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Am J Physiol Heart Circ Physiol 283: H2102-H2109, 2002. First published July 11, 2002; doi:10.1152/ajpheart.01071.2001
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Vol. 283, Issue 5, H2102-H2109, November 2002

The ERK pathway regulates Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport activity in adult rat cardiomyocytes

Delphine Baetz1, Robert S. Haworth2, Metin Avkiran2, and Danielle Feuvray1

1 Laboratoire de Physiologie Cellulaire and Centre National de la Recherche Scientifique, Hôpital Marie Lannelongue-Université Paris XI, 91405 Orsay Cedex, France; and 2 Centre for Cardiovascular Biology and Medicine, King's College London, The Rayne Institute, St. Thomas' Hospital, London 7E1 7EH, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The sarcolemmal Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC) is stimulated by intracellular acidification and acts as an acid extruder. We examined the role of the ERK pathway of the MAPK cascade as a potential mediator of NBC activation by intracellular acidification in the presence and absence of angiotensin II (ANG II) in adult rat ventricular myocytes. Intracellular pH (pHi) was recorded with the use of seminaphthorhodafluor-1. The NH<UP><SUB>4</SUB><SUP>+</SUP></UP> method was used to induce an intracellular acid load. NBC activation was significantly decreased with the ERK inhibitors PD-98059 and U-0126. NBC activity after acidification was increased in the presence of ANG II (pHi range of 6.75-7.00). ANG II plus PD-123319 (AT2 antagonist) still increased NBC activity, whereas ANG II plus losartan (AT1 antagonist) did not affect it. ERK phosphorylation (measured by immunoblot analysis) during intracellular acidification was increased by ANG II, an effect that was abolished by losartan and U-0126. In conclusion, the MAPK(ERK)-dependent pathway facilitates the rate of pHi recovery from acid load through NBC activity and is involved in the AT1 receptor-mediated stimulation of such activity by ANG II.

cardiac ventricular myocytes; angiotensin II


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ISOFORM Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC) was recently cloned from cardiac tissue (9), and functional studies (1, 8, 9, 22, 24, 41) have provided evidence that it is stimulated by intracellular acidification and acts as an acid extruder. In a previous study, we (24) showed that NBC activity, besides that of the Na+/H+ exchanger, accounted for ~38% of the total acid efflux from ventricular myocytes of normal adult rats after intracellular acidification at intracellular pH (pHi) 6.8. Increased sarcolemmal NBC activity and consequent increases in pHi and Na+ have been suggested to be causally involved in pathophysiological responses, notably during ischemia and/or reperfusion (19). However, little is known about the signal transduction mechanisms that cause NBC activation and that might regulate its activity. In contrast to the reported role for intracellular Ca2+ in the control of Na+/H+ exchange activity (the other major pHi alkalinizing transporter) (23), the activity of NBC was not affected by decreasing intracellular Ca2+ or by inhibiting Ca2+/calmodulin protein kinase II (24). In cultured neonatal rat ventricular myocytes, angiotensin II (ANG II) was shown to activate the NBC and the results of the study (21) supported the view that the consequent increase in pHi was independent of the ANG type 1 (AT1) receptor phosphoinositide signaling pathway. They suggested that the AT2 signaling pathway may underlie the response.

Both AT1 and AT2 subtypes of the ANG II receptor are expressed in the ventricular myocardium of many species, including rats (7) and humans (15). Recent observations point to the critical role of ANG II in several pathophysiological processes. Several studies (20, 34) have shown that ANG II is secreted from stretched myocytes and plays an important role in mechanical stretch-induced cardiac hypertrophy. It has been demonstrated that the number of ANG II receptors is increased in adult rat hearts after myocardial infarction (26, 29) and that ANG II may have a profound effect on ventricular remodeling after infarction (38). Both subtypes of ANG II receptors belong to the G protein-coupled receptor superfamily (17, 42). However, there is a low degree of structural homology between the AT1 and AT2 receptors (32-34%) and each subtype appears to couple with its effectors via different intracellular pathways. AT1 receptors are coupled to Gq protein-mediated stimulation of phosphoinositide hydrolysis (42). In addition, mitogen-activated protein (MAP) kinases (MAPK), which are critical components of cellular processes such as growth, differentiation, and apoptosis (3, 4, 43), are activated by ANG II binding to AT1 receptors in various cell types (10, 33). By comparison, little is known about the physiological function(s) of the AT2 receptor, although it has been shown to mediate apoptosis (44) and to have an opposite action to that of AT1 stimulation in various cell types (14, 16).

In the present study, we have investigated the effects of ANG II on sarcolemmal NBC activity in freshly isolated adult rat ventricular myocytes. Our objectives were to determine whether nonselective stimulation of ANG II receptors has an effect on sarcolemmal NBC activity and whether selective stimulation of AT1 or AT2 by ANG II affects sarcolemmal NBC activity. Results of recent studies (4, 27) suggest that intracellular signals transduced via the ERK pathway of the MAPK cascade may be important contributors to Gq protein- coupled receptor-mediated regulation of various transporters. Therefore, we also determined the involvement of the ERK pathway in basal and ANG II-stimulated sarcolemmal NBC activity. To achieve this, we used established techniques for the determination of NBC activity, in conjunction with antagonists of distinct ANG II receptor subtype selectivity and specific kinase inhibitors.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All procedures were in accordance with the regulations of the Ministère de l'Agriculture for the care and use of laboratory animals.

Isolation of rat ventricular myocytes. Briefly, single ventricular myocytes were obtained from hearts of male Wistar rats [250-300 g body wt; anesthetized with thiopental sodium (50 mg/kg body wt ip)] using a combination of enzymatic [0.28 mg/ml collagenase (Yakult) and 0.05 mg/ml protease type XIV (Sigma)] and mechanical dispersion. The composition of the basic solution used for cell isolation and further details of the procedure have been described previously (18). Rod-shaped ventricular myocytes were used on the day of isolation.

Determination of sarcolemmal NBC activity. Sarcolemmal NBC activity was determined in single ventricular myocytes loaded with the pH-sensitive fluorescent dye carboxy-seminaphthorhodafluor-1 (SNARF-1) with a microepifluorescence technique, as described previously (24).

The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered Tyrode superfusing solution contained (in mM) 117 NaCl, 5.4 KCl, 1 CaCl2, 1.2 MgCl2, 11 glucose, and 23 NaHCO3. All HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered solutions were equilibrated with 5% CO2-95% O2 and had a pH of 7.4 at 37°C. Cells (n = 6-7 cells/group, obtained from 4-5 different hearts in each protocol) were subjected to intracellular acidosis induced by transient exposure to 10 mM NH4Cl (5). The rate of acid efflux (JH), calculated at pHi intervals of 0.05 during recovery from intracellular acidosis in the presence of cariporide (1 µM), an inhibitor of Na+/H+ exchange (36), was used as the indicator of NBC activity. Details of the method for calculating acid efflux have been described previously (22, 24). Briefly, acid efflux was estimated using the equation JH = beta T × dpHi/dt, where beta T is the sum of intrinsic buffering power due to intracellular CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (beta CO2). beta CO2 is given by beta CO2 = 2.3 × [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i, where [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i is intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration. [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i is given by [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i = [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]o × 10<SUP>(pH<SUB>i</SUB> − pH<SUB>o</SUB>)</SUP>, where [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]o is extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration and pHo is extracellular pH.

Determination of cellular ERK phosphorylation. Protein samples (~40 µg) from whole cell lysates were separated by SDS-PAGE on 9% polyacrylamide gels (14). pHi decrease- and ANG II-mediated regulation of ERK was determined through the detection of dual phosphorylation of ERK1/2 by immunoblot analysis with phosphospecific antibodies (New England Biolabs), as described previously (14, 39). To confirm equal protein loading, nonphosphospecific antibodies for ERK2 (Santa Cruz Biotechnology) were used. Specific protein bands were detected with enhanced chemiluminescence and autoradiography. Phosphorylation status was quantified with laser densitometry by using the National Institutes of Health Image Analysis System (Scion; Frederick, MD).

Experimental protocols. Within each protocol, there was no significant difference between groups in steady-state pHi. In each protocol, 1 µM cariporide was applied before the cells were exposed to NH4Cl and was present throughout the experiment to inhibit Na+/H+ exchange (36). When the effects of ANG II (Sigma) were studied, this was present before induction of the acid load (using the NH4Cl prepulse method). When used, the nonsubtype-selective ANG II receptor antagonist [Sar1-Leu8]ANG II (Sigma), the AT1-selective antagonist losartan (gift from Merck, Sharp and Dohme) and the AT2-selective antagonist PD-123319 (Sigma) were present from 3 min before the NH4Cl prepulse. When studying the effects of MAPK kinase inhibitors on basal sarcolemmal NBC activity or on sarcolemmal NBC activity in the presence of ANG II, the MAPK kinase-1 (MEK1) inhibitor PD-98059 or the MEK1/2 inhibitor U-0126 (Cell Signaling Technology) dissolved in dimethyl sulfoxide was present from 10 or 30 min before the NH4Cl prepulse, respectively. The maximun concentration of dimethyl sulfoxide in any experiment was 0.1% (vol/vol), which did not affect NBC activity.

Statistical analysis. All values of pHi and JH are quoted as means ± SE along with the number of observations (n). Statistical significance was estimated with Student's t-test or analysis of variance, followed by Student-Newman-Keuls test to locate differences between groups. Differences were considered significant at the level of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Regulation of sarcolemmal NBC activity after acidification. To study sarcolemmal NBC activity alone, 1 µM cariporide was applied throughout the experiments to inhibit Na+/H+ exchange in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered Tyrode solution. Figure 1A illustrates superimposed pHi recoveries from intracellular acidosis in an adult rat ventricular myocyte in the presence and absence of 1 µM cariporide. As shown previously, pHi recovery from intracellular acidification is still observed in the presence of Na+/H+ exchange inhibition, and this occurs via NBC activity (22, 24). This was further confirmed in the presence of the anion blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (22). DIDS (500 µM) completely inhibited pHi recovery from intracellular acidification in the presence of cariporide (Fig. 1B).


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  		Fig. 1.   A: intracellular pH (pHi) recovery from intracellular acidification in CO2-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-buffered Tyrode solution in the absence and presence of cariporide (1 µM). Each single myocyte was acid loaded by the NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (10 mM NH4Cl) prepulse method. The two traces were superimposed to ease comparison. In the presence of cariporide, pHi recovery occurs through Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (NBC) activity. B: cell was acid loaded twice by NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, the first pulse under control conditions (i.e., in presence of cariporide) and the second pulse in the presence of both cariporide and 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS) (500 µM).

MAPKs have been identified as important mediators in a wide array of physiological processes. We examined the role of MAPKs as potential mediators of NBC activation by intracellular acidification. For this purpose, we used PD-98059 (20 µM) and U-0126 (10 µM), two compounds that are selective MAPK signaling cascade inhibitors (2, 12) with a selectivity for MEK over other kinases. Figure 2 shows the typical experiments carried out to investigate the effects of these inhibitors on pHi recovery due to NBC in ventricular myocytes. When PD-98059 (Fig. 2A) or U-0126 (Fig. 2B) was applied to the cell, this resulted in a clear slowing of pHi recovery after an acid load, thus pointing to a slowing down of NBC. This slowing down effect is further stressed in Fig. 2C, where the acid equivalent efflux JH, carried by NBC, is significantly decreased by either PD-98059 or U-0126. In addition, our determinations of ERK phosphorylation after intracellular acidification clearly show that it is increased during the first 3 min and then it decreases. The increase was abolished by pretreatment of cells with the MEK inhibitor U-0126 (Fig. 3).


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  		Fig. 2.   A and B: effects of the MAPK kinase (MEK) inhibitors PD-98059 (20 µM) and U-0126 (10 µM) on pHi recovery due to Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. The myocyte was acid loaded under control conditions (i.e., in the presence of only cariporide) or in the presence of an inhibitor. The two traces were superimposed to ease comparison. C: pHi dependence of the rate of acid efflux (JH) carried by NBC in the absence (open circle , control) or presence of 20 µM PD-98059 (black-triangle) or 10 µM U-0126 (). *P < 0.05 vs. control group (n = 6 cells/group).



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  		Fig. 3.   ERK activity during recovery from acidification. Autoradiograms show representative Western blots with phosphospecific (top) and nonphosphospecific (bottom) ERK antibodies. * P < 0.05 vs. time 0 (4 experiments with cells from 4 hearts).

Effect of ANG II on sarcolemmal NBC activity after acidification. ANG II has been shown to activate NBC in cultured neonatal rat ventricular myocytes (21). So far, no data are available concerning the effect of ANG II on adult ventricular myocytes. Figure 4 illustrates the effect of exposing an adult ventricular myocyte to ANG II (100 nM). ANG II clearly accelerated the recovery of pHi after an intracellular acid load (Fig. 4A). On the other hand, when cells were pretreated with 100 nM [Sar1-Leu8]ANG II, a nonsubtype-selective ANG II receptor antagonist, the application of ANG II had no effect on the rate of recovery from intracellular acidosis (Fig. 4B). This indicates that acceleration of pHi recovery on exposure to ANG II occurs via receptor stimulation. The effects of ANG II on NBC activity are summarized in Fig. 4C. JH was significantly greater in the presence of ANG II over the pHi range that was between 6.75 and 7.00. The increase in JH was prevented in the presence of the nonsubtype-selective ANG II receptor antagonist (not shown).


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  		Fig. 4.   Effects of 100 nM angiotensin II (ANG II) on pHi recovery due to Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. A: representative recordings obtained in a myocyte that was acid loaded twice, the first pulse under control conditions and the second pulse in the presence of ANG II. B: representative recordings obtained in a myocyte under control conditions and in the presence of 100 nM ANG II after exposure of the myocyte to 100 nM [Sar1-Leu8]ANG II. The two traces were superimposed to ease comparison. C: pHi dependence of JH carried by NBC in the absence (open circle ) or presence of 100 nM ANG II (). * P < 0.05 vs. control group (n = 7 cells per group).

Effect of selective stimulation of ANG II AT1 and AT2 receptors on sarcolemmal NBC activity. To investigate which of the two receptor subtypes, AT1 or AT2, could be involved in the ANG II stimulation of NBC activity after acidification, we used the selective AT1 and AT2 antagonists losartan and PD-123319, respectively. Figure 5 shows the JH-versus-pHi relationships constructed using data from such experiments. When cells were pretreated with 100 nM PD-123319 (Fig. 5A), 100 nM ANG II still significantly increased JH over the pHi range 6.75-6.95, indicating the increase in NBC activity. Thus JH at a pHi of 6.95 was increased up to 3.31 ± 0.40 meq · l-1 · min-1 (vs. 1.80 ± 0.18 meq · l-1 · min-1 in control) compared with an increase of up to 3.61 ± 0.31 meq · l-1 · min-1 (nonsignificantly different) when cells were exposed to ANG II without pretreatment with PD-123319. On the other hand, when cells were pretreated with 100 nM losartan (Fig. 5B), 100 nM ANG II had no significant effect on JH throughout the same pHi range. These results indicate that the stimulatory effect of ANG II on NBC activity occurs via ANG II AT1 receptor stimulation.


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  		Fig. 5.   Effects of selective stimulation of ANG II AT1 and AT2 receptors on sarcolemmal NBC activity. A: pHi dependence of JH carried by NBC in control cells (open circle ) and in cells that were exposed to 100 nM ANG II in the presence of 100 nM PD-123319, an AT2 antagonist () (n = 7 cells). B: pHi dependence of JH carried by NBC in control cells (open circle ) and in cells that were exposed to 100 nM ANG II in the presence of 100 nM losartan, the AT1 antagonist () (n = 6 cells). *P < 0.05 vs. control group.

Role of MAP/ERK kinase pathway in stimulatory effect of ANG II on NBC activity. Besides playing a role in NBC activity after acidification, as we have shown above, the MAPK signaling cascade may also be required for ANG II stimulation of NBC. To examine this possibility, we used the highly selective MEK1 and MEK2 inhibitor U-0126 (12). As illustrated in Fig. 6A, pretreatment of cells with U-0126 (10 µM) totally prevented the stimulatory effect of ANG II on the pHi recovery rate from intracellular acidification. This is outlined in Fig. 6B, which shows the pHi dependence of the acid efflux carried by NBC over the pHi range comprised between 6.75 and 7.05. It is clear that when cells were pretreated with 10 µM U-0126, the stimulatory effect of ANG II on NBC activity was abolished.


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  		Fig. 6.   Effect of MEK1/2 inhibition on the stimulatory effect of ANG II on pHi recovery from intracellular acidification. A: representative recordings obtained from a myocyte after pretreatment with 10 µM U-0126 and in the presence of 100 nM ANG II after pretreatment with 10 µM U-0126. The two traces were superimposed to ease comparison. B: pHi dependence of JH carried by NBC in cells pretreated with 10 µM U-0126 (open circle ) and in cells that received 100 nM ANG II and were pretreated with 10 µM U-0126 (n = 7 cells per group).

We sought to determine whether inhibition of the stimulatory effect of ANG II on NBC by U-0126 occurred via AT1 or AT2 subtype receptors. Cells were exposed to ANG II in the presence of either 100 nM PD-123319 or 100 nM losartan during pretreatment with 10 µM U-0126. Figure 7 shows that U-0126 markedly decreased ANG II-stimulated JH through NBC as observed in Fig. 6A. In addition, the comparison of these results with those of Fig. 5A indicates that MEK1/2 inhibition abolishes the selective stimulatory effect of AT1 on NBC activity following acidification. On the other hand, JH carried by NBC from cells exposed to ANG II in the presence of losartan and during pretreatment with U-0126 was not different from the JH measured in cells exposed to ANG II in the presence of losartan over a similar pHi range (see Fig. 5B). Therefore, these results are in favor of an AT1-mediated stimulation of NBC activity that involves the MEK signal transduction pathway. To confirm the role of the ERK pathway in AT1-mediated NBC stimulation, we then tested whether selective AT1 stimulation does indeed increase cellular ERK activity after acidification. Figure 8 shows that in parallel with their effects on sarcolemmal NBC activity, ANG II alone or in the presence of PD-123319 produced significant increase in ERK activity. U-0126 abolished ERK activation by ANG II in both the absence and presence of the AT2 antagonist.


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  		Fig. 7.   Effect of MEK1/2 inhibition on selective stimulation of ANG II AT1 and AT2 receptors on sarcolemmal NBC activity. pHi dependence of JH carried by NBC in cells pretreated with 10 µM U-0126 (open circle ), in cells that were exposed to 100 nM ANG II in the presence of 100 nM PD-123319, an AT2 antagonist, and pretreated with 10 µM U-0126 (), and in cells that were exposed to 100 nM ANG II in the presence of 100 nM losartan, an AT1 antagonist, and pretreated with 10 µM U-0126 () (n = 6 cells per group).



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  		Fig. 8.   Effect of ANG II stimulation, in absence or presence of MEK inhibition, on ERK activity in adult rat ventricular myocytes (3 min after acidification). Cells were exposed to vehicle (control) or 100 nM ANG II in absence or presence of either 100 nM PD-123319 or 100 nM losartan in absence of pretreatment or during pretreatment with MEK inhibitor U-0126 (10 µM). Autoradiograms show representative Western blots with phosphospecific (top) and nonphosphospecific (bottom) ERK antibodies. * P < 0.05 vs. control (4 experiments with cells from 4 hearts).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of this study in adult rat ventricular myocytes is that the ERK pathway of the MAPK cascade regulates sarcolemmal NBC activity during recovery from intracellular acidification and facilitates the AT1 receptor-mediated stimulation of such activity by ANG II.

Regulation of sarcolemmal NBC activity after intracellular acidification. In mammalian cells, the first and best-characterized MAPK cascade is the p42/p44 MAPK cascade, which involves the activation of p42/p44 MAPK (also known as ERK1/2) via direct phosphorylation by the dual-specificity kinases MEK1 and MEK2. This MAPK cascade has been shown to be essential for the propagation of several signals in various cell types (4, 27). We show here that NBC activation by decreasing pHi was inhibited by either the specific MEK1 inhibitor PD-98059 (2) or by the higher affinity MEK1 inhibitor U-0126 (12). Moreover, our molecular data indicate that ERK is phosphorylated after acidification and that this phosphorylation was inhibited by U-0126. Therefore, these results support the notion that the stimulatory effect of intracellular acidification on NBC activation is mediated via the MAPK cascade.

It has been shown (6) that levels of rat NBC-1 mRNA, which is highly expressed in the kidney and also in nonepithelial cells, such as heart cells, remain unchanged after a few days of acidosis. This indicated that functional stimulation of NBC by intracellular acidification is likely to be mediated by a posttranscriptional event, such as phosphorylation (6). Romero et al. (32) indeed reported that rat NBC possesses consensus sites for several kinases, including protein kinases A and C, and for tyrosine phosphorylation. It is worth noting here that a new member of the NBC family has recently been identified (31). This new member, mNBC-3, is expressed uniquely in the skeletal muscle and heart. It could then be hypothesized that this membrane protein, which also possesses several protein kinase consensus phosphorylation sites, may mediate pHi recovery from acidification in the present study. This is, however, unlikely because this muscle-specific mNBC-3 activity is not affected by DIDS (1 mM) (31), whereas 500 µM DIDS inhibited NBC-mediated pHi recovery from acidification in our experiments (Fig. 1). In addition, mNBC-3 activity was shown by Pushkin et al. (31) to be blocked by the Na+/H+ exchange inhibitor EIPA, whereas all our experiments were performed in the presence of the Na+/H+ exchange inhibitor cariporide.

Regulation of sarcolemmal NBC activity by ANG II. Our results clearly show that increased sarcolemmal NBC activity by ANG II in adult rat ventricular myocytes occurred through AT1 receptor stimulation. Indeed, when cells were treated with the AT2 antagonist PD-123319, the application of ANG II still significantly increased acid-equivalent efflux through NBC (e.g., up to 3.74 ± 0.57 vs. 2.06 ± 0.26 meq · l-1 · min-1 under control conditions at pHi 6.9), whereas when cells were treated with losartan, the AT1 antagonist, ANG II stimulation of NBC activity was abolished (1.81 ± 0.35 and 2.39 ± 0.25 meq · l-1 · min-1 in the presence and absence of ANG II, respectively, at the same pHi). This observation is at variance with an earlier report in which ANG II was also shown to activate NBC in neonatal myocytes, an effect that was ascribed to the AT2 receptor (21). The most likely explanation for the discrepancy between this report and our results is that the expression of AT1 and AT2 receptors depends on the developmental stage of the cells. The AT2 receptor is expressed at very high levels in the developing fetus and, although it declines after birth (25), it cannot be excluded that it remains expressed at a relatively high level in cultured neonatal rat ventricular myocytes (21). On the other hand, AT2 receptor expression is low in the cardiovascular system of the adult (25). In addition, the imposed acid load was of very small amplitude in the study that used neonatal myocytes (21) compared with that induced in the present study.

This study demonstrates that not only a MAPK (ERK)-dependent pathway facilitates the rate of pHi recovery from acid load through NBC, but also that this signaling pathway is involved in the stimulatory effect of ANG II. Furthermore, it was most interesting that the MEK1/2 inhibitor U-0126 abolished the effect of AT1 selective stimulation, indicating that the positive effect of AT1 stimulation on NBC activity requires activation of the ERK pathway of the MAPK cascade. The importance of MAPK-dependent pathways, including ERK1/2, in the regulation of another pHi alkalinizing transporter, the Na+/H+ exchanger, has been demonstrated in several recent studies (4, 14, 27, 39). It is worth noting here that sarcolemmal NBC activity did not respond to ANG II stimulation in a similar manner to that described for the sarcolemmal Na+/H+ exchange (14). Simultaneous stimulation of AT1 and AT2 by ANG II was indeed shown not to significantly affect sarcolemmal Na+/H+ exchange activity. On the contrary, the present results show that the application of ANG II significantly increased JH through NBC activation over the pHi range comprised between 6.75 and 7.10 and that this increase was not significantly different from that mediated by selective stimulation of AT1 receptors (see Figs. 4C and 5A). This may have important pathophysiological implications since, under conditions where the effect of AT1 stimulation on Na+/H+ exchanger activity would be counteracted by simultaneous AT2 stimulation (14), sarcolemmal NBC may then be the predominant or even the only alkalinizing transporter functioning to extrude excess acid from myocardial cells.

Pathophysiological implications. Myocardial ischemia-induced acidification (11, 35) and the presence of an endogenous agonist such as ANG II may obviously represent stimulatory events for NBC activity. From our earlier results (19), it was clear that an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent mechanism contributed to pHi recovery after ischemia in adult rat hearts. This mechanism, most likely NBC, appeared rather depressed in a group of diabetic rat hearts. This, together with the reduction in Na+/H+ exchange activity in the group of hearts from diabetic rats (23), resulted in a better recovery of ventricular function on reperfusion. Although puzzling, this was likely to be due to the reduction in Na+ influx, which, secondarily, causes excessive Ca2+ uptake and exacerbates the tissue injury (40). However, this study was performed in isolated rat hearts perfused in the absence of any cardioactive hormone. Yet the renin-angiotensin system is upregulated with diabetes, which may lead to local increases in ANG II (13). It is reasonable to speculate that, in the presence of ANG II during reperfusion, NBC activity would have been stimulated, whereas Na+/H+ exchange activity would not (14). Further studies are necessary to examine whether or not ANG II would compromise recovery of ventricular function of diabetic hearts during postischemic reperfusion.

Finally, it is known that many MAPK-dependent pathways are activated in ischemic-reperfused hearts in vivo (28, 30, 37). A recent study (28) has shown that both ERK1 and -2 are phosphorylated during ischemia, followed by reperfusion. However, this study raised the possibility that protein kinase-mediated activation of the Na+/H+ exchanger through ischemia-reperfusion may also have an inhibitory effect on Na+/H+ exchanger activity early on during reperfusion. Future experiments will examine whether NBC activity may account for pHi recovery in the initial stage of reperfusion when Na+/H+ exchanger activity is transiently inhibited.


    ACKNOWLEDGEMENTS

Part of this study was supported by a grant from the Bonus Qualité Recherche (Université Paris-Sud). D. Baetz is the recipient of a Groupe de Réflexion sur la Recherche Cardiovasculaire grant.


    FOOTNOTES

Address for reprint requests and other correspondence: D. Feuvray, Laboratoire de Physiologie Cellulaire, Bât 443, Université Paris XI, 91405 Orsay cedex, France (E-mail: danielle.feuvray{at}ibaic.u-psud.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 11, 2002;10.1152/ajpheart.01071.2001

Received 6 December 2001; accepted in final form 10 July 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 283(5):H2102-H2109
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


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