SEM of pHi values at steady state in HEPES and HCO3-/CO2 buffers are shown. Note the higher pHi value in myocardium from SHR when only the Na+-H+ exchange mechanism is operative. In contrast, under the physiological HCO3-/CO2 buffer, there is no difference in myocardial pHi in SHR and WKY papillary muscles (n=7 in SHR and n=6 in WKY rats). *P<.05.

If the higher steady state pH_i of SHR myocardium in HEPES buffer were the result of a hyperactive Na⁺-H⁺ exchanger, the blockade of the antiporter should minimize the difference in pH_i between both strains. This is shown in Fig 2, which illustrates that the inhibition of the antiporter with EIPA decreased myocardial pH_i to a greater extent in SHR than in WKY rats. Twenty minutes after treatment with EIPA, pH_i decreased by 0.16±0.01 pH unit in SHR (n=6) versus a decrease of 0.09±0.02 in WKY rats (n=5) (P<.05). The difference in steady state myocardial pH_i was then minimized when the Na⁺-H⁺ exchanger was blocked. These data suggested that in spite of the higher pH_i, the Na⁺-H⁺ exchanger was operating at a higher activity level in SHR. It can be argued that the greater decrease in pH_i induced by EIPA in SHR was the result of a higher proton production. However, the higher steady state myocardial pH_i of SHR in the absence of EIPA makes this possibility very unlikely.

View larger version (15K): [in this window] [in a new window]   Figure 2. Comparison of the effect of EIPA blockade on steady state myocardial pHi in SHR and WKY rats under nominally bicarbonate-free conditions. Muscles (n=6 in SHR and n=5 in WKY rats) were superfused with HEPES buffer bubbled with oxygen. At 0 minutes, 5 µmol/L EIPA was added to the superfusate. Slopes indicate the change in myocardial pHi (pH unit/min) after EIPA in each rat strain. Notice that EIPA induced a greater decrease in the pHi of SHR myocardium than in WKY myocardium, thereby minimizing the initial difference in pHi. *P<.05.

The difference in myocardial pH_i detected between SHR and WKY rats in HEPES buffer was not detected when a bicarbonate/CO₂ buffer was used. In HCO₃^-/CO₂ media, myocardial pH_i levels in SHR and WKY rats were 7.15±0.03 and 7.11±0.05, respectively (P=NS) (Fig 1). This finding suggested that a bicarbonate-dependent mechanism(s) operating in the hypertensive rat was fully compensating for the increased activity of the Na⁺-H⁺ exchanger. This could be accomplished by mechanism(s) that was carrying either HCO₃^- out of the cells or H⁺ into the cells.

The possibility of an enhanced activity of the Cl^--HCO₃^- exchanger in SHR was explored by assessing the effect of SITS on steady state myocardial pH_i in the presence of bicarbonate/CO₂ buffer. Fig 3 shows typical tracings obtained on one muscle from each rat strain. The increase in pH_i induced by SITS in SHR clearly contrasted with the lack of effect in WKY rats. On average, the increase in pH_i induced by SITS was 0.08±0.02 pH unit (P<.05 by paired t test, n=3) in SHR, whereas no significant change was detected in WKY rats (0.04±0.04, n=3). The increase in pH_i induced by SITS in SHR suggested that the blocker was suppressing a mechanism that was extruding HCO₃^- from the cell in this rat strain. This evidence together with the minimization of the differences in myocardial pH_i between hypertensive and normotensive rats under bicarbonate buffer is in favor of the role played by the enhanced activity of the Cl^--HCO₃^- exchanger.

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of SITS blockade on steady state myocardial pHi in SHR and WKY papillary muscles in the presence of CO2/HCO3- buffer. Original tracings obtained from one SHR muscle and one WKY muscle are shown. Notice that after the addition of SITS, myocardial pHi in the SHR gradually increased to a value 0.094 pH unit higher, whereas there was no detectable change in myocardial pHi in WKY rats.

Fig 4 shows the changes in myocardial pH_i observed when the HEPES-buffered superfusate was changed to HCO₃^-/CO₂ buffer, at constant pH_o. After the initial fall induced by CO₂ permeation, pH_i gradually recovered, although the driving force cannot explain a passive HCO₃^- influx. Considering that the equilibrium potential for HCO₃^- ions varied between -35 mV (at the time of peak acidosis) and -20 mV (after pH_i recovery) and estimating a membrane potential value of -80 mV, the equilibrium potential for HCO₃^- ions was more positive than the membrane potential during the whole recovery period, indicating a net outward driving force for HCO₃^- ions. Therefore, mechanisms extruding H⁺ (or the acid equivalent) have to be considered in order to explain the recovery in pH_i.

View larger version (21K): [in this window] [in a new window]   Figure 4. Fall in myocardial pHi induced by introduction of HCO3-/CO2 buffer in SHR and WKY papillary muscle. Note that the initial drop in pHi due to CO2 permeation was similar in both rat strains, despite the fact that myocardial pHi in the presence of HEPES buffer was more alkaline in SHR. *P<.05.

Although the initial pH_i was higher in HEPES buffer in SHR, the fall in pH_i induced by the introduction of the bicarbonate/CO₂ buffer was similar in both rat strains (0.24±0.03 pH unit in SHR and 0.21±0.04 pH unit in WKY rats). This is consistent with the fact that the myocardium of both strains had similar buffer capacities. However, the different pH_i values at the beginning of the recovery preclude the comparisons of the initial velocity of recovery (dpH_i/dt_i). When the velocity of pH_i recovery (dpH_i/dt) of the myocardium of both rat strains was compared at a common pH_i value of 6.99, a faster rate of recovery was found in SHR. In SHR, dpH_i/dt (in pH units per minute) was 0.068±0.02 (n=7), whereas it was 0.014±0.002 (n=6, P<.05) in WKY rats. The enhancement of the rate of proton extrusion in SHR myocardium after the acid load became negligible when the Na⁺-H⁺ exchange was blocked by EIPA was 0.0032±0.002 in SHR (n=5) and 0.0032±0.002 in WKY rats (n=4).

Since the acid-extruding mechanisms are modulated by pH_i, net H⁺ (or acid equivalent) extrusion was estimated as a function of pH_i. As explained in "Materials and Methods," J_H⁺ equal to ß_totxdpH_i/dt and the estimated values plotted as a function of pH_i in SHR and WKY rats are shown in Fig 5. This plot allowed us to compare the H⁺ extrusion at a given pH_i in the myocardium of the two strains of rats. It is evident that for a given pH_i, H⁺ extrusion was greater in the myocardium of the SHR than in the WKY rat; ie, J_H⁺ amounted to 1.58±0.49 mmol/L per minute in SHR and 0.35±0.07 mmol/L per minute in WKY rats at pH_i 7.05 (P<.05). The slopes of the lines relating J_H⁺ and pH_i in the myocardium of both strains were different (-17.46±1.05 in SHR and -7.39±0.33 in WKY rats, P<.05). The lines relating J_H⁺ and pH_i fitted to an equation of the form J_H⁺=-17.46xpH_i+124.61 mmol/L per minute in SHR and J_H⁺ =-7.39xpH_i+52.43 mmol/L per minute in WKY rats. The larger differences in J_H⁺ were detected at the more acidic values of pH_i, but the difference in J_H⁺ progressively vanished as the increase in pH_i took place. This fact is providing additional evidence of the enhanced activity of the antiport in the myocardium of SHR.

View larger version (14K): [in this window] [in a new window]   Figure 5. JH+ as a function of pHi in SHR and WKY papillary muscles. As explained in "Materials and Methods," JH+ values were estimated to be equal to ßtotxdpHi/dt, where ßtot was calculated as the sum of intrinsic buffer capacity plus the buffering power due to intracellular CO2/HCO3-. Note the enhanced acid extrusion in SHR compared with WKY rats at any given pHi. *P<.05.

Since the Na⁺-independent Cl^--HCO₃^- exchanger is the only bicarbonate-dependent mechanism able to acidify the cell and since it is stimulated by increases in pH_i, the possible role played by this exchanger in regulating myocardial pH_i in the SHR was explored. To test the activity of this anion exchanger, the heart muscles from both rat strains were alkalinized with TMA as described in "Materials and Methods," and the recoveries during the alkaline load were compared. In each of three muscles from SHR and from WKY rats, three different pulses of TMA were performed. Fig 6 shows digitized values of pH_i obtained during a TMA pulse on one muscle from SHR and one muscle from WKY rats (top panel). The dpH_i/dt during TMA pulses was measured as pH_i observed during the first minute after the peak of alkalosis, as illustrated by the broken lines. The bottom panel of Fig 6 shows the individual nine results obtained in both rat strains. It can be noted that the recovery from different alkaline loads was more evident in the myocardium from SHR than in the myocardium from WKY rats. Furthermore, when the comparison was made between runs in which pH_i increased to similar values, a lesser increase in pH_i seemed to be necessary to drive the anion exchanger in the myocardium of the hypertensive animals. At a mean pH_i value of 7.49±0.06, dpH_i/dt in SHR was 0.075±0.028 pH unit/min (P<.05, n=3), whereas no significant recovery could be detected in WKY rats (0.027±0.017 pH unit/min, n=3). The enhanced velocity of pH_i recovery in SHR was suppressed after SITS. When the alkaline load induced by the TMA pulse was performed after the blockade of the anion exchanger with 0.1 mmol/L SITS, no significant recovery in myocardial pH_i was detected. At a pH_i value of 7.41±0.04, dpH_i/dt was 0.024±0.013 pH unit/min in SHR (n=3, P=NS). At a similar pH_i (7.47±0.13), dpH_i/dt was 0.004±0.03 pH unit/min (P=NS) in WKY rats (n=3).

View larger version (20K): [in this window] [in a new window]   Figure 6. Comparison of the response to intracellular alkalinization induced by TMA in SHR and WKY papillary muscles. Top, Digitized values of pHi determined on one SHR muscle and one WKY muscle. For comparison, runs in which pHi during TMA pulses attained similar values are shown. Horizontal lines on top of the tracings indicate the period of exposure to TMA. Broken lines illustrate the greater dpHi/dt in SHR compared with WKY muscles when myocardium is alkali-loaded. Bottom, pHi observed after peak alkalosis in the nine runs that were conducted on SHR () and WKY () myocardium.

From the data of pH_i recovery during the alkaline load, the relation between net HCO₃^- efflux (or acid equivalent) (J_HCO3^-) and pH_i was estimated, and the data obtained are shown in Fig 7. The line relating J_HCO3^- to pH_i was shifted leftward, intercepting the x axis at lower pH_i values in SHR (7.25±0.04) than in WKY rats (7.43±0.06) (P<.05), indicating that the bicarbonate-dependent mechanism responsible for the recovery from alkalosis was operative at lower pH_i values in the myocardium of SHR than WKY rats. The slopes of the lines were not different, since J_HCO3^-=27.40xpH_i-196.94 mmol/L per minute in SHR, and J_HCO3^-=26.28xpH_i-193.84 mmol/L per minute in WKY rats. At a given pH_i then, during alkalosis the myocardium from SHR seems to extrude more HCO₃^- than does the myocardium from WKY rats. The electroneutral exchange of Cl^- for HCO₃^- across the membrane of vertebrate cells is a nearly ubiquitous transport mechanism that has been extensively studied in Purkinje fibers³³ and in isolated cardiac myocytes.²¹ The two main characteristics of this system are its Na⁺ independence and its activation at pH_i values above resting pH_i. To check our hypothesis about the involvement of the Cl^--HCO₃^- exchanger in regulating pH_i in SHR, it was necessary to probe the enhanced activity of this exchanger under Na⁺-free conditions. The recoveries from alkalosis under Na⁺-free conditions in experiments similar to those described in Fig 6 were compared in four muscles from SHR and in four from WKY rats. Fig 8 shows tracings from a typical experiment in one muscle from a hypertensive rat. For comparison, the results observed when the myocardium was challenged with the same alkaline load in the presence of extracellular Na⁺ are also shown. It is evident that under Na⁺-free conditions, a marked recovery from the alkaline load was detected. When the rate of pH_i recovery from the alkaline load was compared between SHR and WKY rats, a faster recovery was again detected in SHR. At mean pH_i of 7.30, dpH_i/dt was 0.051±0.02 pH unit/min in SHR (n=4), whereas it amounted to only 0.0195±0.003 pH unit/min in WKY rats (n=4) (P<.05). Therefore, the data support the hypothesis of an enhanced activity of the Na⁺-independent anion exchanger in the myocardium from hypertensive rats.

View larger version (14K): [in this window] [in a new window]   Figure 7. JHCO3- as a function of pHi in SHR and WKY papillary muscles. JHCO3- was estimated to equal ßtotxdpHi/dt (where ßtot was calculated as the sum of intrinsic buffer capacity plus the buffering power due to intracellular CO2/HCO3-; see "Materials and Methods") during the recovery of the TMA pulses. Notice the larger JHCO3- in SHR compared with WKY muscle at any given pHi.

View larger version (16K): [in this window] [in a new window]   Figure 8. Na+ independence of the recovery from an alkali load. Digitized values of pHi during a TMA pulse conducted on papillary muscles of SHR in the presence (left tracing) and in the absence (right tracing) of extracellular Na+ are shown. Removal of extracellular Na+ decreased basal pHi before the TMA pulse, as would be expected after the inhibition of the acid extruder mechanisms. However, removal of extracellular Na+ did not prevent the recovery from the alkalosis induced by TMA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increased steady state myocardial pH_i in the SHR in nominally bicarbonate-free buffer provides evidence for hyperactivity of the Na⁺-H⁺ exchanger in the hypertensive and/or hypertrophic myocardium. This evidence was further strengthened by the following findings: (1) the faster recovery from the intracellular acid load induced by switching from HEPES to CO₂/HCO₃^- buffer and the suppression of this difference when the Na⁺-H⁺ exchanger was inhibited, (2) the greater effect of blocking the Na⁺-H⁺ exchanger with EIPA in SHR myocardium exposed to HEPES-buffer, and (3) the lack of effect of the disulfonic derivative SITS on resting pH_i in SHR myocardium in HEPES buffer bubbled with oxygen, ruling out a possible role of alkalinizing bicarbonate-dependent mechanisms.³²

A still unanswered question in the present study is whether the hyperactivity of the Na⁺-H⁺ exchanger that we detected is a characteristic of the hypertensive animals or of the hypertrophic myocardium itself. Further experiments are necessary to dissect hypertension from hypertrophy in order to elucidate this problem. In connection with this, a recent study by de la Sierra et al⁵ showed that an increased Na⁺-H⁺ exchange and decreased Na⁺-K⁺-Cl^- cotransport activities in erythrocytes from patients with essential hypertension were significantly correlated with left ventricular mass, independent of the blood pressure levels.

Cellular growth and the effects of growth factors are known to be linked to intracellular alkalinization via stimulation of the Na⁺-H⁺ exchanger.^{34 35} Na⁺-H⁺ hyperactivity in hypertension can account for the observed increases in [Na⁺]_i³⁶ and can also provide a molecular basis for cellular growth, particularly if it is accompanied by a decrease in Na⁺ pump activity.³⁷ The fact that in the presence of physiological bicarbonate buffer, no significant changes in myocardial pH_i were detected in our experiments cannot be argued to deny an increase in [Na⁺]_i due to the hyperactivity of Na⁺-H⁺ exchange. The increase in [Na⁺]_i can still be present even if the effect of the hyperactivity of Na⁺-H⁺ exchange on pH_i was blunted by the enhanced activity of the Cl^--HCO₃^- exchanger. The increase in [Na⁺]_i will lead to an increase in [Ca²⁺]_i via the Na⁺-Ca²⁺ exchanger. The increase in [Ca²⁺]_i could be a signal for promoting cellular growth by activation of protein kinase C and/or protooncogene induction.³⁸

The possibility of an enhanced activity of the Na⁺-H⁺ exchanger has been widely explored in several blood cell types from hypertensive humans and animals.^{6 7 8 9 10 11 12 13} In vivo studies suggest a hyperactivity of the Na⁺-H⁺ exchanger in skeletal muscle of hypertensive patients³⁹ and SHR.⁴⁰ We are aware of two studies in which measurements of myocardial pH_i were performed in hypertensive or hypertrophic rats.^{14 15} Neither of these studies showed changes in steady state pH_i in the hypertensive animal, but we have to consider that these studies were not designed to study the possible hyperactivity of the exchanger and that both of them were performed with bicarbonate/CO₂ buffer. As we mentioned before, an increased activity of the Na⁺-independent Cl^--HCO₃^- exchanger will blunt the increase in pH_i.

Although our data report for the first time the enhanced activity of the Na⁺-H⁺ exchanger in the myocardium of the SHR, we do not elucidate the cellular mechanism for the enhanced exchanger activity. In a recent study,⁴¹ a polyclonal serum specific for NHE-1 was raised in the rabbit and was used to study NHE-1 expression in cultures of vascular and skeletal myocytes from SHR and WKY rats and also from extracts of crude membrane from hearts. No significant changes in the amount of NHE-1 protein were detected in SHR cells relative to WKY cells. These data and others showing more extensive phosphorylation of the exchanger^{13 42} suggest that the increased activity results from an increased turnover number per Na⁺-H⁺ exchanger molecule.

An enhanced Na⁺-H⁺ exchange activity may be the result of an increased intracellular H⁺ activity, induced either by a diminished buffer capacity or by an increased proton generation. These possibilities seem to be unlikely since (1) no significant differences in buffer capacities were detected between the normotensive and hypertensive animals, in accordance with previous findings,^{11 41} and (2) in the SHR, the steady state myocardial pH_i in HEPES buffer was higher and not lower, as would be expected if proton production were increased. However, an overcompensation of an increased H⁺ production by an hyperactive antiporter is a possibility to be considered. In addition, the data displayed in Fig 5 show that at any given pH_i, J_H⁺ is greater in the hypertensive than in the normotensive rat.

It has been postulated that changes in [Ca²⁺]_i are linked to changes in Na⁺-H⁺ exchange activity, with the rise in [Ca²⁺]_i being the primary cause of enhanced Na⁺-H⁺ exchange activity.^{43 44} However, [Ca²⁺]_i has to be elevated far beyond the values found in hypertension to induce an activation of the antiport.⁴⁵ An increased activity of PKC would also induce hyperactivity of the Na⁺-H⁺ exchanger.⁴⁶ Although no evidence of altered PKC activity in essential hypertension has yet been provided,¹³ it looks attractive if the possibility of an overexpression of tissue angiotensin-converting enzyme resulting in angiotensin-mediated hypertrophy⁴⁷ is considered. In this regard, it may be of interest that multiple clinical studies have suggested that pharmacological inhibition of angiotensin-converting enzyme may be superior to other blood pressure–lowering interventions for inducing regression of cardiac hypertrophy.⁴⁸

A very interesting finding in the present study was that the increased activity of the myocardial Na⁺-H⁺ exchanger was masked by the enhanced activity of the Na⁺-independent Cl^--HCO₃^- exchanger. This electroneutral exchange of intracellular HCO₃^- for extracellular Cl^- across the plasma membrane of vertebrate cells was first described in erythrocytes.⁴⁹ It has been extensively studied in cardiac muscle (for further references, see Reference 33³³ ), and it has been implicated in the defense against alkali load. This exchanger appears to be activated only at pH_i above resting values.³³ Although we do not know of any study involving this exchanger in myocardium from hypertensive animals, it is interesting that an enhanced activity of the transporter has been detected in red blood cells from hypertensive patients.⁵⁰ The authors hypothesized that the decreased pH_i of red blood cells from hypertensive subjects was the result of the enhanced activity of this anion exchanger. It is evident from our data that pH_i has to surpass values of 7.4 in order to detect HCO₃^- efflux in the myocardium of the WKY rats. However, lower pH_i values were inducing significant HCO₃^- efflux in the myocardium of SHR. The cellular mechanisms by which the hypertensive rat (or the hypertrophic myocardium) is presenting an increased activity of this anion exchanger accompanying the enhanced activity of the Na⁺-H⁺ exchange is not apparent to us. The driving force for this exchanger results from an inward gradient for Cl^- exceeding that for HCO₃^- under normal conditions. If we assume constant extracellular ionic concentrations and constant membrane potential, either a decrease in intracellular Cl^- or an increase in intracellular HCO₃^- would increase the activity of the exchanger. An increased [HCO₃^-]_i with the resulting increase in pH_i could result from an enhanced activity of the Na⁺-H⁺ exchanger. However, our data comparing the activity of the anion exchanger in the myocardium from normotensive and hypertensive rats at similar values of pH_i do not support this possibility. The enhanced activity of the anion exchanger in SHR seems to be independent of changes in pH_i. We are not aware of reports about a decrease in intracellular Cl^- in hypertension that could explain the hyperactivity of this exchanger.

In conclusion, we report an increased activity of the Na⁺-H⁺ exchanger in the hypertrophic myocardium of the SHR. This hyperactivity of the antiporter is not a consequence of a decreased pH_i, and it is masked under physiological conditions in which the HCO₃^-/CO₂ buffer is used, by the Na⁺-independent Cl^--HCO₃^- anion exchanger. The role played by an enhanced anion exchanger activity compensating the hyperactive Na⁺-H⁺ exchanger is based on the following pieces of evidence: (1) Steady state myocardial pH_i in SHR was higher than in WKY rats under nominally bicarbonate-free conditions, but the difference diminished in the presence of HCO₃^-/CO₂ buffer. (2) The blockade of bicarbonate-dependent mechanisms by SITS increased steady state pH_i only in SHR. (3) Challenging the anion exchanger with an alkaline load (TMA pulse) induced a recovery in pH_i significantly faster in SHR than in WKY rats, and this difference was not detected after SITS blockade. (4) The enhanced recovery of myocardial pH_i in the SHR after an alkaline load was Na⁺ insensitive.

	Selected Abbreviations and Acronyms

 

BCECF-AM = 2'-7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein BDM = 2,3-butanedione monoxime EIPA = 5-(N-ethyl-N-isopropyl)amiloride hydrochloride JHCO3- = net HCO3- flux JH+ = net H+ flux NHE-1 = most common Na+-H+ exchanger isoform NMG = N-methyl-D-glucamine SHR = spontaneously hypertensive rat(s) TMA = trimethylamine WKY = Wistar-Kyoto

	Acknowledgments

 
N.G. Pérez is the recipient of a postdoctoral fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. Drs Camilión de Hurtado and Cingolani are Established Investigators of CONICET, Argentina. We thank Dr Eduardo Marban (Johns Hopkins University, Baltimore, Md) for his valuable comments on the manuscript.

Received December 30, 1994; accepted August 28, 1995.

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				S. Engelhardt, L. Hein, U. Keller, K. Klambt, and M. J. Lohse Inhibition of Na+-H+ Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in {beta}1-Adrenergic Receptor Transgenic Mice Circ. Res., April 19, 2002; 90(7): 814 - 819. [Abstract] [Full Text] [PDF]

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