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(Circulation Research. 2000;86:622.)
© 2000 American Heart Association, Inc.

Cellular Biology

Stimulation of Myocardial Na⁺-Independent Cl^--HCO₃^- Exchanger by Angiotensin II Is Mediated by Endogenous Endothelin

Maria C. Camilión de Hurtado, Bernardo V. Alvarez, Irene L. Ennis, 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, Calle 60 y 120, 1900 La Plata, Argentina.

	Abstract

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Abstract—Experiments were performed in isolated cat papillary muscles loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in the esterified form to study the effect of endothelin-1 (ET-1) on the activity of the Na⁺-independent Cl^--HCO₃^- exchanger. Exposure to ET-1 (10 nmol/L) raised pH_i by 0.13±0.03 U (P<0.05) in papillary muscles superfused with nominally HCO₃^--free solution, whereas no significant change was detected under CO₂/HCO₃^--buffered medium. However, if ET-1 was applied to muscles pretreated with the anion exchanger inhibitor 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid, pH_i increased by 0.09±0.02 U (P<0.05) in the presence of CO₂/HCO₃^- buffer. The rate of pH_i recovery from trimethylamine hydrochloride–induced intracellular alkaline load was enhanced so that net HCO₃ efflux increased about three times in the presence of ET-1 (2.74±0.25 versus 9.66±1.29 mmol · L^-1 · min^-1 at pH_i 7.55, P<0.05). This effect was canceled by previous exposure to either 50 nmol/L PD 142,893 (nonselective endothelin receptor blocker) or 300 nmol/L BQ 123 (selective blocker of ET_A receptors). BQ 123 also abolished angiotensin II–induced activation of the Na⁺ independent Cl^--HCO₃^- exchanger. These results show that ET-1 increases the activity of the Na⁺-independent Cl^--HCO₃^- exchanger in cardiac tissue through the ET_A receptors. Furthermore, our data suggest that the previously described angiotensin II–induced stimulation of the anion exchanger activity is mediated by endogenous ET-1.

Key Words: endothelin-1 • angiotensin II • anion exchanger • receptors, ET_A • Na⁺-H⁺ exchanger

	Introduction

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Endothelin-1 (ET-1) has not only vasoconstrictive action but also a wide spectrum of pharmacological effects on cardiac tissue, including the increase of muscle contractility,^{1 2} hypertrophic action,^{3 4} and stimulation of the Na⁺-H⁺ exchanger (NHE) activity.^{5 6} These ET-1 cardiac actions are all exerted through the activation of type A ET receptors (ET_A). ET_A receptors belong to the seven-membrane domain type of receptors linked to the hydrolysis of membrane phosphoinositides and protein kinase C (PKC) activation.

During the course of our experiments, we observed that ET-1 did not produce the expected increase in pH_i due to NHE activation if applied in the presence of HCO₃. This observation suggested that ET-1 might be simultaneously activating an acidifying HCO₃-dependent mechanism. We have previously reported that angiotensin II (Ang II) stimulates the activity of the Na⁺-independent Cl^--HCO₃^- exchanger (AE) in cardiac tissue.⁷ Because Ang II acts through a signal transduction process that may be similar to that of ET-1, the possibility of ET-1 activating an acidifying HCO₃-dependent mechanism seemed likely.

Evidence indicates that ET-1 is a mediator of many of the Ang II effects. Ang II stimulates the expression of preproendothelin (ppET-1) mRNA and protein in several cell types, including cardiomyocytes.^{8 9 10 11} Ang II–induced cardiomyocyte hypertrophy has been shown to involve paracrine/autocrine release of ET-1 in neonatal rat cell cultures.^{11 12 13} The administration of a selective ET_A receptor antagonist abrogated the hypertensive response to Ang II infusion in the rat.¹⁴ Recently, we demonstrated that the stimulatory effect of Ang II on the cardiac NHE is mediated by an autocrine/paracrine mechanism that involves ET acting on the ET_A receptors.¹⁵ In this study, we present evidence that the ET-1 stimulatory effect on AE activity occurs via the ET_A receptors in cardiac tissue, and that this is the same mechanism as that underlying the Ang II–induced stimulation of AE activity.⁷

	Materials and Methods

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Experiments were carried out on isometrically contracting (0.2 Hz) isolated cat papillary muscles (0.29±0.03 mm² mean cross-sectional area) superfused with either HEPES- or CO₂/HCO₃^--buffered solutions, as previously described.⁷ Atenolol and prazosin (final concentration 1 µmol/L each) were added to the buffers to prevent adrenoceptor activation by possible catecholamine release from the nerve endings.

Measurements of pH_i were made after loading the muscles with the acetoxymethyl ester form of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM, Molecular Probes).⁷ BCECF fluorescence was excited at 450 and 495 nm, and the fluorescence emission was monitored after passage through a 535±5-nm filter. To limit photobleaching, a neutral-density filter (1% transmittance) was placed in the excitation light path, and sampling intervals were selected during the protocol (3 seconds in duration, every 15 seconds). At the start of the experiments, fluorescence at H⁺-sensitive wavelength was 10 to 15 times greater than basal autofluorescence. Values of pH_i were estimated from the ratio of BCECF fluorescence signals (F₄₉₅/F₄₅₀) after subtraction of background autofluorescence. At the end of each experiment, the emission fluorescence ratios were calibrated in situ with the high K⁺–nigericin method.¹⁶

Basal pH_i was noted after stabilization for 15 minutes with HEPES- or CO₂/HCO₃^--buffered solution, and the selected peptide, 500 nmol/L Ang II or 10 nmol/L ET-1 (Biochemical Research International), was then applied. To determine the effects of ET-1 and Ang II on the AE activity operating in its "forward" mode,^{7 17} intracellular alkalosis was induced by exposure to 40 mmol/L trimethylamine hydrochloride. AE activity was characterized by the rate of pH_i recovery from the alkaline loads.^{7 17} The pH_i recoveries during the whole trimethylamine (TMA) pulses were analyzed by fitting the pH_i versus time records to an exponential decay function. The rate of pH_i change (dpH_i/dt) at any selected pH_i value was obtained by evaluating the derivative of the exponential fit at that selected pH_i. Net HCO₃ fluxes (J_HCO3-) were determined by multiplying dpH_i/dt by the total intracellular buffering power, the latter comprising the intrinsic (ß_i) and the CO₂-dependent (ß_CO2) buffering power. ß_i was computed from the initial change in pH_i after TMA washout in the presence of acid-extruding inhibition.¹⁷ At the midpoint of pH_i change, ß_i values were used to determine ß_i as a function of pH_i in the flux calculations. ß_CO2 was assumed to be 2.3x [HCO₃^-]_i.^{17 18} Selective receptor blockers were applied 10 minutes before the agonist at the final concentrations of 50 nmol/L PD 142,893, 300 nmol/L BQ 123 (both from Biochemical Research International), and 10 µmol/L losartan (kind gift from Dupont Merck, West Point, Pa). SITS (Sigma Chemical Co) was applied at 0.1 mmol/L, 20 minutes before ET-1.

Statistics. Data are expressed as mean±SEM. Statistical analysis was performed by t test and ANOVA followed by Bonferroni’s test, as appropriate. The probability of the null hypothesis at <5% (P<0.05) was considered significant.

	Results

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Figure 1 shows that the effect of ET-1 on pH_i is dependent on the presence or absence of HCO₃ in the medium. In the nominal absence of HCO₃ (HEPES-buffered medium), 10 nmol/L ET-1 consistently evoked an intracellular alkalinization that amounted to an average rise in myocardial pH_i of 0.13±0.02 U (n=5, P<0.05) over the predrug value (Figure 1A). An initial transient acidification was observed in all of these experiments. This transient acidification peaked in 2.3±0.3 minutes and caused a maximal decrease in pH_i by 0.035±0.007 U (P<0.05). A similar acidifying effect of ET-1 in chick embryo and neonatal rat cardiomyocytes has been previously reported by Kohmoto et al.¹⁹ The mechanism underlying the initial decrease in pH_i is not known.

View larger version (17K): [in this window] [in a new window]   Figure 1. Alkalinizing effect of ET-1 is abolished in the presence of HCO3. A, Left panel shows time course of changes of pHi evoked by ET-1 in a representative experiment performed with a cat papillary muscle superfused with a nominal HCO3-free medium (HEPES buffer). Right panel shows individual (n=5) and mean values of pHi before and after 20 minutes of exposure to ET-1. B, Left panel shows absence of change in pHi when ET-1 was applied in the presence of HCO3 in a representative experiment. Right panel shows individual (n=6) and mean pHi values before and after ET-1.

Figure 1B shows that when 10 nmol/L ET-1 was applied in the presence of HCO₃, no significant change in pH_i was detected. Figure 1 also shows, in accordance with previous reports,^{18 20 21} that myocardial pH_i is slightly more alkaline in HCO₃-buffered medium than in HEPES-buffered medium. It may be argued that the higher initial pH_i in the presence of HCO₃ could prevent a further rise in pH_i on ET-1 treatment. However, the data in Figure 1 (right panels) show that the lack of pH_i response to ET-1 in the presence of HCO₃ was independent of the initial pH_i value.

The simultaneous activation of a HCO₃-dependent acidifier mechanism may explain the absence of an effect of ET-1 on pH_i when HCO₃ is present. If such a mechanism were present, an increased efflux of HCO₃ would counterbalance the increased NHE activity and no change in pH_i would be observed. One candidate for this mechanism is the AE, which has been shown to be present in cardiac cells^{17 22} and to contribute to the maintenance of resting (steady-state) pH_i.^{23 24} The activity of AE has been reported to be sensitive to the stilbene disulfonate inhibitors DIDS and SITS.^{25 26} . The resting pH_i of papillary muscles superfused with HCO₃ buffer increased from 7.07±0.03 to 7.12±0.03 U (n=8, P<0.05) after the application of SITS (0.1 mmol/L). This indicates that, within the conditions of our experiments, a background acid loading is carried by the AE over the physiological pH_i range.

Not only does SITS inhibit the AE, but the alkalinizing Na⁺-HCO₃^- cotransport is also sensitive to stilbene derivatives. The fact that resting pH_i increased after SITS application suggests that the cotransport is not very active at physiological pH_i values.

A novel acid-loading mechanism, a HCO₃-independent Cl^--OH^- exchanger, has been reported to contribute to pH_i regulation in isolated guinea pig ventricular myocytes.²⁷ However, SITS did not cause any significant change in resting pH_i when applied in the absence of HCO₃ (7.02±0.02 versus 7.03±0.01 U, n=4), making it unlikely that the Cl^--OH^- exchanger participated in the background acid loading of our preparations.

The response of pH_i to ET-1 in the presence of HCO₃ changed from a nondetectable effect to an intracellular alkalinization when the peptide was applied to SITS-pretreated muscles. Under this experimental condition, ie, blockade of HCO₃-dependent mechanisms by SITS, the rise in myocardial pH_i induced by ET-1 amounted, on average, to 0.09±0.02 U (n=5, P<0.05). Therefore, AE inhibition by SITS revealed the predominant alkalinizing effect of NHE stimulation by ET-1 in the presence of HCO₃.

In cardiac cells, the recovery of pH_i after an intracellular alkaline load involves the participation of AE activity. The extrusion of internal HCO₃ in exchange for external Cl^- causes the return of pH_i to the control value, provided enough time is allowed. It is conceivable that the recovery of pH_i from intracellular alkalosis would be accelerated in the presence of ET-1. Figure 2A shows the pH_i records of two representative experiments. In one, the recovery of pH_i from TMA-induced intracellular alkalosis was assessed under control conditions (no ET-1 present); in the other, the TMA pulse was applied after 20 minutes of exposure to ET-1. After the initial increase in pH_i caused by the TMA pulse, there is a recovery of myocardial pH_i toward the basal value. Similar peak intracellular alkalization was attained in the absence and in the presence of ET-1, but the initial rate of pH_i recovery was augmented in the presence of the peptide. This effect was consistently observed in all of the experiments (n=6). On average, myocardial pH_i rose from 7.15±0.02 to a peak alkaline value of 7.59±0.02 during the TMA pulse in control (n=10) and from a value of 7.11±0.02 to 7.55±0.02 in the presence of ET-1. The rate of pH_i recovery from the alkaline load increased 2- to 3-fold in the presence of ET-1, depending on the values of pH_i examined. A HCO₃-independent Cl^--OH^- exchange was also shown to be involved in the recovery of pH_i from intracellular alkaline loads in isolated guinea pig ventricular myocytes.²⁸ The lack of pH_i recovery from TMA-induced intracellular alkalosis in the absence of HCO₃, previously reported by us⁷ and other investigators,^{17 29} argues against the possibility that the Cl^--OH^- exchanger participates in the effects of ET-1 that are described in the present study.

View larger version (18K): [in this window] [in a new window]   Figure 2. ET-1 accelerates the recovery of pHi from an intracellular alkaline load. A, Tracings show changes in myocardial pHi during TMA-induced intracellular alkalinization with and without ET-1. Exposure to 40 mmol/L trimethylamine hydrochloride induced a rapid increase in pHi that reached similar peak values in the absence and presence of ET-1. Recovery toward control pHi was accelerated in the presence of ET-1. B, JHCO3- was determined as the product of dpHi/dt times the total intracellular buffering power (see Materials and Methods). ßi (mmol/L per pH unit), estimated as a function of pHi, was =166-19.6 (pHi). Control group (open bars, n=10); ET-1 (closed bars, n=6). Note the break in the ordinate scale. *Values significantly different from control (P<0.05, ANOVA).

Values of J_HCO3- carried by the AE were calculated at resting pH_i and at various selected pH_i values during the recovery from intracellular alkalinization. Figure 2B shows the estimated values in the absence and presence of ET-1. Consistent with the pH_i dependence of AE activity,^{17 22 30} J_HCO3- increased with the increase of pH_i. Ludt et al³¹ proposed that phosphorylation of the AE by PKC might stabilize the exchanger into a conformation with increased sensitivity to high pH_i. Evidence has been presented showing that the NH₂-terminal domain of the AE3 isoform present in cardiac tissue contains several potential PKC consensus phosphorylation sites.³²

ET-1–induced acceleration of the recovery from alkalosis was canceled by previous application of the nonselective ET receptor antagonist PD 142,893 (50 nmol/L). The rate of pH_i recovery in the presence of PD 142,893 plus ET-1 was not different from the rate under control conditions. During pH_i recovery, mean J_HCO3- values were estimated to be 2.58±0.69, 2.15±0.58, and 1.69±0.35 mmol · L^-1 · min^-1 at pH_i 7.55, 7.50, and 7.45 in the presence of ET-1 plus PD 142,893 (n=3). In control conditions, the J_HCO3- values estimated at the same pH_i values were 2.74±0.25, 2.21±0.19, and 1.73±0.16, respectively. To further ascertain the type of ET receptors involved in the effects of ET-1, the selective ET_A receptor antagonist BQ 123 was used. Figure 3A shows that ET-1–induced acceleration of pH_i recovery from intracellular alkalosis was prevented by the blockade of ET_A receptors, suggesting that these types of receptors are involved in the effect of ET-1 on myocardial AE activity. J_HCO3- values during pH_i recovery, assessed at pH_i 7.45, 7.50, and 7.55 with and without ET-1, are shown in Figure 1B.

View larger version (19K): [in this window] [in a new window]   Figure 3. ET-1–induced acceleration of pHi recovery from an intracellular alkaline load is mediated through ETA receptors. A, Tracings show that previous blockade of ETA receptors with BQ 123 suppressed the acceleration of pHi recovery otherwise caused by ET-1. B, JHCO3- values at selected pHi in control and in the presence of BQ 123+ET-1 (hatched bars, n=4). Control data belong to the same group of experiments shown in Figure 2. Note that the ordinate scales are different.

We recently showed that Ang II increases the activity of the AE in cardiac tissue.⁷ This activation was prevented by the blockade of the AT₁ receptor. Because an increasing number of Ang II effects are shown to be mediated by the participation of endogenous ET-1,^{11 12 13 14 15} we decided to explore whether this peptide was also involved in the stimulatory effect of Ang II on AE activity. To test this possibility, we examined the effect of Ang II on pH_i recovery after blocking ET_A receptors with BQ 123. As shown in Figure 4, the acceleration of pH_i recovery and the increase in J_HCO3- were canceled when Ang II was applied after the blockade of ET_A receptors, indicating that both endogenous ET and ET_A receptors are involved in the stimulation of AE activity by Ang II.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effect of Ang II on AE activity is abolished by the blockade of ETA receptors. A, Left panel shows recovery of pHi from TMA-induced intracellular alkalosis in a representative experiment in a control group. Right panel shows estimated mean JHCO3- values at different pHi. B, Left panel shows pHi recovery from imposed alkalosis in a muscle exposed to Ang II in a representative experiment. Right panel shows mean values of JHCO3- at selected pHi (n=3). Note the break in the ordinate scale. *Values significantly different from control (P<0.05, ANOVA). C, Left panel shows a representative experiment illustrating that the acceleration of pHi recovery caused by Ang II was abolished by BQ 123. Right panel shows mean values of JHCO3- in the presence of BQ 123 plus Ang II (n=4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results show that ET-1 produces a stimulatory effect on the activity of AE in cardiac tissue. ET-1 failed to raise pH_i in the presence of HCO₃, and AE inhibition by SITS changed the lack of pH_i response to ET-1 in HCO₃ into an alkalinizing effect. Furthermore, ET-1 increased the rate of pH_i recovery from an alkaline load, and ET receptor antagonists abolished the acceleration of pH_i recovery induced by ET-1.

The experimental model used in our study was the isolated cat papillary muscle. In this multicellular preparation, the possibility that pH_i signals arising from endothelial cells may have influenced the pH_i measurements cannot be completely ruled out. However, in the present study, increases of pH_i correlated with increases of developed force in muscle (and vice versa) after TMA application and washout (data not shown). The present study and our previous experiments^{7 18 33 34} validate the measurements of myocardial pH_i in our setup and would be indicative that most, if not all, of the measured pH_i changes were those occurring in cardiac myocytes. An important conclusion derived from these findings is that although there are no significant changes in the steady-state pH_i after ET-1 in CO₂/HCO₃^- buffer, the recovery of pH_i after any alkaline or acid load will be accelerated due to the simultaneous activation of the acid- and alkaline-loading mechanisms.

The AE, first identified as a component of the band 3 protein in erythrocytes,³⁵ has been implicated as the mechanism responsible for the recovery of pH_i from intracellular alkalinization because such recovery is eliminated in the absence of HCO₃.^{7 17 29} The mechanism has been extensively characterized in myocardial tissue and shown to be independent of extracellular sodium^{17 33} but strictly dependent on the presence of chloride ions in the extracellular space.¹⁷ Many neurohumoral factors have been reported to modulate the activity of this mechanism, namely ATP,³⁶ ß-adrenoceptor agonists,³⁷ aldosterone,²⁵ vasopressin,³⁸ and Ang II.^{7 39} To our knowledge, this is the first direct demonstration that ET-1 activates the cardiac AE. Furthermore, our data seem to indicate that the underlying mechanism of action of Ang II on AE activity is mediated through crosstalk between the Ang II and ET-1 pathways.

ET-1 belongs to a family of three 21-amino acid peptides (ET-1, ET-2, and ET-3) with highly homologous amino acidic sequences. The first original report described ET-1 as a vasoactive compound,⁴⁰ but lately a wide variety of effects on myocardial cells have been described. ET-1 has been reported to have inotropic and chronotropic actions,^{1 2 5 41 42} release atrial natriuretic peptide,⁴³ and promote cardiomyocyte hypertrophy.^{3 4} There are two fully characterized ET receptor subtypes, ET_A and ET_B, although pharmacological evidence has been presented to support the existence of other subtypes of the ET_A receptors.^{44 45} BQ 123, a potent selective antagonist of ET_A receptors,⁴⁶ has been shown to cancel the stimulation of the AE activity induced by ET-1, indicating that the ET_A receptors are involved in the effects described in the present study.

In addition to providing evidence that AE activation by ET-1 masks the changes in pH_i by NHE stimulation, our data suggest that the activation of the AE by Ang II⁷ is due to endogenous ET-1. Although this new information appears to be exciting, we must consider that the results could also be explained by nonspecific blockade of AT₁ receptors by BQ 123. We cannot rule out this possibility, but the selectivity of BQ 123 for the ET_A receptors has been established.^{11 46} In addition, it has been reported that Ang II–mediated hypertension in the rat was blunted by an ET-1 receptor blocker different from the one used in the present study.¹⁴

The origin of ET-1 after Ang II is open to speculation. The original description of ET-1 by Yanagasawa et al⁴⁰ explained that the peptide is produced by endothelial cells. There is mounting evidence that ppET-1 mRNA is expressed in endothelial cells and in other cell types, including cardiomyocytes.^{9 10 11} Ang II induces rapid (within 5 minutes) expression of ppET-1 mRNA in endothelial cells.⁹ In cultured cardiomyocytes, induction of ppET-1 mRNA was found to peak in 30 minutes.¹¹ In our experiments, the papillary muscles had been exposed to Ang II for at least 20 minutes before the activity of the AE was assessed. This appears to be enough time for ET-1 to attain effective concentration at the biophase of ET-1 receptors. Other studies on the involvement of ET-1 in the effects of Ang II, including hypertensive action,¹⁴ stimulation of hypertrophy,^{11 12 13} and NHE activity,¹⁵ support these findings. Therefore, it is tempting to hypothesize that endogenous ET-1 does play a role in the effects of Ang II.

	Acknowledgments

 
This study was supported in part by a grant (PIP 4058) from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Bernardo V. Alvarez was a recipient of a predoctoral fellowship from CONICET of Argentina. Irene L. Ennis was a recipient of a predoctoral fellowship from La Plata University, Argentina. Maria C. Camilión de Hurtado and Horacio E. Cingolani are Established Investigators, CONICET, Argentina.

	Footnotes

 
This manuscript was sent to Masao Endoh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received July 27, 1999; accepted January 21, 2000.

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