Vol. 283, Issue 5, H2102-H2109, November 2002
The ERK pathway regulates Na+-HCO
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 |
The sarcolemmal
Na+-HCO
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
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 |
THE ISOFORM
Na+-HCO 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 |
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-buffered Tyrode superfusing solution
contained (in mM) 117 NaCl, 5.4 KCl, 1 CaCl2, 1.2 MgCl2, 11 glucose, and 23 NaHCO3. All
HCO-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 = 
T × dpHi/dt, where T is the sum of
intrinsic buffering power due to intracellular
CO2/HCO (CO2). CO2 is
given by CO2 = 2.3 × [HCO]i, where
[HCO]i is intracellular
HCO concentration.
[HCO]i is given by [HCO]i = [HCO]o × 
, where
[HCO]o is extracellular
HCO 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 |
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-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).
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
A: intracellular pH (pHi) recovery
from intracellular acidification in
CO2-HCO-buffered Tyrode solution in the
absence and presence of cariporide (1 µM). Each single myocyte was
acid loaded by the NH (10 mM NH4Cl)
prepulse method. The two traces were superimposed to ease comparison.
In the presence of cariporide, pHi recovery occurs through
Na+-HCO cotransporter (NBC) activity.
B: cell was acid loaded twice by NH, 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).
View larger version (19K):
[in this window]
[in a new window]
|
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
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
( , control) or presence of 20 µM PD-98059
( ) or 10 µM U-0126 ( ).
*P < 0.05 vs. control group (n = 6 cells/group).
|
|
View larger version (27K):
[in this window]
[in a new window]
|
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).
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of 100 nM angiotensin II (ANG II) on
pHi recovery due to Na+-HCO
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 () 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 · min1
(vs. 1.80 ± 0.18 meq · l1 · min1
in control) compared with an increase of up to 3.61 ± 0.31 meq · l1 · min1
(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.
View larger version (10K):
[in this window]
[in a new window]
|
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
() 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 () 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.
View larger version (16K):
[in this window]
[in a new window]
|
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 () 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.
View larger version (10K):
[in this window]
[in a new window]
|
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 (), 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).
|
|
View larger version (29K):
[in this window]
[in a new window]
|
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 |
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 · l1 · min1
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 · l1 · min1
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-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 |
1.
Aiello, EA,
Vila Petroff MG,
Mattiazzi AR,
and
Cingolani HE.
Evidence for an electrogenic Na+-HCO symport in rat cardiac myocytes.
J Physiol
512:
137-148,
1998[Abstract/Free Full Text].
2.
Alessi, DR,
Cuenda A,
Cohen P,
Dudley DT,
and
Saltiel AR.
PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem
270:
27489-27494,
1995[Abstract/Free Full Text].
3.
Aoki, H,
Richmond M,
Izumo S,
and
Sadoshima J.
Specific role of the extracellular signal-regulated kinase pathway in angiotensin II-induced cardiac hypertrophy in vitro.
Biochem J
347:
275-284,
2000[ISI][Medline].
4.
Bianchini, L,
L'Allemain G,
and
Pouysségur J.
The p42/p44 mitogen-activated protein kinase cascade is determinant in mediating activation of the Na+/H+ exchanger (NHE1 isoform) in response to growth factors.
J Biol Chem
272:
271-279,
1997[Abstract/Free Full Text].
5.
Boron, WF,
and
De Weer P.
Intracellular transients in squid giant axons caused by CO2, NH3 and metabolic inhibitors.
J Gen Physiol
67:
91-112,
1976[Abstract/Free Full Text].
6.
Burnham, CE,
Flagella M,
Wang Z,
Amlal H,
Shull GE,
and
Soleimani M.
Cloning, renal distribution, and regulation of the rat Na+-HCO cotransporter.
Am J Physiol Renal Physiol
274:
F1119-F1126,
1998[Abstract/Free Full Text].
7.
Busche, S,
Gallinat S,
Bohle RM,
Reinecke A,
Seebeck J,
Franke F,
Fink L,
Zhu M,
Sumners C,
and
Unger T.
Expression of angiotensin AT1 and AT2 receptors in adult rat cardiomyocytes after myocardial infarction. A single-cell reverse transcriptase chain reaction study.
Am J Pathol
157:
605-611,
2000[Abstract/Free Full Text].
8.
Camilion de Hurtado, MC,
Perez NG,
and
Cingolani HE.
An electrogenic sodium-bicarbonate cotransport in the regulation of myocardial intracellular pH.
J Mol Cell Cardiol
27:
231-242,
1995[ISI][Medline].
9.
Choi, I,
Romero MF,
Khandoudi N,
Bril A,
and
Boron WF.
Cloning and characterization of a human electrogenic Na+-HCO cotransporter isoform (hhNBC).
Am J Physiol Cell Physiol
276:
C576-C584,
1999[Abstract/Free Full Text].
10.
Duff, JL,
Berk BC,
and
Corson MA.
Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells.
Biochem Biophys Res Commun
188:
257-264,
1992[ISI][Medline].
11.
El Banani, H,
Bernard M,
Baetz D,
Cabanes E,
Cozzone P,
Lucien A,
and
Feuvray D.
Changes in intracellular sodium and pH during ischaemia-reperfusion are attenuated by trimetazidine. Comparison between low- and zero-flow ischaemia.
Cardiovasc Res
47:
688-696,
2000[Abstract/Free Full Text].
12.
Favata, MF,
Horiuchi KY,
Manos EJ,
Daulerio AJ,
Stradley DA,
Feeser WS,
Van Dyk DE,
Pitts WJ,
Earl RA,
Hobbs F,
Copeland RA,
Magolda RL,
Scherle PA,
and
Trzaskos JM.
Identification of a novel inhibitor of mitogen-activated protein kinase kinase.
J Biol Chem
273:
18623-18632,
1998[Abstract/Free Full Text].
13.
Fiordaliso, F,
Li B,
Latini R,
Sonnenblick EH,
Anversa P,
Leri A,
and
Kajstura J.
Myocyte death in streptozotocin-induced diabetes in rats is angiotensin II dependent.
Lab Invest
80:
513-527,
2000[ISI][Medline].
14.
Gunasegaram, S,
Haworth RS,
Hearse DJ,
and
Avkiran M.
Regulation of sarcolemmal Na+/H+ exchanger activity by angiotensin II in adult rat ventricular myocytes. Opposing actions via AT1 versus AT2 receptors.
Circ Res
85:
919-930,
1999[Abstract/Free Full Text].
15.
Haywood, GA,
Gullestad L,
Katsuya T,
Hutchinson HG,
Pratt RE,
Horiuchi M,
and
Fowler MB.
AT1 and AT2 angiotensin receptor gene expression in human heart failure.
Circulation
95:
1201-1206,
1997[Abstract/Free Full Text].
16.
Huang, XC,
Richards EM,
and
Sumners C.
Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors.
J Biol Chem
271:
15635-15641,
1996[Abstract/Free Full Text].
17.
Inagami, T,
Iwai N,
Sasaki K,
Yamano Y,
Bardhan S,
Chaki S,
Guo DF,
and
Furuta H.
Cloning, expression, and regulation of angiotensin II receptors.
In: Cellular and Molecular Biology of the Renin-Angiotensin System. Ann Arbor, MI: CRC, 1993, p. 273-291.
18.
Jourdon, P,
and
Feuvray D.
Calcium and potassium currents in ventricular myocytes isolated from diabetic rats.
J Physiol
470:
411-429,
1993[Abstract/Free Full Text].
19.
Khandoudi, N,
Bernard M,
Cozzone P,
and
Feuvray D.
Mechanisms of intracellular pH regulation during postischemic reperfusion of diabetic rat hearts.
Diabetes
44:
196-202,
1995[Abstract].
20.
Kijima, K,
Matsubara H,
Murasawa S,
Maruyama K,
Mori Y,
Ohkubo N,
Komuro I,
Yazaki Y,
Iwasaka T,
and
Inada M.
Mechanical stretch induces enhanced expression of angiotensin II receptor subtypes in neonatal rat cardiac myocytes.
Circ Res
79:
887-897,
1996[Abstract/Free Full Text].
21.
Kohout, TA,
and
Rogers TB.
Angiotensin II activates the Na+-HCO symport through a phosphoinositide-independent mechanism in cardiac cells.
J Biol Chem
35:
20432-20438,
1995.
22.
Lagadic-Gossmann, D,
Buckler KJ,
and
Vaughan-Jones RD.
Role of bicarbonate in pHi recovery from intracellular acidosis in the guinea-pig ventricular myocyte.
J Physiol
458:
361-384,
1992[Abstract/Free Full Text].
23.
Le Prigent, K,
Lagadic-Gossmann D,
and
Feuvray D.
Modulation by pHo and intracellular Ca2+ of Na+-H+ exchange in diabetic rat isolated ventricular myocytes.
Circ Res
80:
253-260,
1997[Abstract/Free Full Text].
24.
Le Prigent, K,
Lagadic-Gossmann D,
Mongodin E,
and
Feuvray D.
HCO-dependent alkalinizing transporter in adult rat ventricular myocytes: characterization and modulation.
Am J Physiol Heart Circ Physiol
273:
H2596-H2603,
1997[Abstract/Free Full Text].
25.
Matsubara, H.
Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases.
Circ Res
83:
1182-1191,
1998[Abstract/Free Full Text].
26.
Meggs, LG,
Coupet J,
Huang H,
Cheng W,
Li P,
Capasso JM,
Homcy CJ,
and
Anversa P.
Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.
Circ Res
72:
1149-1162,
1993[Abstract/Free Full Text].
27.
Moor, AN,
and
Fliegel L.
Protein kinase-mediated regulation of the Na+/H+ exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways.
J Biol Chem
274:
22985-22992,
1999[Abstract/Free Full Text].
28.
Moor, AN,
Gan XT,
Karmazyn M,
and
Fliegel L.
Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium.
J Biol Chem
276:
16113-16122,
2001[Abstract/Free Full Text].
29.
Nio, Y,
Matsubara H,
Murasawa S,
Kanasaki M,
and
Inada M.
Regulation of gene transcription of angiotensin II receptor subtypes in myocardial infarction.
J Clin Invest
95:
46-54,
1995[ISI][Medline].
30.
Omura, T,
Yoshiyama M,
Shimada T,
Shimizu N,
Kim S,
Iwao H,
Takeuchi K,
and
Yoshikawa J.
Activation of mitogen-activated protein kinases in in vivo ischemia/reperfused myocardium in rats.
J Mol Cell Cardiol
31:
1269-1279,
1999[ISI][Medline].
31.
Pushkin, A,
Abduladze N,
Lee I,
Newman D,
Hwang J,
and
Kurtz I.
Cloning, tissue distribution, genomic organization, and functional characterization of NBC3, a new member of the sodium bicarbonate cotransporter family.
J Biol Chem
274:
16569-16575,
1999[Abstract/Free Full Text].
32.
Romero, MF,
Fong P,
Berger UV,
Hediger MA,
and
Boron WF.
Cloning and functional expression of rNBC, an electrogenic Na+-HCO cotransporter from rat kidney.
Am J Physiol Renal Physiol
274:
F425-F432,
1998[Abstract/Free Full Text].
33.
Sadoshima, J,
Qiu Z,
Morgan JP,
and
Izumo S.
Angiotensin II and other hypertrophic stimuli mediated by G-protein coupled receptors activate tyrosine kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes. The critical role of Ca2+-dependent signaling.
Circ Res
76:
1-15,
1995[Abstract/Free Full Text].
34.
Sadoshima, J,
Xu Y,
Slayter HS,
and
Izumo S.
Autocrine release of angiotensin II mediated stretch-induced hypertrophy of cardiac myocytes in vitro.
Cell
75:
977-984,
1993[ISI][Medline].
35.
Sandmann, S,
Yu M,
Kaschina E,
Blume A,
Bouzinova E,
Aalkjaer C,
and
Unger T.
Differential effects of angiotensin AT1 and AT2 receptors on the expression, translation and function of the Na+-H+ exchanger and Na+-HCO symporter in the rat heart after myocardial infarction.
J Am Coll Cardiol
37:
2154-2165,
2001[Abstract/Free Full Text].
36.
Scholz, W,
Albus U,
Counillon L,
Gögelein H,
Lang HJ,
Linz W,
Weichert A,
and
Schölkens BA.
Protective effect of HOE 642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischemia and reperfusion.
Cardiovasc Res
29:
260-268,
1995[ISI][Medline].
37.
Shimizu, N,
Yoshiyama M,
Omura T,
Hanatani A,
Kim S,
Takeuchi K,
Iwao H,
and
Yoshikawa J.
Activation of mitogen-activated protein kinases and activator protein-1 in myocardial infarction in rats.
Cardiovasc Res
38:
116-124,
1998[Abstract/Free Full Text].
38.
Sladek, T,
Sladkova J,
Kolar F,
Papousek F,
Cicutti N,
Korecky B,
and
Rakusan K.
The effect of AT1 receptor antagonist on chronic cardiac response to coronary artery ligation in rats.
Cardiovasc Res
31:
568-576,
1996[ISI][Medline].
39.
Snabaitis, AK,
Yokoyama H,
and
Avkiran M.
Roles of mitogen-activated protein kinases and protein kinase C in 
1A-adrenoreceptor-mediated stimulation of the sarcolemmal Na+-H+ exchanger.
Circ Res
86:
214-220,
2000[Abstract/Free Full Text].
40.
Tani, M,
and
Neely JR.
Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts.
Circ Res
65:
1045-1056,
1989[Abstract/Free Full Text].
41.
Terzic, A,
Pucéat M,
Clément-Chomienne O,
and
Vassort G.
Phenylephrine and ATP enhance an amiloride insensitive bicarbonate-dependent alkalinising mechanism in rat single cardiomyocytes.
Naunyn Schmiedebergs Arch Pharmacol
346:
597-600,
1992[ISI][Medline].
42.
Timmermans, PB,
Wong PC,
Chiu AT,
Herblin WF,
Benfield P,
Carini DJ,
Lee RJ,
Wexler RR,
Saye JA,
and
Smith RD.
Angiotensin II receptors and angiotensin II receptor antagonists.
Pharmacol Rev
45:
205-251,
1993[ISI][Medline].
43.
Xia, Z,
Dickens M,
Raingeaud J,
Davis RJ,
and
Greenberg ME.
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
270:
1326-1331,
1995[Abstract/Free Full Text].
44.
Yamada, T,
Horiuchi M,
and
Dzau VJ.
Angiotensin II type 2 receptor mediates programmed cell death.
Proc Natl Acad Sci USA
93:
156-160,
1996[Abstract/Free Full Text].
Am J Physiol Heart Circ Physiol 283(5):H2102-H2109
0363-6135/02 $5.00
Copyright © 2002 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
