| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Physiologisches Institut, Justus Liebig Universität Giessen, D-35392 Giessen, Germany
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
We investigated
the question of whether inhibition of the
Na+/H+ exchanger (NHE) during ischemia is
protective due to reduction of cytosolic Ca2+ accumulation
or enhanced acidosis in cardiomyocytes. Additionally, the role of the
Na+-HCO3
symporter (NBS) was
investigated. Adult rat cardiomyocytes were exposed to simulated
ischemia and reoxygenation. Cytosolic pH [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)], Ca2+ (fura 2), Na+ [sodium-binding
benzolfuran isophthatlate (SBFI)], and cell length were
measured. NHE was inhibited with 3 µmol/l HOE 642 or 1 µmol/l 5-(N-ethyl-N-isopropyl)-amiloride
(EIPA), and NBS was inhibited with HEPES buffer. During anoxia
in bicarbonate buffer, cells developed acidosis and intracellular Na
and Ca (Nai and Cai, respectively) overload.
During reoxygenation cells underwent hypercontracture (44.0 ± 4.1% of the preanoxic length). During anoxia in bicarbonate buffer,
inhibition of NHE had no effect on changes in intracellular pH
(pHi), Nai, and Cai, but it
significantly reduced the reoxygenation-induced hypercontracture (HOE:
61.0 ± 1.4%, EIPA: 68.2 ± 1.8%). The sole inhibition of
NBS during anoxia was not protective. We conclude that inhibition of
NHE during anoxia protects cardiomyocytes against reoxygenation injury
independently of cytosolic acidification and Cai overload.
hypercontracture; pH control; calcium; sodium-hydrogen exchanger
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
IT HAS BEEN SHOWN
that the ischemic-reperfused myocardium can be protected when the
Na+/H+ exchanger (NHE) is inhibited (27,
31). Pronounced protection is achieved, however, only if the
inhibitor of NHE is applied before or with the onset of ischemia
(7, 15, 17, 25). The mechanism of this protection is still
unknown, but the following hypotheses have been put forward.
First, inhibition of NHE reduces cytosolic Na+ accumulation
(2) and subsequent Ca2+ overload, which may
result from reduced activity of the Na+/Ca2+
exchanger (NCE) in forward mode operation or from inhibition of its
reverse mode operation (37). Second, inhibition of NHE enhances
acidification of the ischemic myocardial cell. Finally, reduced
Ca2+ overload and enhanced acidification is protective for
the myocardial cell in ischemia-reperfusion (20, 21, 34).
The present study was undertaken to test these hypotheses at the
cellular level. Additionally, we investigated whether the
protective effect of NHE inhibition can be reproduced or enhanced by
inhibition of another Na+-dependent H+
extrusion system: the Na+/HCO3
symporter (NBS). The role of NBS in the pathophysiology of
ischemic myocardium (22) is still poorly understood, and
it is unknown if sole inhibition of NBS can be as protective as sole
inhibition of NHE.
As an experimental model we used isolated ventricular cardiomyocytes from the adult rat heart, which were exposed to a sequence of anoxia at extracellular pH (pHo) 6.4 (simulated ischemia) and reoxygenation at pHo 7.4 (simulated reperfusion). This model has been characterized in previous studies (33, 35). It was investigated, first, if the presence during simulated ischemic conditions of one of the chemically distinct NHE inhibitors HOE 642 (32) or 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) alters the development of intracellular acidosis, Na+, or Ca2+ overload. Second, the results were compared for circumstances permitting bicarbonate-dependent sarcolemmal H+ transport and for circumstances that do not, i.e., the role of NBS was analyzed. Third, effects of intraischemic NHE inhibition on ischemic cells were differentiated from its effects on reoxygenated cells, specifically on reoxygenation-induced hypercontracture. The last point of this analysis is based on previous investigations on this model, which demonstrated that reoxygenation-induced hypercontracture is elicited by the coincidence of Ca2+ overload, accumulated during ischemic conditions, and recovery of energy production within the reoxygenated cardiomyocyte (34).
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Isolation of cardiomyocytes. Ventricular heart muscle cells were isolated from 200- to 250-g adult male Wistar rats and plated in medium 199 with 4% fetal calf serum on glass coverslips that had been preincubated overnight with 4% fetal calf serum (28, 29). Four hours after plating the coverslips, the coverslips were washed with medium 199. As a result of the wash, damaged cells were removed, leaving a homogeneous population of rod-shaped quiescent cardiomyocytes (>95%) attached to the coverslip. From each isolation, two to three coverslips were used. On each coverslip, four to six cells were investigated. Only cells exhibiting a rod-shaped morphology and no signs of sarcolemmal blebbing were used for the experiments. These cells were found to have a low resting free cytosolic Ca2+ concentration ([Ca2+]i).
Ca2+, Na+, Mg2+, pH, and cell length measurements. To measure Ca2+, Na+, Mg2+, or H+ concentrations, cardiomyocytes were loaded in medium 199 at 35°C for 30 min with acetoxymethyl esters of fura 2 (2.5 µmol/l), Mg-fura 2 (1.5 µmol/l), sodium-binding benzolfuran isophthatlate (SBFI, 10 µmol/l), or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF, 1.5 µmol/l). After loading, cells were washed twice with medium 199 followed by incubation in medium 199 for 30 min to allow hydrolysis of the acetoxymethyl esters within the cell. The fluorescence from dye-loaded cells was 20-30 times higher than background fluorescence from unloaded cells.
The coverslip with loaded cells was introduced into a gas-tight, temperature-controlled (37°C), transparent perfusion chamber positioned in the light path of an inverted microscope (Diaphot TMD, Nikon, Düsseldorf, Germany). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura 2 and SBFI, 337 and 375 nm for Mg-fura 2, or 440 and 490 nm for BCECF was performed with an AR-Cation Measurement System adapted to the microscope (Spex Industries, Grasbrunn, Germany). Emitted light (490-510 nm for fura 2, SBFI; e.g., Mg-fura 2 and 520-560 nm for BCECF) from a 10 × 10-mm area within a single fluorescent cell was collected by the photomultiplier of the Spex system. The light signal was recorded and analyzed by an IBM PC/AT-based data analysis system (model DM3000CM, Spex Industries). Simultaneously to the measurement of the fluorescence, the microscopic image of the cell was recorded with a video camera and stored on tape. From these recordings, changes of cell length were determined. In the case of a hypercontracted cell, the dimension of the cell along its previous longitudinal axis was determined. The loading protocols used were selected from a number of variations because they provided the highest yield in fluorescence and minimal dye compartmentation. To assess the extent of intracellular dye compartmentation, cells were chemically "skinned" with digitonin as described previously (4). This test showed that the fluorescent signal from intracellular stores did not exceed 10% for fura 2, 15% for SBFI, 15% for Mg-fura 2, and 12% for BCECF compared with the signal from whole cells. Furthermore, the extent of dye compartmentation did not differ significantly between control cells and cells after anoxia and reoxygenation. For the purpose of the present study, therefore, correction of the data for this small extent of dye compartmentation seemed unnecessary. Because of the saturation limit of fura 2, Ca2+ data were usually expressed in arbitrary units of fluorescence ratios of the emitted light of the two corresponding excitation wavelengths. To facilitate understanding, calibration protocols were performed to obtain numerical relationships between selected ratio values and ion concentrations. The fura 2 signal was calibrated according to the method described by Li et al. (23). For this purpose, the cells were exposed to 5 µmol/l ionomycin in modified Tyrode solution (pH 7.4; for composition see Media) containing either 3 mmol/l Ca2+ or 5 mmol/l EGTA to obtain the maximum (Rmax) and the minimum (Rmin) ratio of fluorescence, respectively. To prevent morphological alterations during calibration, cells were depleted of ATP with 1 mmol/l KCN. The [Ca2+]i was calculated according to Grynkiewicz et al. (13) with the use of pH-dependent dissociation constant (Kd) values for fura 2, determined in intact cardiomyocytes by constructing calibration curves. Calibrations under protocols 1-3 were performed separately for intracellular pH (pHi) of normoxic control cells and for the end-anoxic pHi of anoxic cells. Calibrations for pHi
6, as occurred at the end of anoxia
under protocol 4, are not possible because the fura 2 signal
loses Ca2+ sensitivity at such low pH values. Calibration
of the BCECF ratio signal was performed as previously described
(19), with 10 µg/ml nigericin, a
K+-H+ ionophore, and incubation media with
various pH values. SBFI ratio was calibrated according to Harootunian
et al. (14), with 6 µmol/l gramicidin D and incubation
in media containing various Na+ concentrations and pH values.
Media.
The perfusion chamber (1 ml filling volume) placed on the microscope
stage was perfused at a flow rate of 0.5 ml/min with modified,
glucose-free normoxic bicarbonate-buffered solution at 37°C
containing (in mmol/l) 118 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, and 22.0 NaHCO3; the medium was gassed
with 5% CO2-95% O2, and the resulting pH was
7.4. In the anoxic medium the bicarbonate concentration was reduced to
2.2 mmol/l, resulting in a pH of 6.4 when medium was gassed with 5%
CO2-95% N2. NaCl concentration of the anoxic
medium was elevated to 137.8 mmol/l to equalize Na+
concentrations of anoxic and normoxic media. Medium was made anoxic by
autoclaving as described previously (1). To exclude the
function of the HCO3-dependent, pH-regulating
mechanism, a bicarbonate-free, HEPES-buffered medium was used. The
HEPES-buffered solution contained (in mmol/l) 140 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, and 25 HEPES; pH was adjusted at 7.4. For anoxic
incubations, pH was adjusted at 6.4, and media were equilibrated with
100% N2. The pH of normoxic medium was adjusted to 7.4 and
equilibrated with air. For the ammonium-prepulse experiments, we added
10 mM NH4Cl to the normoxic HEPES-buffered solution.
Simulated ischemia-reoxygenation. Four sets of experiments were used. In all of them, the anoxic period was 80 min at pHo 6.4 and 10 min reoxygenation at pHo 7.4 at 37°C. Under control conditions (protocol 1), cardiomyocytes were perfused with bicarbonate-buffered medium during simulated ischemia and reoxygenation. Under protocol 2, NHE was inhibited only during anoxia with 3 µmol/l HOE 642, which was washed out with the onset of reoxygenation. Under protocols 3 and 4, cells were superfused with HEPES-buffered medium, to exclude the NBS. Under protocol 4, NHE was additionally inhibited only during anoxia with 3 µmol/l HOE 642. To test the decline of the pHi in cardiomyocytes under normoxic conditions, we changed medium pH from 7.4 to 6.4 and perfused the normoxic cells for about 80 min with medium pH 6.4.
To exclude the protective side effects of HOE 642 independently from NHE inhibition, additional experiments following protocol 2 were performed with EIPA (1 µmol/l).Materials. Medium 199 was purchased from Boehringer Mannheim, fetal calf serum was from GIBCO, acetoxymethyl esters of fura 2 and BCECF were from Paesel and Lorey, EIPA was from RBI, and HOE 642 was a gift from Dr. H. J. Lang from Hoechst AG. All other chemicals were from Merck or Sigma and of the highest purity available.
Statistics. Data are given as mean values ± SE from n individual cells investigated in separate experiments. Statistical comparisons were performed by one-way ANOVA and the Student-Newman-Keuls test for post hoc analysis. When only two experimental groups had to be compared, Student's t-test was applied. Differences with P < 0.05 were regarded as statistically significant.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Effect of HOE 642 and EIPA on pHi recovery after an
ammonium prepulse.
It was first tested whether 3 µmol/l HOE 642, described as a blocking
dose of the NHE inhibitor (30), had indeed such an effect
on the cardiomyocytes. An ammonium-prepulse test in normoxic HEPES
buffer was used. Cardiomyocytes were superfused for 5 min with 10 mM
NH4+ in HEPES buffer (pH 7.4) with following washout of
NH4+ for another 15 min (Table
1). This creates cytosolic acidification from which the cells spontaneously recover. Treatment of the cells with
3 µmol/l HOE 642 only during the washout period completely abolished
recovery from acidosis, indicating that this dosage blocks the NHE.
These experiments were repeated with 1 µmol/l EIPA. The data show
(Table 1) that 1 µmol/l EIPA is also sufficient to abolish
completely the pHi recovery after the ammonium prepulse. Therefore, HOE 642 and EIPA were administrated during anoxia at these
tested concentrations.
|
Effect of HOE 642 on pHi during simulated ischemia and
reperfusion in HEPES and bicarbonate-buffered medium.
Under normoxic conditions at pHo of 7.4, the
pHi of cells submitted subsequently to protocol
1 was 7.09 ± 0.03 (n = 22), for protocol 2 it was 7.11 ± 0.04 (n = 28), for protocol 3 it was 7.16 ± 0.02 (n = 25), and for protocol 4 it was
7.15 ± 0.03 (n = 25). When the cells were exposed
to 80 min of anoxia in medium with pH 6.4 in bicarbonate-buffered
medium (protocol 1), pHi declined to 6.46 ± 0.04 (n = 22) (Fig.
1). Addition of 3 µmol/l HOE 642 to the
anoxic bicarbonate-buffered medium (protocol 2) had no significant effect on the development of intracellular acidosis. After
80 min of anoxia, pHi in these cells was 6.38 ± 0.04 (n = 28; not significant vs. protocol 1).
When anoxia was performed in HEPES-buffered medium (protocol
3), pHi declined to 6.45 ± 0.03;
(n = 25; not significant vs. protocol 1).
When HOE 642 was present in anoxic, HEPES-buffered medium
(protocol 4), the cytosolic acidification was markedly
enhanced. After 80 min of anoxia, pHi was 5.44 ± 0.07 (n = 25; P < 0.05 vs. protocols
1-3). Superfusion with bicarbonate-buffered normoxic medium
at pH 6.4 produced intracellular acidification of the cardiomyocytes
but not as strongly as under anoxic conditions. During 80 min of
normoxia at pH 6.4, the pHi of the cardiomyocytes decreased
from 7.05 ± 0.01 (n = 20) to 6.79 ± 0.02 (n = 20).
|
Time of rigor contracture and cytosolic
Mg2+ concentration.
After extensive loss of high-energy phosphates, cardiomyocytes develop
rigor contracture (5), causing shortening of the cells by
one-third of their resting length within 1 min. Under control
conditions in the present study, cells developed rigor contracture
after 36.5 ± 5.9 min (n = 38) of anoxia.
Inhibition during anoxia of NHE or NBS or both did not influence the
onset of rigor contracture (Table 2).
|
|
Changes in [Ca2+]i
during anoxia and reoxygenation.
The ratio of fura 2 fluorescence was monitored to evaluate changes in
[Ca2+]i during simulated ischemia
and reoxygenation. According to the calibration protocol, the initial
ratio of 0.4 (arbitrary units) in normoxic cells corresponds to a
[Ca2+]i of 72 nmol/l. During anoxia at
pHo 6.4 (protocol 1), the cells underwent rigor
shortening followed by a progressive rise of the fura 2 ratio,
indicating accumulation of Ca2+ in the cytosol. After 80 min the fura 2 ratio reached a value of 2.16 ± 0.09 (arbitrary
units) (n = 42), which corresponds to a
[Ca2+]i of 1.9 µmol/l. This level
represents severe Ca2+ overload. When cells were
reoxygenated in medium with pH 7.4, the fura 2 ratio declined to the
initial control value within 10 min (Fig.
3).
|
Effect of inhibition of NHE on reoxygenation-induced
hypercontracture.
During anoxia, the cell length was reduced by about one-third of the
initial length due to rigor shortening. This rigor shortening was
similar under all experimental conditions; an average cell retained
70.0 ± 1.3% of their initial length. Reoxygenation of cells
after anoxia led to additional, irreversible reduction of cell length,
i.e., hypercontracture (21). In Fig.
4, the changes in cell length induced by
reoxygenation are shown. When cells were reoxygenated under the
conditions of protocol 1, they rapidly developed
hypercontracture, resulting in 44.0 ± 4.1% (n = 42) of initial cell length. Inhibition of NHE during simulated ischemia under protocol 2 protected against the subsequent
reoxygenation-induced hypercontracture. The length of these cells was
only slightly reduced to 61.0 ± 1.4% (n = 35;
P < 0.05 vs. protocol 1) of initial cell
length. Inhibition of NBS during simulated ischemia under protocol 3 did not influence the degree of subsequent
reoxygenation-induced hypercontracture. Under protocol 4,
where NHE and the NBS were inhibited simultaneously,
reoxygenation-induced hypercontracture was partly prevented in a degree
similar to protocol 2. The data show that inhibition of NHE
alone or simultaneous inhibition of NHE and NBS during simulated
ischemia can protect adult cardiomyocytes against hypercontracture
elicited by subsequent reoxygenation. Anoxic inhibition of NBS alone is
not protective.
|
Effect of NHE inhibition on cytosolic
Na+ loading.
The aforementioned results showed that the sole inhibition of NHE
during anoxia at pHo 6.4 (protocol 2) can
effectively protect cardiomyocytes from hypercontracture otherwise
occurring upon reoxygenation (protocol 1). We therefore
extended our experimental analysis of protocol 2 versus
protocol 1, with respect to possible effects of NHE
inhibition on cytosolic Na+ loading. Changes in cytosolic
Na+ during anoxia and reoxygenation were analyzed with
SBFI. Under protocol 1, the SBFI ratio rose from an initial
value of 0.6 ± 0.01 (n = 32) to a value of
0.77 ± 0.02 (n = 32) after 80 min of anoxia,
corresponding to a rise of intracellular Na+ concentration
from 15 to 84 mmol/l. Treatment of the cells with HOE 642 in
bicarbonate containing buffer (protocol 2) did not significantly influence the rate of rise as well as the end-anoxic value of the SBFI ratio (0.76 ± 0.02; n = 28) (Fig. 5). During reoxygenation, the SBFI ratio falls to its normoxic control level within 10 min under either protocol. These results agree with those on
changes in [Ca2+]i and pHi,
reported above, which were also not different between protocols 1 and 2.
|
Effect of anoxic inhibition of NHE with EIPA on
[Ca2+]i, pHi,
and reoxygenation-induced hypercontracture.
In an additional set of experiments, the chemically distinct inhibitor
EIPA was used instead of HOE 642. The data in Table 3 demonstrate that EIPA does not
influence the anoxic calcium loading nor the acidification of the
cytosol. Neither was the time of rigor contracture different from the
anoxic control cells (data not shown). The cardiomyocytes exposed to
EIPA during anoxia were nevertheless protected against
reoxygenation-induced hypercontracture (Table 3).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The aim of the present study was to investigate whether inhibition of the NHE and/or the NBS during simulated ischemia can protect cardiomyocytes during simulated ischemia or upon reoxygenation. The main finding is that an inhibition of the NHE with HOE 642 or EIPA solely during ischemic conditions protects cardiomyocytes against reoxygenation-induced hypercontracture independently of ischemic Ca2+ overload and cytosolic acidification. Additional inhibition of the NBS significantly enhances ischemic cytosolic acidosis but does not provide additional protection against reoxygenation-induced hypercontracture. The sole inhibition of the NBS during ischemic conditions shows no protective effects.
The model of isolated cardiomyocytes exposed to anoxia in medium with pH 6.4 and subsequent reoxygenation in normoxic medium with pH 7.4 has been characterized in earlier studies (20, 21, 34). Depleted from oxygen, the cells develop a deficit of energy that causes a rigor-mediated partial shortening of the myofibrils. H+, Na+, and Ca2+ accumulate in the cytosol. This ionic imbalance is rapidly reversed when cells are reenergized during reoxygenation. When reoxygenation is performed on cells that have already developed severe Ca2+ overload, they rapidly undergo hypercontracture. This results from the resupply of energy to the myofibrils at elevated [Ca2+]i (20, 34), predominantly occurring in the form of rapid Ca2+ oscillations. Reoxygenation-induced hypercontracture has been demonstrated in studies in vitro (10, 16, 36) and in vivo (3) to contribute substantially to lethal reperfusion injury of myocardial cells and thus to the postischemic extent of myocardial infarction. In the isolated cell model, hypercontracture develops without disruption of the sarcolemma because the cells do not mutually exchange force.
When anoxia was performed in bicarbonate-buffered media (pHo = 6.4), the pHi of the cardiomyocytes declined from 7.1 to 6.4 (protocol 1). In the presence of the NHE inhibitors HOE 642 or EIPA the development of intracellular acidosis was not significantly altered (protocol 2; see Table 2). This was the case even though these drugs at the chosen concentration are shown to completely inhibit NHE-dependent proton extrusion in this cell model. For protocols 1 and 2, the development of anoxic Na+ overload was also compared and not found different (see Fig. 5 and Table 2). In another set of experiments, bicarbonate buffer was replaced with HEPES buffer throughout simulated ischemia to abolish activity of the NBS (protocol 3). In HEPES buffer, the pHi decline during anoxia was not significantly altered. Only when the change of buffer was combined with the presence of HOE 642, i.e., when both NBS and NHE were blocked, intracellular acidosis was markedly enhanced, leading to pHi 5.5 at the end of anoxia (protocol 4). These results show that during anoxia, these two proton extruders can mutually compensate their actions in the investigated model. Under all protocols, pHi rapidly recovered when cells were reoxygenated in media with pH 7.4. The recovery of pHi was slightly delayed under protocol 3 and markedly under protocol 4 when the cardiomyocytes had to recover from an enhanced acidosis.
Within the investigated period of simulated ischemia, the cardiomyocytes underwent rigor shortening. This caused a reduction in cell length by approximately one-third. As shown before, rigor shortening is due to extensive loss of energy (5). It develops rapidly, i.e., in less than a minute, which allows one to define a "rigor time" (Table 2) during anoxia. Rigor times coincide with the points in time when the Mg-fura 2 signal starts to rise rapidly, indicative of a rapid rise in cytosolic free Mg2+. The concentration of Mg2+ in the cytosol increases during ischemia as a result of ATP degradation (6). Rigor times were not significantly different among the four investigated anoxic protocols. This indicates that anoxic energy depletion proceeds at similar pace under all protocols. Consequently, differences in energy depletion cannot be responsible for differences in the outcome of the different protocols.
It has been observed consistently that rigor time coincides with or precedes immediately the onset of cytosolic Ca2+ accumulation (21, 35). Onset or rate of cytosolic Ca2+ accumulation during anoxia was not affected by either inhibition of NHE or NBS alone. The rise of the fura 2 ratio, used to follow Ca2+ overload, was attenuated when both routes for H+ extrusion were blocked simultaneously (protocol 4), but this observation has to be interpreted with caution because of the pH dependence of the fura 2 ratio.
When cells were reoxygenated after prolonged ischemic conditions, they rapidly underwent hypercontracture. Hypercontracture is caused by an uncontrolled activation of the contractile machinery, which is elicited when the cells are reenergized in the presence of severe cytosolic Ca2+ overload (20). We showed previously (21) that reoxygenated cardiomyocytes can be protected against reoxygenation-induced hypercontracture through a preservation of ischemic cytosolic acidosis during the initial phase of reoxygenation. The results of protocols 1, 3, and 4 are consistent with these previous results because they show that 1) cardiomyocytes develop reoxygenation-induced hypercontracture when pHi recovers rapidly upon reoxygenation (protocols 1 and 3) and 2) hypercontracture is significantly reduced when pHi recovery is markedly delayed (protocol 4). The protection against hypercontracture of protocol 2, however, cannot be explained on these grounds. Under protocol 2, pHi recovered with similar rapidity as under protocol 1, but the observed protection against hypercontracture was similar to protocol 4 where pHi recovery was delayed. Therefore, the ability of protocol 2 to protect against reoxygenation-induced hypercontracture cannot be attributed to a prolongation of cytosolic acidosis. Neither is its success explained by intraischemic differences in acidosis, Na+ or Ca2+ overload, or energy depletion (see above). This indicates that the presence of NHE inhibitor HOE 642 exerts a protective effect, which is independent of these parameters. To corroborate this point and to exclude effects related to the particular chemical structure of HOE 642, the decisive experiment of protocol 2 was repeated with the chemically distinct NHE inhibitor EIPA. We found that EIPA acts in the same manner as HOE 642. Taken together, these results indicate that intraischemic inhibition of the NHE provides protection by a mechanism independent of the development of cytosolic acidification, Na+ overload, and subsequent Ca2+ overload.
Our results are in apparent opposition to the findings of others, who reported a reduction in Ca2+ or Na+ overload when the NHE was inhibited in ischemic myocardium (2, 8, 15, 24, 26). A possible explanation for this difference may be that most studies were performed with amiloride, which is known to exert inhibitory side effects also on the sarcolemmal Na+/Ca2+ exchanger (18) and L-type Ca2+ channel (11). In some of the studies, the NHE blockers were tested only in bicarbonate-free perfusates (21, 26), i.e., conditions comparable to protocol 4. The findings of Choy et al. (8), who observed a reduction in cytosolic Na+ loading, cannot be compared with our results because they tested the effect of HOE 642 in combination with a cardioplegic solution with high potassium, which causes depolarization of the membrane and multiple changes in ionic transport mechanisms. The results of Hendrikx et al. (15) show, by histochemical methods, a reduction of Ca2+ precipitates in mitochondria in ischemic-reperfused myocardium pretreated with HOE 694, indicating reduction of cell injury. Cytosolic free Ca2+ during ischemia was not determined. In general, it seems appropriate to say that none of these studies went beyond an observational association of protection with changes in cytosolic Na+ and Ca2+, i.e., the causal role of either parameter was not tested independently. In any case, our study shows that NHE inhibitors exert a protective effect on cardiomyocytes beyond effects possibly associated with the intraischemic changes in cytosolic cation control observed in other models.
In the investigated model, the results of our study contradict the favored hypothesis that the presence of NHE inhibitors throughout ischemia protects myocardial cells under ischemia-reperfusion conditions, because it changes the cytosolic H+ and Na+ balance and consequently the cytosolic Ca2+ balance under the ischemic conditions. One may avoid that conclusion by assuming that these changes occur only in a small subsarcolemmal space and have therefore escaped our analysis, but it would be difficult to relate such small local ionic changes to the distinct protection against reoxygenation-induced hypercontracture demonstrated in the presence study. It is more plausible to hypothesize that the protective mechanism of the NHE inhibitors is due to an effect unrelated to NHE ion transport function. One may speculate that the NHE molecule is structurally or functionally linked to cytoskeletal elements (12, 38) involved in the process of hypercontracture development.
| |
ACKNOWLEDGEMENTS |
|---|
The technical help of D. Schreiber and H. Holzträger is gratefully acknowledged.
| |
FOOTNOTES |
|---|
The study was supported by the European Union through a grant of the Biomed-2 program and Grant LA 1159/2-1 of Deutsche Forschungsgemeinschaft.
Address for reprint requests and other correspondence: H. M. Piper, Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129 D-35392 Giessen, Germany (E-mail: michael.piper{at}physiologie.med.uni-giessen.de)
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.
Received 15 November 1999; accepted in final form 15 May 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Allshire, A,
Piper HM,
Cuthbertson KSR,
and
Cobbold PH.
Cytosolic free calcium in single rat heart cells during anoxia and reoxygenation.
Biochem J
244:
381-385,
1987[Web of Science][Medline].
2.
Anderson, SE,
Murphy E,
Steenbergen C,
London RE,
and
Cala PM.
Na+/H+ in myocardium: effects of hypoxia and acidification on Na+ and Ca2+.
Am J Physiol Cell Physiol
259:
C940-C948,
1990
3.
Barrabes, JA,
Garcia-Dorado D,
Ruiz-Meana M,
Piper HM,
Solares J,
Gonzales MA,
Oliveras J,
Pilar Herrejon M,
and
Soler Soler J.
Myocardial segment shrinkage during coronary reperfusion in situ.
Pflügers Arch
431:
519-526,
1996[Web of Science][Medline].
4.
Borzak, S,
Kell RA,
Krämer BK,
Motoba Y,
Marsh JD,
and
Reers M.
In situ calbration of fura 2 and BCECF fluorescence in adult rat ventricular myocytes.
Am J Physiol Heart Circ Physiol
259:
H973-H981,
1990
5.
Bowers, KC,
Allshire AP,
and
Cobbold PH.
Bioluminescent measurement in single cardiomyocytes of sudden cytosolic ATP depletion coincident with rigor.
J Mol Cell Cardiol
24:
213-218,
1992[Web of Science][Medline].
6.
Budinger, GRS,
Duranteau J,
Chandel NS,
and
Schumacker PT.
Hibernation during hypoxia in cardiomyocytes.
J Biol Chem
273:
3320-3326,
1998
7.
Bugge, E,
and
Ytrehus K.
Inhibition of sodium-hydrogen exchange reduces infarct size in the isolated rat heart-a protective additive to ischaemic preconditioning.
Cardiovasc Res
29:
269-274,
1995[Web of Science][Medline].
8.
Choy, IO,
Schepkin VD,
Budinger TF,
Obayashi DY,
Young JN,
and
DeCampli WM.
Effects of specific sodium/hydrogen exchange inhibitor during cardioplegic arrest.
Ann Thorac Surg
64:
94-99,
1997
9.
Doering, AE,
and
Lederer WJ.
The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells.
J Physiol (Lond)
466:
481-499,
1993
10.
Ganote, CE,
McGarr J,
Liu SY,
and
Kaltenbach JP.
Oxygen induced enzyme release. Assessment of mitochondrial function in anoxic myocardial injury and effects of the mitochondrial uncoupling agent 2,4,-dinitrophenol (DNP).
J Mol Cell Cardiol
12:
387-408,
1980[Web of Science][Medline].
11.
Garcia, ML,
King VF,
Shevell JL,
Slaugther RS,
Suarez-Kurtz G,
Winquist RJ,
and
Kaczorowski GJ.
Amilorid analogs inhibit L-type calcium channels and display calcium entry blocker activity.
J Biol Chem
265:
3763-3771,
1990
12.
Grinstein, S,
Woodside M,
Waddell TK,
Downey GP,
Orlowski J,
Pouyssegur J,
Wong DCP,
and
Foskett JK.
Focal localisation of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH.
EMBO J
12:
5209-5218,
1993[Web of Science][Medline].
13.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985
14.
Harootunian, AT,
Kao JP,
Eckert BK,
and
Tsien RY.
Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes.
J Biol Chem
264:
19458-19467,
1989
15.
Hendrikx, M,
Mubagwa K,
Verdonk F,
Overloop K,
Van Heck P,
Vanstapel F,
Van Lommel A,
Verbecken E,
Lauweryns J,
and
Flameng W.
New Na+/H+ exchange inhibitor HOE 694 improves postischemic function, high-energy phosphate resynthesis and reduces Ca2+ overload in isolated perfused rabbit heart.
Circulation
89:
2787-2798,
1994
16.
Hohl, C,
Ansel A,
Altschuld R,
and
Brierly GP.
Contracture of isolated rat heart cells on anaerobic to aerobic transition.
Am J Physiol Heart Circ Physiol
242:
H1022-H1030,
1982.
17.
Klein, HH,
Pich S,
Bohle RM,
Wollenweber J,
and
Nebendahn K.
Myocardial protection by Na+/H+ exchange inhibition in ischemic reperfused porcine hearts.
Circulation
92:
912-917,
1992
18.
Kleyman, TR,
and
Cragoe EJ.
Antiarrhythmic activity of amilorid: mechanisms.
J Membr Biol
105:
1-21,
1988[Web of Science][Medline].
19.
Koop, A,
and
Piper HM.
Protection of energy status of hypoxic cardiomyocytes by mild acidosis.
J Mol Cell Cardiol
24:
55-65,
1992[Web of Science][Medline].
20.
Ladilov, YV,
Siegmund B,
Balser C,
and
Piper HM.
Simulated ischemia increases the susceptibility of rat cardiomyocytes to hypercontracture.
Circ Res
80:
69-75,
1997
21.
Ladilov, YV,
Siegmund B,
and
Piper HM.
Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na+/H+ exchange.
Am J Physiol Heart Circ Physiol
268:
H1531-H1539,
1995
22.
Lagadic-Gossmann, D,
Buckler KJ,
and
Vaughan-Jones KJ.
Role of bicarbonate in pH recovery from intracellular acidosis in the guinea-pig ventricular myocyte.
J Physiol (Lond)
458:
361-384,
1992
23.
Li, Q,
Altschuld RA,
and
Stokes BT.
Quantitation of intracellular free calcium in single adult cardiomyocytes by fura 2 fluorescence microscopy: calibration of fura 2 ratios.
Biochem Biophys Res Commun
147:
120-126,
1987[Web of Science][Medline].
24.
Murphy, E,
Perlman M,
London RE,
and
Steenbergen C.
Amiloride delays the ischemia-induced Rise in cytosolic free calcium.
Circ Res
68:
1250-1258,
1991
25.
Myers, ML,
Mathur S,
Li GH,
and
Karmazyn M.
Sodium-hydrogen exchange inhibitors improve postischemic recovery of function in the perfused rabbit heart.
Cardiovasc Res
29:
209-214,
1995[Web of Science][Medline].
26.
Pike, MM,
Luo CS,
Clark MD,
Kirk KA,
Kitakaze M,
Madden MC,
Cragoe EJ, Jr,
and
Pohost GM.
NMR measurement of Na+ and cellular energy in ischemic rat heart: role of Na+/H+ exchange.
Am J Physiol Heart Circ Physiol
265:
H3017-H3026,
1993.
27.
Piper, HM,
Balser C,
Ladilov YV,
Schäfer M,
Siegmund B,
and
Garcia-Dorado D.
The role of Na+/H+ exchange in ischemic reperfusion.
Basic Res Cardiol
91:
191-202,
1996[Web of Science][Medline].
28.
Piper, HM,
Probst I,
and
Schwartz P.
Culturing of calcium stable adult cardiac myocytes.
J Mol Cell Cardiol
14:
397-412,
1982[Web of Science][Medline].
29.
Piper, HM,
Volz A,
and
Schwartz P.
Adult ventricular rat heart muscle cells.
In: Cell Cuture Techniques In Heart And Vessel Research, edited by Piper HM.. Berlin: Springer, 1990, p. 36-60.
30.
Ru
, U,
Balser C,
Scholz W,
Albus U,
Lang HJ,
Weichert A,
Schölkens BA,
and
HGögelein
Effects of the Na+/H+-exchange inhibitor HOE 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes.
Pflügers Arch
433:
26-34,
1996[Web of Science][Medline].
31.
Scholz, W,
and
Albus U.
Potential of selective sodium-hydrogen exchange inhibitors in cardiovascular therapy.
Cardiovasc Res
29:
184-188,
1995[Web of Science][Medline].
32.
Scholz, W,
Albus U,
Counillon L,
Lang HJ,
Linz W,
Weichert A,
and
Schölkens BA.
Protective effects of HOE 642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischemia and reperfusion.
Cardiovasc Res
29:
260-268,
1995[Web of Science][Medline].
33.
Siegmund, B,
Koop A,
Klietz T,
Schwartz P,
and
Piper HM.
Sarcolemmal integrity and metabolic competence of cardiomyocytes under anoxia-reoxygenation.
Am J Physiol Heart Circ Physiol
258:
H385-H391,
1990.
34.
Siegmund, B,
Schlack W,
Ladilov YV,
Balser C,
and
Piper HM.
Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture.
Circulation
96:
4372-4379,
1997
35.
Siegmund, B,
Zude R,
and
Piper HM.
Recovery of anoxic-reoxygenated cardiomyocytes from severe Ca2+ overload.
Am J Physiol Heart Circ Physiol
263:
H1262-H1269,
1992
36.
Stern, MD,
Chien AM,
Capogrossi MC,
Pelto DJ,
and
Lakatta E.
Direct observation of the "oxygen paradox" in single rat ventricular myocytes.
Circ Res
56:
899-903,
1985
37.
Tani, M,
and
Neely JR.
Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+-Na+ and Na+-Ca2+ exchange.
Circ Res
65:
1045-1056,
1989
38.
Vexler, ZS,
Symons M,
and
Barber DL.
Activation of Na+/H+-Exchange is necessary for Rho-A-induced stress fiber formation.
J Biol Chem
271:
22281-22284,
1996
This article has been cited by other articles:
|
|
 |
|
  S. C. Peters and H. M. Piper Reoxygenation-induced Ca2+ rise is mediated via Ca2+ influx and Ca2+ release from the endoplasmic reticulum in cardiac endothelial cells Cardiovasc Res, January 1, 2007; 73(1): 164 - 171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Abdallah, A. Gkatzoflia, H. Pieper, E. Zoga, S. Walther, S. Kasseckert, M. Schafer, K.D. Schluter, H.M. Piper, and C. Schafer Mechanism of cGMP-mediated protection in a cellular model of myocardial reperfusion injury Cardiovasc Res, April 1, 2005; 66(1): 123 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ten Hove, M. G. J. Nederhoff, and C. J. A. Van Echteld Relative contributions of Na+/H+ exchange and Na+/HCO3- cotransport to ischemic Nai+ overload in isolated rat hearts Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H287 - H292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ruiz-Meana, D. Garcia-Dorado, P. Pina, J. Inserte, L. Agullo, and J. Soler-Soler Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions Am J Physiol Heart Circ Physiol, August 7, 2003; 285(3): H999 - H1006. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G Allen and X.-H. Xiao Role of the cardiac Na+/H+ exchanger during ischemia and reperfusion Cardiovasc Res, March 15, 2003; 57(4): 934 - 941. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
) during anoxia under protocol 1 (A) and 2 (B).
