© 1999 American Heart Association, Inc.
Cellular Biology |
Regulation of Sarcolemmal Na+/H+ Exchanger Activity by Angiotensin II in Adult Rat Ventricular Myocytes
Opposing Actions via AT1 Versus AT2 Receptors
From the Centre for Cardiovascular Biology and Medicine, King’s College London, UK.
Correspondence to Dr Metin Avkiran, Cardiovascular Research, The Rayne Institute, St Thomas’ Hospital, Lambeth Palace Rd, London SE1 7EH, UK. E-mail metin.avkiran{at}kcl.ac.uk
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Abstract |
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Abstract—Increased sarcolemmal Na+/H+ exchanger activity has been implicated as a mediator of the cardiac actions of angiotensin II. We studied the receptor subtypes and signaling pathways involved in the regulation of sarcolemmal Na+/H+ exchanger activity by angiotensin II in adult rat ventricular myocytes. Cells were loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, and acid efflux rates estimated during recovery from intracellular acidosis were used to quantify exchanger activity. Sarcolemmal Na+/H+ exchanger activity was not affected by angiotensin II alone but was increased by angiotensin II plus PD123319 (AT2 antagonist). In contrast, angiotensin II plus losartan (AT1 antagonist) or CGP42112A (AT2 agonist) did not affect exchanger activity. The increase in Na+/H+ exchanger activity induced by angiotensin II plus PD123319 was blocked by losartan, PD98059 (extracellular signal–regulated kinase inhibitor), GF109203X (protein kinase C inhibitor), and tyrphostin AG1478 (epidermal growth factor receptor kinase inhibitor). Extracellular signal–regulated kinase phosphorylation and activity, measured by immunoblot analysis and an immune-complex kinase assay, respectively, were increased significantly by angiotensin II plus PD123319; these increases were blocked by losartan and PD98059. The increase in extracellular signal–regulated kinase phosphorylation induced by angiotensin II plus PD123319 was blocked also by GF109203X and tyrphostin AG1478. These data show that AT1 stimulation increases sarcolemmal Na+/H+ exchanger activity in adult rat ventricular myocytes and that this response requires extracellular signal-regulated kinase activation through a protein kinase C– and epidermal growth factor receptor–mediated mechanism. The positive effect of AT1 stimulation on Na+/H+ exchanger activity is counteracted by simultaneous AT2 stimulation through a mechanism that does not involve direct inhibition of the exchanger or attenuation of extracellular signal–regulated kinase activation.
Key Words: angiotensin • myocyte • Na+/H+ exchanger • signal transduction • extracellular signal–regulated kinase
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Introduction |
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The sarcolemmal Na+/H+ exchanger (NHE) of cardiac myocytes consists of the ubiquitous NHE1 isoform of this multigene family1 and plays an important role in mediating recovery of intracellular pH (pHi) from acidosis.2 Although sarcolemmal NHE activity is regulated primarily by pHi and is increased in response to acidosis, it is also subject to modulation by a number of neurohormonal stimuli,3 such as
1-adrenergic
agonists4 endothelin 15 and
thrombin.6 These agonists appear to stimulate sarcolemmal
NHE activity, through their respective Gq
protein–coupled receptors, by increasing the exchanger’s affinity for
intracellular H+, which is the primary mechanism
underlying receptor-mediated regulation of NHE1.7
Several recent studies have suggested that angiotensin II
also may stimulate sarcolemmal NHE activity and that a relative
intracellular alkalosis that arises from increased
H+ extrusion through the exchanger may underlie
the positive inotropic action of this peptide.8 9 However,
studies with ventricular myocytes isolated from adult rat
and rabbit hearts have revealed that (1) H+
efflux rate via the exchanger is increased only moderately by
angiotensin II and only over a very limited
pHi range (
6.95 to 7.00),9 which
is contrary to observations made with other mediators that stimulate
exchanger activity via Gq protein–coupled
receptors,4 6 and (2) the degree of intracellular
alkalosis and the magnitude of the positive inotropic effect induced by
angiotensin II are markedly smaller than those induced by
endothelin 1,8 another vasoactive peptide that has been
shown to stimulate the exchanger.5 Thus, the role of
angiotensin II as an important modulator of sarcolemmal NHE
activity appears open to question.
In light of the above, it is interesting to note that (1) both AT1 and AT2 subtypes of the angiotensin receptor are expressed in the ventricular myocardium of many species, including rat,10 rabbit,11 and human,12 13 and (2) in a variety of cell types in culture, AT1 and AT2 mediate opposing actions, particularly in growth regulation.14 15 16 Furthermore, in some preparations,17 18 AT2 stimulation has been shown to counteract AT1-mediated activation of extracellular signal–regulated kinases 1 and 2 (ERK1/ERK2). Because this pathway of the mitogen activated protein kinase signaling cascade is a key mediator of growth factor–induced stimulation of NHE1,19 it is possible that AT1 and AT2 may mediate opposing actions also in the regulation of sarcolemmal NHE activity.
In an effort to gain a better understanding of angiotensin II–mediated regulation of sarcolemmal NHE activity, the objectives of the present study were to determine, in adult rat ventricular myocytes, whether (1) simultaneous stimulation of AT1 and AT2 by angiotensin II alters sarcolemmal NHE activity, (2) selective stimulation of AT1 or AT2 by angiotensin II alters sarcolemmal NHE activity, (3) the ERK pathway is involved in any regulation of the exchanger by angiotensin II, and (4) protein kinase C (PKC) and/or the epidermal growth factor (EGF) receptor are upstream mediators of any effects of angiotensin II on ERK and sarcolemmal NHE activity.
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Materials and Methods |
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Studies in Adult Myocytes
Cell Isolation
Ventricular myocytes were isolated from hearts of adult male Wistar rats by enzymatic digestion4 6 20 21 for the study of drug effects on sarcolemmal NHE activity or cellular ERK phosphorylation and activity.
Determination of Sarcolemmal NHE Activity
A microepifluorescence technique was used to record
pHi in single myocytes loaded with cSNARF-1, and
the rate of acid efflux (JH), calculated at
pHi intervals of 0.05 during recovery from
intracellular acidosis, was used as the indicator of NHE
activity.4 6 20 21 Cells (n=7 to 10 per group,
obtained from 6 to 10 hearts in each protocol) were subjected to
acidosis by transient exposure to NH4Cl (first
acid pulse), which was repeated 15 minutes later (second acid
pulse).4 6 Within each protocol, there was no significant
difference between groups in basal or minimal pHi
(Table
). In control cells, both acid pulses occurred in the absence of
drug. When the effects of angiotensin II (Sigma) were
studied, this was present during the second pulse. When used, the
AT2-selective antagonist PD123319
(Research Biochemicals International) and/or the
AT1-selective antagonist
losartan (gift from Merck Sharp and Dohme, Inc) were
present from 3 minutes before the second acid pulse. When studying
the effects of angiotensin II plus PD123319 in the presence
of a kinase inhibitor (mitogen-activated protein
kinase kinase [MEK] inhibitor PD98059, PKC
inhibitor GF109203X, or EGF receptor kinase
inhibitor tyrphostin AG1478; Calbiochem-Novabiochem Corp),
this was present from 10 minutes before the second acid pulse.
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Determination of Cellular ERK Phosphorylation
and Activity
Angiotensin II–mediated regulation of ERK was
studied by the following 2 approaches: (1) determination, by Western
analysis, of ERK phosphorylation on both
threonine and tyrosine residues of the regulatory TEY motif, and (2)
measurement, by an immune-complex kinase assay, of ERK activity. Drug
exposure protocols were identical to those used to study sarcolemmal
NHE activity.
Determination of Cellular AT2 Expression
AT2 expression was determined by Western
analysis, using a rabbit polyclonal AT2
antibody22 23 (gift from Dr R.M. Carey, University of
Virginia Health Sciences Center, Charlottesville, VA). Membrane protein
from HEK293 cells stably transfected with AT2 was
a gift from Dr A.J. Balmforth (University of Leeds, UK).
Studies in Neonatal Myocytes
Ventricular myocytes isolated from hearts of
2-day-old Wistar rats of mixed sex were cultured in 6-well plates (on
coverslips for microepifluorescence studies) for 2 to 3 days
and transferred to serum-free medium 24 hours before use. Drug effects
on cellular ERK phosphorylation and sarcolemmal NHE
activity were determined as described for adult cells.
Statistical Analysis
Data are presented as mean±SEM. To assess changes in
JH within groups (ie, between the first and
second acid pulses), a paired t test was used. For
intergroup comparisons of the change in JH
at pHi 6.90
(
JH6.9) or of ERK
phosphorylation/activity, data were subjected to ANOVA,
followed by the Dunnett test (to compare every group with the control
group) or Student-Newman-Keuls test (to compare every group with every
other). P<0.05 was considered significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Studies in Adult Myocytes
Effect of Simultaneous Stimulation of AT1 and AT2 on Sarcolemmal NHE Activity
Angiotensin II at 10 to 1000 nmol/L had no significant effect on JH throughout the pHi range 6.75 to 7.10 (Figure 1
). Therefore,
JH6.9 did not differ significantly from
control (0.5±0.6 mmol/L · min–1) in
the groups that received 10 (0.2±0.8 mmol/L ·
min–1), 100 (0.2±0.4 mmol/L ·
min–1), or 1000 (0.5±0.3 mmol/L ·
min–1) nmol/L angiotensin II. These
data indicate that simultaneous stimulation of
AT1 and AT2 by
angiotensin II does not significantly affect sarcolemmal
NHE activity. Consistent with this, exposure of myocytes to 100
nmol/L angiotensin II for 10 minutes did not significantly
affect resting pHi (7.28±0.03 and 7.29±0.04,
respectively, before and after angiotensin II; n=3).
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) and second (•) acid pulses are shown in control cells
(A) and in cells that were exposed to angiotensin II at 10
(B), 100 (C), or 1000 (D) nmol/L throughout the second pulse (n=8 cells
per group, obtained from 6 hearts). ANG indicates
angiotensin II.
Effect of Selective Stimulation of AT1 on Sarcolemmal
NHE Activity
Figure 2 shows
representative recordings of
pHi during the consecutive acid pulses in single
myocytes as well as the
JH-versus-pHi
relationships constructed using data from 8 such experiments in each of
the 3 key groups of this subsection of the study. In control
experiments (Figure 2A), the profiles of
pHi recovery from intracellular acidosis were
similar after both pulses; consequently, the
JH-versus-pHi curves
were superimposed, indicating that temporal changes in NHE activity do
not occur in the absence of drug exposure. Similar results were
obtained when cells were exposed to 100 nmol/L angiotensin
II during the second acid pulse (Figure 2B), as described above.
However, when cells were pretreated with 100 nmol/L PD123319, 100
nmol/L angiotensin II accelerated pHi
recovery from intracellular acidosis and produced a rightward shift of
the JH-versus-pHi
curve, such that over the pHi range 6.75 to 7.00,
JH was significantly greater in the
presence of angiotensin II (Figure 2C).
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Figure 3A shows
JH at pHi 6.90
(JH6.9) during the first and the second
acid pulses in the various study groups and illustrates the
dose-dependent action of PD123319 in revealing the NHE-stimulatory
effect of angiotensin II. As shown, 100 nmol/L
angiotensin II significantly increased
JH6.9 only in the presence of pretreatment
with 30 or 100 nmol/L PD123319; importantly, 100 nmol/L PD123319 alone
did not significantly affect JH6.9,
indicating that the increase in NHE activity did not arise from a
direct exchanger-stimulatory action of the AT2
antagonist. In these experiments, although
JH6.9 was not significantly different
from control in cells that received angiotensin II alone or
PD123319 alone, PD123319 dose-dependently revealed a stimulatory
response to angiotensin II (Figure 3B). The ability
of angiotensin II to increase sarcolemmal NHE activity only
in the presence of the AT2 antagonist
suggests that this response may arise from selective
AT1 stimulation.
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) and second (
) acid pulses
(A) and
To determine the dose-dependency of the NHE stimulatory effect of
angiotensin II in the presence of AT2
blockade, we also studied the effects of various concentrations of
angiotensin II (10, 30, or 100 nmol/L) in combination with
100 nmol/L PD123319. As shown in Figure 4A, under these conditions,
angiotensin II produced a significant increase in
JH6.9 at all 3 concentrations. The dose
dependency of the response is illustrated in Figure 4B, which
shows that JH6.9 was significantly
greater than control in the groups that received 30 or 100 nmol/L
angiotensin II in combination with 100 nmol/L PD123319.
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To confirm that the NHE stimulatory effect of the
PD123319/angiotensin II combination that was observed in
the studies described above was indeed mediated via
AT1, we sought to determine whether this effect
could be inhibited by losartan, an
AT1-selective antagonist. As shown in
Figure 5A, the combination of 100 nmol/L
PD123319 and 100 nmol/L angiotensin II once again
significantly increased JH6.9, confirming
our earlier findings. This effect, however, was partially inhibited by
10 nmol/L losartan and was completely abolished by 30 and 100
nmol/L losartan. As shown in Figure 5B, relative to
control, JH6.9 was significantly
increased by the PD123319/angiotensin II combination, but
this effect was inhibited, in a dose-dependent manner, by
losartan. The combination of 100 nmol/L PD123319 and 100 nmol/L
losartan was without effect on JH
in the absence of angiotensin II. Furthermore, the
inhibitory effect of losartan on the response to
the PD123319/angiotensin II combination was sustained
throughout the pHi range 6.75 to 7.05 (data not
shown). These findings provide further support for the hypothesis that
selective stimulation of AT1 increases
sarcolemmal NHE activity.
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Effect of Selective Stimulation of AT2 on Sarcolemmal
NHE Activity
The inability of angiotensin II to increase
sarcolemmal NHE activity unless AT2 is blocked
suggests that stimulation of this receptor subtype opposes
AT1-mediated activation of the exchanger. To
determine whether this negative regulatory effect is mediated by a
direct inhibition of the exchanger, we next determined the effect of
selective stimulation of AT2 on sarcolemmal NHE
activity. This was achieved by exposing cells either to 100 nmol/L
angiotensin II in the presence of increasing concentrations
(10 to 100 nmol/L) of losartan or to the
AT2-selective agonist CGP42112A (20 nmol/L).
These experiments revealed that neither angiotensin II in
combination with losartan nor CGP42112A had any effect on
JH throughout the pHi
range studied (data not shown). These findings indicate that the
negative effect of AT2 stimulation on
AT1-mediated activation of the sarcolemmal NHE is
likely to occur not by direct inhibition of the exchanger but by
inhibition of the signaling pathway(s) that mediate the
AT1 response.
AT2 Expression in Adult Rat Ventricular
Myocytes
The pharmacological data presented above suggest that
AT1 and AT2 mediate
opposing actions in the regulation of sarcolemmal NHE activity in adult
rat ventricular myocytes. Contrary to this, however, some
radioligand binding studies suggest that
AT2 may be absent in adult rat
myocardium.24 25 To determine whether
AT2 is expressed at protein level in the adult
rat ventricular myocytes used in the present study, we
used Western analysis with an AT2
antibody that has been recently characterized by Wang et
al.23 The immunoblot illustrated in Figure 6 reveals that this antibody detected a
protein of 44 kDa, which is believed to represent
AT2,23 in protein extracts from 3
independent adult rat ventricular myocyte preparations, as
well as in a membrane protein sample from HEK293 cells stably
transfected with AT2. This finding is
consistent with the recent immunocytochemical data of Wang et
al23 and earlier radioligand binding studies
in rat ventricular tissue10 26 and supports
the existence of functional AT2 in adult rat
ventricular myocytes.
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Role of the ERK Pathway in the Regulation of Sarcolemmal NHE
Activity by Angiotensin II
In studying the role of the ERK pathway in the regulation of
sarcolemmal NHE activity by angiotensin II, we initially
examined whether combinations of angiotensin II and
PD123319 produce progressive activation of this pathway in parallel
with their effects on sarcolemmal NHE activity. As shown in Figure 7, in the presence of 100 nmol/L
PD123319, angiotensin II produced a dose-dependent increase
in ERK1/ERK2 phosphorylation, which mirrored its
dose-dependent effects on sarcolemmal NHE activity (see Figure 4B
). This finding is consistent with a causal
association between activation of the ERK pathway and stimulation of
NHE activity.
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To determine whether activation of the ERK pathway is a necessary step
in AT1-mediated stimulation of sarcolemmal NHE
activity, we then tested whether PD98059 (which selectively inhibits
MEK, the upstream activator of ERK1/ERK2)27 28
inhibits this response. As shown in Figure 8A, JH6.9 was
again significantly increased by the PD123319/angiotensin
II combination; however, this effect was abolished in the presence of
10 or 50 µmol/L PD98059, although even the higher concentration
of the MEK inhibitor was without significant effect when
given alone. Thus, as shown in Figure 8B, JH6.9 was significantly greater than
control in the group that received the PD123319/angiotensin
II combination but was unaffected by this combination in the presence
of PD98059 or by PD98059 alone. Indeed,
JH6.9 in response to the
PD123319/angiotensin combination was significantly smaller
in the presence of either concentration of the MEK
inhibitor than in their absence. This finding suggests that
AT1-mediated stimulation of the sarcolemmal NHE
requires ERK activation.
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To confirm the role of the ERK pathway in
AT1-mediated NHE stimulation, we next tested
whether selective AT1 stimulation does indeed
increase cellular ERK1/ERK2 phosphorylation and
activity in our system and whether the interventions that we have shown
to inhibit AT1-mediated NHE stimulation (such as
losartan and PD98059) inhibit any such increase. Additionally,
we compared the effects of selective AT1
stimulation versus simultaneous
AT1/AT2 stimulation, to
determine whether the different effects of these stimuli on sarcolemmal
NHE activity could arise from different magnitudes of ERK activation.
Figure 9 shows the effects of the
various interventions on myocyte ERK1/ERK2
phosphorylation (Figure 9A) and activity (Figure 9B). As shown, ERK phosphorylation and activity
were both increased significantly by selective
AT1 stimulation, and these effects were abolished
by losartan and by PD98059 (losartan or PD98059 alone
did not significantly affect ERK activity; data not shown). The
parallel effects of these interventions on ERK
phosphorylation/activity (Figure 9) and
sarcolemmal NHE activity (see Figures 5 and 8) support
the hypothesis that ERK activation is required for
AT1-mediated stimulation of the exchanger.
Notably, however, ERK activation by simultaneous
AT1 and AT2 stimulation was
similar in magnitude to that by selective AT1
stimulation in both assays (Figure 9A and 9B). These data
suggest that the ability of AT2 stimulation to
oppose AT1-mediated NHE activation does not arise
from an attenuation of ERK1/ERK2 activation.
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Upstream Regulators of AT1-Mediated Actions
Previous studies suggest that AT1-mediated
activation of the ERK pathway is PKC dependent in cultured neonatal rat
ventricular myocytes,29 but protein tyrosine
kinase dependent (via EGF receptor transactivation) in adult rat
vascular smooth muscle cells30 and neonatal rat cardiac
fibroblasts.31 To delineate the roles of these pathways in
AT1-mediated activation of the ERK pathway in
adult rat ventricular myocytes, they were exposed to
angiotensin II plus PD123319 in the presence of 1
µmol/L GF109203X (PKC inhibitor) or 250 nmol/L tyrphostin
AG1478 (EGF receptor kinase inhibitor). As shown in Figure 10, both kinase inhibitors
abolished the increase in ERK phosphorylation induced
by angiotensin II plus PD123319, suggesting roles for both
PKC and the EGF receptor as upstream mediators of the ERK response to
AT1 stimulation. This finding led us to determine
the effects of GF109203X and tyrphostin AG1478 also on
AT1-mediated stimulation of sarcolemmal NHE
activity. Figure 11 illustrates that,
in parallel with their inhibitory effects on ERK activation
(Figure 10), GF109203X and tyrphostin AG1478 also inhibited
AT1-mediated NHE activation. This is
consistent with our conclusion above that ERK activation is
necessary for AT1-mediated stimulation of the
exchanger.
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Studies in Neonatal Myocytes
Because angiotensin receptor expression in rat
myocardium is subject to developmental
regulation,10 the effects of angiotensin II on
ERK and NHE activity may differ between neonatal and adult myocytes. To
ascertain whether this is the case, we determined the effects of
angiotensin II, given alone or together with PD123319, on
cellular ERK and sarcolemmal NHE activity also in neonatal rat
ventricular myocytes maintained in culture. Figure 12A shows that, in this preparation,
angiotensin II increased cellular ERK activity to a similar
extent regardless of the presence or absence of the
AT2-selective antagonist. This is
consistent with the previous findings of Sadoshima et
al32 in a similar neonatal myocyte preparation and with
our observations in adult ventricular myocytes as described
above (Figure 9). Distinct from our observations in adult
myocytes (Figure 3), however, angiotensin II induced
no increase in sarcolemmal NHE activity in neonatal myocytes, in the
presence of up to 100 nmol/L PD123319 (Figure 12B).
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Discussion |
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The present study provides the first evidence in adult ventricular myocytes that AT1 and AT2 mediate opposing actions in determining a physiological response to angiotensin II, namely an increase in sarcolemmal NHE activity. To delineate the roles of AT1 and AT2 in the regulation of sarcolemmal NHE activity, we have used the endogenous agonist angiotensin II (which has similar affinity for both receptor subtypes) in combination with PD123319 (an AT2-selective antagonist) and/or losartan (an AT1-selective antagonist). Our data show that, in freshly isolated adult rat ventricular myocytes, angiotensin II increases sarcolemmal NHE activity only in the presence of AT2 blockade and that this effect is inhibited by AT1 blockade. These observations (which are distinct from those made in rat aortic smooth muscle cells33 ) suggest that, in adult rat ventricular myocytes, (1) AT1 stimulation increases sarcolemmal NHE activity, and (2) this effect is opposed by AT2 stimulation. In view of the signaling pathways that AT1 shares in common with other Gq protein–coupled receptors that mediate increased sarcolemmal NHE activity in this cell type (such as
1-adrenergic,4
endothelin,5 and thrombin6 receptors), it is
probable that AT2 may attenuate exchanger
activation by other neurohormonal stimuli also, although confirmation
of this awaits further investigation. On a more fundamental level, our data support the existence of functional AT1 and AT2 on adult rat ventricular myocytes, an issue that has been subject to question.34 35 Although it may be argued that the regulation of sarcolemmal NHE activity by angiotensin II in the present study could have a paracrine basis through the release from contaminating nonmyocyte cells of unknown NHE-regulatory factor(s), this is unlikely for several reasons. First, all visible cells that adhered to the coverslip at the bottom of the cell chamber had the morphological characteristics of cardiac myocytes. Second, cell density in the chamber was very low, ensuring considerable dilution of any paracrine factor(s) that are released from contaminating nonmyocyte cells. Finally, the myocytes under study were continuously superfused at 3.5 mL/min (equivalent to a complete change of the chamber volume every 2 seconds), thereby ensuring that any paracrine factor(s) released into the superfusion medium would be rapidly removed. Thus, it is highly likely that, in the present study, angiotensin receptor–mediated regulation of sarcolemmal NHE activity was mediated through the interaction of angiotensin II with its receptors on the myocytes themselves.
Although this is the first report of opposing actions of angiotensin receptor subtypes in adult cardiac myocytes, analogous observations have been reported previously with regard to angiotensin II–mediated regulation of several physiological processes of cardiovascular relevance. The first such reports showed that AT2 mediates an antiproliferative effect and counteracts the growth-promoting action of AT1 stimulation in vascular smooth muscle cells18 and coronary endothelial cells.14 More recently, evidence has been obtained that AT2 stimulation opposes AT1-mediated hypertrophic effects in neonatal rat ventricular myocytes,16 induction of new protein synthesis in adult rat hearts,36 and chronotropic effects in adult mouse hearts.37 Further information on the roles of AT2 in cardiovascular regulation will undoubtedly arise from studies in transgenic mice, in which the AT2 gene has been either disrupted38 39 or overexpressed,37 which have already revealed that AT2 attenuates AT1-mediated pressor responses to angiotensin II.37 38 39 Taken together, these findings (1) challenge the notion that AT1 is the primary mediator of the cardiovascular actions of angiotensin II (see also recent review by Matsubara40 ) and (2) illustrate that cardiovascular responses to angiotensin II may vary significantly depending on the relative availability of AT1 and AT2. Indeed, in the present study, angiotensin II in the presence of an equimolar concentration of PD123319 increased sarcolemmal NHE activity in adult myocytes but failed to do so in neonatal myocytes. This may reflect the greater density of AT2 in neonatal myocardium,10 although roles for developmental changes in other pertinent signaling mechanisms cannot be excluded.
In the present study, we have also investigated the mechanisms through which the opposing actions of AT1 versus AT2 on sarcolemmal NHE activity in adult ventricular myocytes may be mediated. The obvious possibility that AT2-mediated pathways may directly inhibit sarcolemmal NHE activity, thereby resulting in functional antagonism of the AT1-mediated response, was excluded on the basis that selective AT2 stimulation was without effect on exchanger activity. This finding indicates that the ability of AT2 stimulation to oppose NHE activation via AT1 is likely to be mediated by inhibition of the signaling pathway(s) that mediate the AT1 response. Although NHE1 is subject to regulation by multiple signaling pathways in various cell types (see recent reviews in References 41 and 4241 42 ), we elected to concentrate initially on the role of the ERK pathway, on the basis of several recent findings. First, work in cultured fibroblasts has shown that the ERK pathway is a critical mediator of NHE1 activation by exogenous stimuli such as thrombin,19 which we have shown to increase sarcolemmal NHE activity in adult rat ventricular myocytes.6 Second, AT1 stimulation has been shown to induce ERK activation in cultured neonatal ventricular myocytes,32 as well as in other cell types.17 43 Finally, AT2 stimulation appears to oppose AT1-mediated ERK activation in various cell types in culture, such as vascular smooth muscle cells18 and brain neuronal cells.17
In our studies, (1) PD98059, a pharmacological inhibitor of the ERK pathway, inhibited the AT1-mediated increase in sarcolemmal NHE activity; (2) selective AT1 stimulation, which increased sarcolemmal NHE activity, also produced increases in ERK1/ERK2 phosphorylation and activity; and (3) losartan and PD98059, which inhibited the AT1-mediated increase in sarcolemmal NHE activity, also inhibited the AT1-mediated increases in ERK1/ERK2 phosphorylation and activity. Such findings strongly suggest that ERK activation is mechanistically involved in AT1-mediated stimulation of sarcolemmal NHE activity in adult rat ventricular myocytes. Furthermore, our observation in adult myocytes that simultaneous AT1 and AT2 stimulation produced an increase in myocyte ERK1/ERK2 phosphorylation and activity similar to that of selective AT1 stimulation suggests that (1) AT2-mediated inhibition of the AT1-mediated increase in sarcolemmal NHE activity in this cell type does not arise from an attenuation of ERK activation and (2) ERK activation is not sufficient to increase sarcolemmal NHE activity. The latter conclusion is supported also by our observations in neonatal ventricular myocytes, in which angiotensin II induced significant increases in ERK1/ERK2 phosphorylation without parallel increases in sarcolemmal NHE activity, even in the presence of up to 100 nmol/L PD123319. It appears, therefore, that, although ERK activation is necessary for AT1-mediated stimulation of sarcolemmal NHE activity, it is not the sole signaling mechanism that mediates this response.
In the broader context of angiotensin receptor–mediated regulation of myocardial ERK activity, it is important to note that our common finding in adult and neonatal rat ventricular myocytes, that simultaneous AT1 and AT2 stimulation and selective AT1 stimulation both produce a similar activation of the ERK pathway, is consistent with earlier data. Thus, Masaki et al37 and Sadoshima et al32 have previously shown that, in wild-type adult mouse hearts and in neonatal rat ventricular myocytes, respectively, angiotensin II alone produces a significant increase in ERK1/ERK2 activity that is comparable in magnitude with that produced by the combination of angiotensin II and PD123319.
In the present study, we have also investigated the potential roles of PKC and the EGF receptor as upstream mediators of the effects of AT1 stimulation on ERK and NHE activity in adult rat ventricular myocytes. Our observation that the PKC inhibitor GF109203X and the EGF receptor kinase inhibitor tyrphostin AG1478 could each inhibit AT1-mediated activation of both ERK and NHE suggests that, in this cell type, PKC activation and EGF receptor transactivation are both necessary to achieve such responses. Notably, analogous findings with regard to the regulation of myocyte ERK activity by a different stimulus have recently been reported by Seko et al,44 who showed that activation of the ERK pathway by pulsatile stretch in neonatal myocytes could be blocked equally effectively by the PKC inhibitor calphostin C and by the tyrosine kinase inhibitor genistein. One possible mechanism that might explain our data is a requirement for PKC activity for AT1-mediated transactivation of the EGF receptor. Such a requirement has been shown to exist in HEK293 cells for transactivation of the EGF receptor by another member of the Gq protein–coupled receptor family, namely, the m1 muscarinic receptor.45 Determination of the role of PKC in any AT1-mediated transactivation of the EGF receptor in adult rat ventricular myocytes requires further study.
Our present results do not allow delineation of the mechanism(s) through which AT2 stimulation opposes the AT1-mediated increase in sarcolemmal NHE activity. One potential mechanism is AT2-mediated inhibition of exchanger-stimulatory AT1 signaling downstream from ERK1/ERK2 activation. In this regard, although ERK1/ERK2 can phosphorylate in vitro a fusion protein that contains the carboxyl-terminal region of NHE1,46 the work of Bianchini et al19 suggests that ERK-mediated stimulation of NHE1 activity in intact cells is not by direct phosphorylation but most likely involves intermediary proteins. A candidate NHE-regulatory intermediary protein, the regulation of which by AT1 and AT2 in adult rat ventricular myocytes warrants investigation, is the 90-kDa ribosomal S6 kinase, given that this enzyme lies downstream of ERK1/ERK2 in vascular smooth muscle cells and has been proposed as a putative NHE1 kinase.47 Another potential mechanism that is consistent with our findings is an AT2-mediated activation of distinct signaling pathways that do not affect basal NHE activity but oppose ERK1/ERK2-mediated stimulation of the exchanger. Potential such pathways include those mediated via p38 kinase,48 protein kinase D,49 and a calcineurin homologous protein,50 given that these pathways have been proposed to have negative NHE-regulatory roles in other cell types.
It is important to consider briefly the potential physiological/pathophysiological and therapeutic implications of the novel opposing actions of AT1 and AT2 reported here. First, our findings may help explain the relative inefficacy of angiotensin II in increasing sarcolemmal NHE activity (eg, see Matsui et al9 versus Yokoyama et al4 ) and enhancing myocardial inotropic status.8 They may also have relevance to the opposing effects of AT1 versus AT2 in the induction of myocyte hypertrophy,16 given that upregulation of NHE has recently been associated with hypertrophy in both in vivo51 and in vitro52 53 models. Finally, in considering the potential therapeutic implications of our findings, it should be noted that sarcolemmal NHE activity is believed to be a critical determinant of the severity of cardiac injury and dysfunction, including the induction of ventricular fibrillation, in myocardial ischemia and reperfusion (for a recent review, see Avkiran54 ). On the basis of the present data, it is reasonable to speculate that selective antagonists of AT1 may possess cardioprotective efficacy. This may arise from inhibition of sarcolemmal NHE activation during ischemic episodes, not only by angiotensin II itself (through blockade of positive-regulatory AT1) but also by other neurohormonal stimuli (through enhanced stimulation of negative-regulatory AT2), particularly given that treatment with AT1 antagonists appears to increase the plasma angiotensin II concentration.55 Indeed, in view of the evidence that the AT2:AT1 ratio is increased in the failing human heart,12 13 it is possible that such a cardioprotective mechanism may have contributed to the reduction in sudden cardiac death that has recently been reported in heart failure patients treated with losartan.56
In conclusion, the present study has shown, for the first time, that AT1 stimulation increases sarcolemmal NHE activity in adult rat ventricular myocytes and that this effect is opposed via AT2-mediated pathways. Our work has also revealed that (1) ERK activation is necessary but not sufficient for AT1-mediated NHE activation, (2) activation of ERK and NHE by AT1 stimulation both occur via a PKC- and EGF receptor–mediated pathway, and (3) the AT2-mediated inhibitory effect on AT1-mediated stimulation of NHE activity does not occur via direct NHE inhibition or an attenuation of ERK activation. In view of the potential impact that changes in sarcolemmal NHE activity have on cardiac function in health and disease, further investigation of the molecular signaling mechanisms that mediate such regulation of the exchanger by angiotensin receptors appears warranted.
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Acknowledgments |
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Part of this work was supported by a grant from the Merck Medical School Grants Committee. S.G. was the recipient of a Prize Studentship from the Wellcome Trust (045435/Z/95/Z). R.S.H. is supported by a project grant (PG/98197), and M.A. holds a Senior Lectureship Award (BS/93002) from the British Heart Foundation.
Received March 3, 1999; accepted August 30, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Fliegel L, Dyck JRB. Molecular biology of the cardiac sodium/hydrogen exchanger. Cardiovasc Res. 1995;29:155–159.[Medline] [Order article via Infotrieve]
2.
Karmazyn M, Moffat MP. Role of
Na+/H+ exchange in cardiac
physiology and pathophysiology: mediation of myocardial reperfusion
injury by the pH paradox. Cardiovasc Res. 1993;27:915–924.
3. Pucéat M, Vassort G. Neurohumoral modulation of intracellular pH in the heart. Cardiovasc Res. 1995;29:178–183.[Medline] [Order article via Infotrieve]
4.
Yokoyama H, Yasutake M, Avkiran M.
1-Adrenergic stimulation of sarcolemmal
Na+/H+ exchanger activity
in rat ventricular myocytes: evidence for selective
mediation by the 1A-adrenoceptor subtype.
Circ Res. 1998;81:1078–1085.
5.
Krämer BK, Smith TW, Kelly RA. Endothelin and
increased contractility in adult rat
ventricular myocytes: role of intracellular alkalosis
induced by activation of the protein kinase C-dependent
Na+-H+ exchanger.
Circ Res. 1991;68:269–279.
6.
Yasutake M, Haworth RS, King A, Avkiran M. Thrombin
activates the sarcolemmal
Na+/H+ exchanger: evidence
for a receptor-mediated mechanism involving protein kinase C.
Circ Res. 1996;79:705–715.
7.
Noel J, Pouyssegur J. Hormonal regulation,
pharmacology, and membrane sorting of vertebrate
Na+/H+ exchanger isoforms.
Am J Physiol. 1995;268:C283–C296.
8. Ito N, Kagaya Y, Weinberg EO, Barry WH, Lorell BH. Endothelin and angiotensin II stimulation of Na+/H+ exchange is impaired in cardiac hypertrophy. J Clin Invest. 1997;99:125–135.[Medline] [Order article via Infotrieve]
9. Matsui H, Barry WH, Livsey CV, Spitzer KW. Angiotensin II stimulates sodium-hydrogen exchange in adult rabbit ventricular myocytes. Cardiovasc Res. 1995;29:215–221.[Medline] [Order article via Infotrieve]
10.
Suzuki J, Matsubara H, Urakami M, Inada M. Rat
angiotensin II (type IA) receptor mRNA regulation and
subtype expression in myocardial growth and hypertrophy.
Circ Res. 1993;73:439–447.
11.
Baker KM, Campanile CP, Trachte GJ, Peach MJ.
Identification and characterization of the rabbit
angiotensin II myocardial receptor. Circ Res. 1984;54:286–293.
12.
Haywood GA, Gullestad L, Katsuya T, Hutchinson HG,
Pratt RE, Horiuchi M, Fowler MB. AT1 and
AT2 angiotensin receptor gene
expression in human heart failure. Circulation. 1997;95:1201–1206.
13.
Asano K, Dutcher DL, Port D, Minobe WA, Tremmel KD,
Roden RL, Bohlmeyer TJ, Bush EW, Jenkin MJ, Abraham WT, Raynolds MV,
Zisman LS, Perryman MB, Bristow MR. Selective downregulation of the
angiotensin II AT1-receptor subtype
in failing human ventricular myocardium.
Circulation. 1997;95:1193–1200.
14. Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651–657.
15.
Yamada T, Horiuchi M, Dzau VJ. Angiotensin
II type 2 receptor mediates programmed cell death. Proc Natl Acad
Sci U S A. 1996;93:156–160.
16.
Booz GW, Baker KM. Role of type 1 and type 2
angiotensin receptors in angiotensin II-induced
cardiomyocyte hypertrophy.
Hypertension. 1996;28:635–640.
17.
Huang X-C, Richards EM, Sumner 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. 1996;271:15635–15641.
18.
Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W,
Morishita R, Zhang L, Horiuchi M, Pratt RE, Dzau VJ. The
angiotensin II type 2 (AT2) receptor
antagonizes the growth effects of the AT1
receptor: gain-of-function study using gene transfer. Proc Natl
Acad Sci U S A. 1995;92:10663–10667.
19.
Bianchini L, L’Allemain G, Pouyssegur 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. 1997;272:271–279.
20. Haworth RS, Yasutake M, Brooks G, Avkiran M. Cardiac Na+/H+ exchanger during postnatal development in the rat: changes in mRNA expression and sarcolemmal activity. J Mol Cell Cardiol. 1997;29:321–332.[Medline] [Order article via Infotrieve]
21.
Shipolini AR, Yokoyama H, Galinanes M, Edmondson SJ,
Hearse DJ, Avkiran M.
Na+/H+ exchanger activity
does not contribute to protection by ischemic preconditioning
in the isolated rat heart. Circulation. 1997;96:3617–3625.
22.
Ozono R, Wang Z-Q, Moore AF, Inagami T, Siragy HM,
Carey RM. Expression of the subtype 2 angiotensin
(AT2) receptor protein in rat kidney.
Hypertension. 1997;30:1238–1246.
23.
Wang Z-Q, Moore AF, Ozono R, Siragy HM, Carey RM.
Immunolocalization of subtype 2 angiotensin II (AT2)
receptor protein in rat heart. Hypertension. 1998;32:78–83.
24.
Fareh J, Touyz RM, Schiffrin EL, Thibault G. Cardiac
type-1 angiotensin II receptor status in
deoxycorticosterone acetate-salt hypertension in rats.
Hypertension. 1997;30:1253–1259.
25.
Meggs LG, Coupet J, Huang H, Cheng W, Li P, Capasso JM,
Homcy CJ, Anversa P. Regulation of angiotensin II receptors
on ventricular myocytes after myocardial infarction in
rats. Circ Res. 1993;72:1149–1162.
26.
Lopez JJ, Lorell BH, Ingelfinger JR, Weinberg EO,
Schunkert H, Diamant D, Tang S. Distribution and function of cardiac
angiotensin AT1- and AT2- receptor subtypes in
hypertrophied rat hearts. Am J Physiol. 1994;267:H844–H852.
27.
Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A
synthetic inhibitor of the mitogen-activated
protein kinase cascade. Proc Natl Acad Sci U S A. 1995;92:7686–7689.
28.
Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD
098059 is a specific inhibitor of the activation of
mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem. 1995;270:27489–27494.
29.
Zou Y, Komuro I, Yamazaki T, Aikawa R, Kudoh S,
Shiojima I, Hiroi Y, Mizuno T, Yazaki Y. Protein kinase C, but not
tyrosine kinases or Ras, plays a critical role in
angiotensin II-induced activation of REaf-1 kinase and
extracellular signal-regulated protein kinases in cardiac myocytes.
J Biol Chem. 1996;271:33592–33597.
30.
Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa
T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F,
Inagami T. Calcium-dependent epidermal growth factor receptor
transactivation mediates the angiotensin II-induced
mitogen-activated protein kinase activation in vascular smooth
muscle cells. J Biol Chem. 1998;273:8890–8896.
31.
Murasawa S, Mori Y, Nozawa Y, Gotoh N, Shibuya M,
Masaki H, Maruyama K, Tsutsumi Y, Moriguchi Y, Shibazaki Y, Tanaka Y,
Iwasaka T, Inada M, Matsubara H. Angiotensin II type 1
receptor-induced extracellular signal-regulated protein kinase
activation is mediated by
Ca2+/calmodulin-dependent
transactivation of epidermal growth factor receptor. Circ
Res. 1998;82:1338–1348.
32.
Sadoshima J, Qiu Z, Morgan JP, 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
Ca++-dependent signaling. Circ Res. 1995;76:1–15.
33.
Ye M, Flores G, Battle D. Angiotensin II
and angiotensin-(1–7) effects on free cytosolic sodium,
intracellular pH, and the
Na+-H+ antiporter in
vascular smooth muscle. Hypertension. 1996;27:72–78.
34.
Fareh J, Touyz RM, Schiffrin EL, Thibault G.
Endothelin-1 and angiotensin II receptors in cells from
hypertrophied heart: receptor regulation and intracellular
Ca2+ modulation. Circ Res. 1996;78:302–311.
35.
Kijima K, Matsubara H, Murasawa S, Maruyama K, Mori Y,
Ohkubo N, Komuro I, Yazaki Y, Iwasaka T, Inada M. Mechanical stretch
induces enhanced expression of angiotensin II receptor
subtypes in neonatal rat cardiac myocytes. Circ Res. 1996;79:887–897.
36.
Bartunek J, Weinberg EO, Tajima M, Rohrbach S, Lorell
BH. Angiotensin II type 2 receptor blockade amplifies the
early signals of cardiac growth response to angiotensin II
in hypertrophied hearts. Circulation. 1999;99:22–25.
37. Masaki H, Kurihara T, Yamaki A, Inomata N, Nozawa Y, Mori Y, Murasawa S, Kizima K, Maruyama K, Horiuchi M, Dzau VJ, Takahashi H, Iwasaka T, Inada M, Matsubara H. Cardiac-specific overexpression of angiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects. J Biol Chem. 1998;101:527–535.
38. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan BLM, Inagama T. Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature. 1995;377:748–750.[Medline] [Order article via Infotrieve]
39. Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK. Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor gene in mice. Nature. 1995;377:744–747.[Medline] [Order article via Infotrieve]
40.
Matsubara H. Pathophysiological
role of angiotensin II type 2 receptor in
cardiovascular and renal diseases. Circ Res. 1998;83:1182–1191.
41.
Orlowski J, Grinstein S.
Na+/H+ exchangers of
mammalian cells. J Biol Chem. 1997;272:22373–22376.
42.
Wakabayashi S, Shigekawa M, Pouyssegur J. Molecular
physiology of vertebrate
Na+/H+ exchangers.
Physiol Rev. 1997;77:51–74.
43.
Liao D-F, Duff JL, Daum G, Pelech SL, Berk BC.
Angiotensin II stimulates MAP kinase kinase activity in
vascular smooth muscle cells: role of Raf. Circ Res. 1996;79:1007–1014.
44. Seko Y, Takahashi N, Tobe K, Kadowaki T, Yazaki Y. Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes. Biochem Biophys Res Commun. 1999;259:8–14.[Medline] [Order article via Infotrieve]
45. Tsai W, Morielli AD, Peralta EG. The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity. EMBO J. 1997;16:4597–4605.[Medline] [Order article via Infotrieve]
46. Wang H, Silva NLCL, Lucchesi PA, Haworth RS, Wang K, Michalak M, Pelech S, Fliegel L. Phosphorylation and regulation of the Na+/H+ exchanger through mitogen-activated protein kinase. Biochemistry. 1997;36:9151–9158.[Medline] [Order article via Infotrieve]
47.
Takahashi E, Abe J, Berk BC. Angiotensin II
stimulates p90rsk in vascular smooth muscle
cells: a potential Na+-H+
exchanger kinase. Circ Res. 1997;81:268–273.
48.
Kusuhara M, Takahashi E, Peterson TE, Abe J, Ishida M,
Han J, Ulevitch R, Berk BC. p38 kinase is a negative regulator of
angiotensin II signal transduction in vascular smooth
muscle cells: effects on
Na+/H+ exchange and ERK1/2.
Circ Res. 1998;83:824–831.
49. Haworth RS, Sinnett-Smith J, Rozengurt E, Avkiran M. Protein kinase D inhibits plasma membrane Na+/H+ exchanger activity. Am J Physiol. In press.
50.
Lin X, Barber DL. A calcineurin homologous protein
inhibits GTPase-stimulated Na-H exchange. Proc Natl Acad Sci
U S A. 1996;93:12631–12636.
51. Takewaki S, Kuro-o M, Hiroi Y, Yamazaki T, Noguchi T, Miyagishi A, Nakahara K, Aikawa M, Manabe I, Yazaki Y, Nagai R. Activation of Na+-H+ antiporter (NHE-1) gene expression during growth, hypertrophy and proliferation of the rabbit cardiovascular system. J Mol Cell Cardiol. 1995;27:729–742.[Medline] [Order article via Infotrieve]
52.
Yamazaki T, Komuro I, Kudoh S, Zou Y, Nagai R, Aikawa
R, Uozumi H, Yazaki Y. Role of ion channels and exchangers in
mechanical stretch-induced cardiomyocyte
hypertrophy. Circ Res. 1998;82:430–437.
53.
Schluter K-D, Schafer M, Balser C, Taimor G, Piper HM.
Influence of pHi and creatine phosphate on
-adrenoceptor-mediated cardiac hypertrophy. J Mol
Cell Cardiol. 1998;30:763–771.[Medline]
[Order article via Infotrieve]
54. Avkiran M. Rational basis for use of Na+/H+ exchange inhibitors in myocardial ischemia. Am J Cardiol. 1999;83:10G–18G.[Medline] [Order article via Infotrieve]
55.
Christen Y, Waeber B, Nussberger J, Porchet M, Borland
RM, Lee RJ, Maggon K, Shum L, Timmermans P, Brunner HR. Oral
administration of DUP-753, a specific angiotensin II
receptor antagonist, to normal male volunteers: Inhibition
of pressor response to exogenous angiotensin I and
angiotensin II. Circulation. 1991;83:1333–1342.
56. Pitt R, Segal R, Martinez FA, Meurers G, Cowley AJ, Thomas I, Deedwania PC, Ney DE, Snavely DB, Chang PI. Randomised trial of losartan versus captopril in patients over 65 with heart failure (Evaluation of Losartan in the Elderly Study, ELITE). Lancet. 1997;349:747–752.[Medline] [Order article via Infotrieve]
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 A. K. Snabaitis, H. Yokoyama, and M. Avkiran Roles of Mitogen-Activated Protein Kinases and Protein Kinase C in {alpha}1A-Adrenoceptor-Mediated Stimulation of the Sarcolemmal Na+-H+ Exchanger Circ. Res., February 4, 2000; 86(2): 214 - 220. [Abstract] [Full Text] [PDF]
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