From the Department of Biochemistry, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada and the
§ Department of Pharmacology and Toxicology, University of
Western Ontario, London, Ontario N6A 5C1, Canada
Received for publication, January 18, 2001, and in revised form, March 2, 2001
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ABSTRACT |
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The activity of the
Na+/H+ exchanger has been implicated as
an important contributing factor in damage to the myocardium that occurs during ischemia and reperfusion. We examined regulation of the
protein in ischemic and reperfused isolated hearts and isolated
ventricular myocytes. In isolated myocytes, extracellular signal-regulated kinases were important in regulating activity of the
exchanger after recovery from ischemia. Ischemia followed by
reperfusion caused a strong inhibitory effect on NHE1 activity that
abated with continued reperfusion. Four major protein kinases of size
90, 55, 44, and 40 kDa phosphorylated the
Na+/H+ exchanger. The
Na+/H+ exchanger-directed kinases demonstrated
dramatic increases in activity of 2-10-fold that was induced by 3 different models of ischemia and reperfusion in intact hearts and
isolated myocytes. p90rsk was identified as the 90-kDa protein
kinase activated by ischemia and reperfusion while ERK1/2 was
identified as accounting for some of the 44-kDa protein kinase
phosphorylating the Na+/H+ exchanger. The
results demonstrate that MAPK-dependent pathways including
p90rsk and ERK1/2 and are important in regulating the
Na+/H+ exchanger and show their dramatic
increase in activity toward the Na+/H+
exchanger during ischemia and reperfusion of the myocardium. They also
show that ischemia followed by reperfusion have important inhibitory
effects on Na+/H+ exchanger activity.
The Na+/H+ exchanger isoform 1 (NHE1)1 is an integral
membrane protein that exchanges one intracellular proton for one
extracellular sodium ion in response to intracellular acidification,
thereby regulating internal pH (pHi) in most mammalian cells (1, 2). Of the six known isoforms of the Na+/H+
exchanger (NHE1-NHE6), NHE1 is the major isoform present in the myocardium (3). Ionic homeostasis is vital for normal myocardial function and the regulation of exchanger activity in the heart by
hormones and growth factors via protein kinase-mediated signal transduction, plays an important role in maintenance of this
homeostasis (3, 4). The NHE1 protein is ~815 amino acids in length
and is thought to consist of a membrane domain with 12 integral
membrane segments and a long intracellular carboxyl terminus of 315 amino acids. It is within this intracellular carboxyl terminus that regulation of exchanger activity is postulated to occur by
phosphorylation on distal serine/threonine residues (1, 2). Recently,
serine 703 has been identified as one important amino acid which is
phosphorylated by the protein kinase p90rsk (5, 6). However,
earlier work has demonstrated growth factor induced phosphorylation of
several different peptides of the carboxyl-terminal region of the
Na+/H+ exchanger (7, 8). We have also shown
that MAPK (ERKs) are involved in hormonal regulation of activity of the
exchanger in skeletal muscle (9) and more recently we have shown that
both MAPK (ERKs) and p90rsk are involved in regulation of
exchanger activity in the healthy rat myocardium (10). Another protein
kinase, Ca2+/calmodulin-dependent protein
kinase II (CaM kinase II) has been shown to phosphorylate the
COOH-terminal domain in vitro (11), but it is not known if
CaM kinase II directly regulates the exchanger activity in intact cells.
It is well known that the activity of the
Na+/H+ exchanger during ischemia and
reperfusion produces numerous secondary effects that lead to the
exacerbation of tissue injury. The Na+/H+
exchanger removes protons either during ischemia or during reperfusion which causes excess intracellular Na+. This results in
either inhibition of the Na+/Ca2+ exchangers
ability to extrude Ca2+, or reversal in activity of the
Na+/Ca2+ exchanger and accumulation of
Ca2+. The increased intracellular Ca2+ results
in a variety of detrimental effects to the heart. It has been shown
that inhibition of Na+/H+ exchanger activity,
via the use of potent amiloride analogs as well as more recently
developed NHE1-specific inhibitors, can prove beneficial to recovery
from ischemia and reperfusion events (4, 12, 13). Various lines of
evidence suggest that the Na+/H+ exchanger may
be in an activated state during ischemia and/or reperfusion of the
myocardium. The protein kinase pathways that could be involved in regulation of the
antiporter during ischemia and ischemia reperfusion are not known. It
is known that multiple subfamilies of the mitogen-activated protein
kinase (MAPK) superfamily such as extracellular signal-regulated kinases (ERKs), p38MAPK, and c-Jun NH2-terminal
kinases (JNKs) are activated in ischemic-reperfused hearts in
vivo (21, 22). In contrast, some studies only implicate p38 and
JNK (stress kinases) activation and not ERKs during ischemia, ischemia
reperfusion, oxidative damage, and other stresses to the heart
(23-26). However, it has been shown that not only can JNKs and p38 be
activated during ischemia reperfusion, but MAPK (ERKs) can also be
activated (27). Recently it has been shown that cardiac myocytes
exposed to hydrogen peroxide (H2O2) show increased Na+/H+ exchanger activity via
activation of MAPK-dependent pathways (ERK1/2) (28). It is
clearly possible that these signaling pathways may play one or more
roles in regulation of the exchanger under ischemia and reperfusion
conditions especially since MAPK-dependent pathways have
been shown to be important in regulation of the Na+/H+ exchanger in the myocardium (10).
To date, no studies have shown definitively which kinases from the
ischemic and reperfused heart phosphorylate the distal amino acids in
the carboxyl terminus of the Na+/H+ exchanger.
The purpose of our study was to examine regulation of the
Na+/H+ exchanger in the myocardium during
ischemia and reperfusion. We examined the ability of multiple protein
kinases from ischemic and ischemia-reperfused myocardium to
phosphorylate the cytoplasmic domain of the
Na+/H+ exchanger. This present study provides
the first evidence of protein kinase-mediated regulation of the
Na+/H+ exchanger in response to ischemia and
ischemia reperfusion. The results also suggest that regulation of the
myocardial Na+/H+ exchanger may be important in
mitigating the damaging effects that ischemia and reperfusion have on
the myocardium.
Materials--
PD98059, a MEK1 inhibitor, and SB202190, a p38
inhibitor, were from Calbiochem-Novabiochem Corp. (La Jolla, CA).
Plasmid pGEX-3X, glutathione-Sepharose 4B affinity column, and protein
A-Sepharose CL-4B beads were from Amersham Pharmacia Biotech AB
(Uppsala, Sweden). Protein G-PLUS agarose was from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-ERK-1/2 (rabbit polyclonal),
anti-phospho-ERK (mouse monoclonal), anti-RSK1 (goat polyclonal),
anti-p38 (rabbit polyclonal), anti-CaM kinase II (goat polyclonal),
anti-JNK2 (rabbit polyclonal), and anti-MEK1 (rabbit polyclonal) were
from Santa Cruz Biotechnology. Anti-RSK (p90rsk) (mouse
monoclonal) was from Transduction Laboratories (Lexington, KY). MF-20
(mouse monoclonal) was purchased from the Developmental Studies
Hybridoma Bank (Iowa City, IA). [ Construction and Purification of Glutathione
S-Transferase-Na+/H+ Exchanger
Fusion Protein--
The carboxyl-terminal 178 amino acids of the
rabbit cardiac Na+/H+ exchanger were expressed
as described previously (9) as a fusion protein with GST (PCRA) using
the plasmid pGEX-3X. The Escherichia coli TOPP 2 strain was
induced with 1 mM
isopropylthio- Animals and Ischemic Reperfusion of Hearts--
Adult
Harlan Sprague-Dawley rats were purchased from either Charles
River Canada (St. Constant, PQ) or Harlan Sprague-Dawley (Indianapolis, IN) and maintained in the Health Sciences Animal Care
facility of the University of Western Ontario in accordance with the
guidelines of the Canadian Council on Animal Care (Ottawa, ON). Rats
were killed by decapitation and the hearts were immediately removed and
perfused by way of the aorta at 37 °C with oxygenated Krebs-Henseleit buffer consisting of 120 mM sodium
chloride, 4.63 mM potassium chloride, 1.17 mM
potassium phosphate monobasic, 1.25 mM calcium chloride,
1.2 mM magnesium chloride, 20.0 mM sodium bicarbonate, and 8.0 mM glucose, pH 7.4, at a flow rate of
10 ml/min (29). All hearts were equilibrated for 30 min, after which
ischemia was induced for 30 min by completely stopping the flow with or
without subsequent reperfusion for 30 min. Control hearts were perfused
normally for 60 min. In another set of experiments, hearts were allowed
to equilibrate for 10 min. This was followed by 30 min of buffer
perfusion with or without 40 µM PD98059 (a MAPK kinase
(MEK1) inhibitor), then 30 min of no flow (ischemia), followed by 15 min of reperfusion with or without 40 µM PD98059. At the
end of the perfusions hearts were "freeze clamped" and stored at
minus 80 °C.
Preparation of Cell Extracts from Control, Ischemic, and
Ischemic-reperfused Adult Rat Myocardium--
The heart tissue was
homogenized at a high setting with a Polytron homogenizer for 30 s
in 2.5 (v/w) of extraction buffer containing 50 mM
tetrasodium pyrophosphate, 50 mM sodium fluoride, 50 mM sodium chloride, 5 mM EDTA, 5 mM
EGTA, 0.1 mM sodium orthovanadate, 0.1% Triton X-100, 10 mM HEPES, pH 7.4, and a mixture of protease inhibitors. The
homogenate was centrifuged at 6,000 × g × 60 min at 4 °C. The supernatant was then centrifuged at 10,000 × g × 60 min at 4 °C. Western blot analysis was done
using commercially available antibodies against ERK 1/2, phospho-ERK,
p38, JNK2, p90rsk, MEK1, and MF-20 (cardiac sarcomeric myosin
heavy chain). Ischemic-reperfused hearts were also perfused in the
presence of the drug PD98059, to partially inhibit the MAPK (ERK1/2)
pathway by inhibiting MEK1. Heart cell extracts from these experiments
were prepared as above.
In-gel Kinase Assays--
To identify protein kinases that
phosphorylated the Na+/H+ exchanger fusion
protein, cell extracts (80 µg of protein) from control, ischemic, and
ischemic-reperfused myocardium (with and without 40 µM
PD98059) were separated by 10% SDS-PAGE in a gel containing 1 mg/ml
substrate. In-gel kinase assays were performed as previously described
(10). The gels were dried for autoradiography and visualization of
phosphorylation. Equal amounts of protein were used for the in-gel
kinase assays. We confirmed that equal amounts of protein were applied
to the gels by Coomassie Blue staining of identically run gels or by
Western blot analysis of identically run samples with an antibody
against MF-20. Protein concentrations were measured using the Bio-Rad
DC protein assay. For one series of experiments samples in
IGKAs were treated with 5 µM SB202190 during incubation
with labeled ATP to inhibit p38 kinase.
Immunoprecipitation of ERK1/2, p90rsk, p38, JNK2 and MEK1
from Control, Ischemic, and Ischemic-reperfused Heart Cell
Extracts--
For immunoprecipitations, ERK-1/2, p38, RSK1, JNK2, and
MEK1 antibodies were used. Extracts (1 ml) were pretreated by
incubating with protein A-Sepharose CL-4B beads (in experiments with
ERK-1/2, anti-p38, JNK2, and MEK1 antibodies) or protein G-PLUS agarose beads (in experiments with anti-RSK1) for 30 min. The samples were then
centrifuged for 1-2 min at 7000 rpm at 4 °C to remove nonspecifically adsorbed proteins bound to the resins. In addition, Sepharose beads used for immunoprecipitation were pretreated to reduce
nonspecific binding. The beads were then incubated with heart cell
extract for 2 h at 4 °C and washed with extraction buffer. For
immunoprecipitation of protein kinases, 30 µl of 200 µg/ml stock
anti-ERK-1/2, MEK1, JNK2, or anti-p38 was added to pretreated heart
cell extract and this mixture was rotated for 2 h at 4 °C. The
antibody-antigen complex was then added to pretreated protein
A-Sepharose beads and rotated for 1 h at 4 °C. The beads were
next washed extensively in extraction buffer and solubilized in
SDS-PAGE gel sample buffer. For the remaining immunoprecipitation, 10 µl of 200 µg/ml stock anti-RSK1 was added to protein G-PLUS agarose
beads overnight at 4 °C. The next day the heart cell extract was
added to this antibody-bead complex and allowed to bind for 4 h at
4 °C. The beads were then washed extensively in extraction buffer
and solubilized in SDS-PAGE gel sample buffer. The immunoprecipitates were all analyzed by Western blotting and by in-gel kinase assay for
their ability to phosphorylate the Na+/H+
exchanger fusion protein.
Primary Cultures of Isolated Neonatal Myocytes--
Primary
myocyte cultures were prepared from neonatal Harlan Sprague-Dawley rat
heart ventricles as described previously (10). Isolated primary
myocytes were plated onto glass coverslips for physiologic studies, or
onto PrimariaTM (Falcon) culture dishes or flasks for
collection of cell extracts. Myocytes were maintained for 4-5 days in
medium containing Dulbecco's modified Eagle's medium/Ham's
F-12 supplemented with 10% fetal bovine serum, 10 µg/ml transferrin,
10 µg/ml insulin, 10 ng/ml selenium, 50 units/ml penicillin, 50 µg/ml streptomycin, 2 mg/ml bovine serum albumin, 5 µg/ml linoleic
acid, 3 mM pyruvic acid, 0.1 mM minimum
essential medium non-essential amino acids, 10% MEM vitamin, 0.1 mM bromodeoxyuridine, 100 µm L-ascorbic acid, and 30 mM HEPES, pH 7.1. Cells were serum-starved overnight
prior to all experiments.
Ischemia and Ischemia Reperfusion Conditions in Cardiac
Myocytes--
Neonatal cardiac myocytes were made ischemic by
incubation at pH 6.2 in Krebs-Ringer-HEPES (KRH) buffer containing 130 mM sodium chloride, 2.5 mM potassium chloride,
2.5 mM potassium cyanide (to inhibit oxidative
phosphorylation), 1 mM potassium phosphate monobasic, 1.2 mM magnesium sulfate, 2 mM calcium chloride,
and 20 mM HEPES for 4 h at 37 °C (modified from
Bond et al. (30)). This ischemic treatment is referred to as
I1. In other experiments, 10 mM
2-deoxy-D-glucose was added to this medium to inhibit
glycolytic ATP production and result in a more severe ischemia
(referred to as I2) (30). To simulate reperfusion, the
metabolic inhibitor was removed by "washout" with KRH buffer at pH
7.4 (without potassium cyanide) and incubation for 30 min at 37 °C.
Control myocytes were incubated with KRH buffer, pH 7.4 (without
potassium cyanide), for the same time period as the ischemic myocytes.
Following the experiment, myocytes were washed with ice-cold
phosphate-buffered saline and an extraction buffer containing 50 mM tetrasodium pyrophosphate, 50 mM sodium
fluoride, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 0.1 mM sodium orthovanadate, 0.1%
Triton X-100, 10 mM HEPES, pH 7.4, and a mixture of
protease inhibitors (31). The cells were then frozen on dry ice for 5 min, allowed to thaw on ice for 15 min, scraped, and transferred into
microcentrifuge tubes. The myocyte extracts were sonicated for
10 s on ice and then centrifuged at 10,000 rpm for 30 min at
4 °C. Protein kinases from the extracts were analyzed by Western blot analysis using antibodies against ERK 1/2, phospho-ERK, and MF-20
(cardiac sarcomeric myosin heavy chain) and for their ability to
phosphorylate the Na+/H+ exchanger fusion
protein using the in-gel kinase assay described earlier.
Measurement of Intracellular pH during Ischemia and Ischemia
Reperfusion Conditions in Isolated Neonatal Cardiac
Myocytes--
Myocytes were grown on glass coverslips and the
acetoxymethyl ester of
2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein was used to measure
steady-state pHi as described previously (9, 32). Briefly,
pHi was measured using the dual excitation single emission
ratio technique using a temperature-controlled Shimadzu RF5000
spectrofluorophotometer. Excitation wavelengths were at 452 and 503 nm
with emission at 524 nm. Myocytes were serum-starved before all
pHi measurements. Cells on coverslips were placed into a holder
device and inserted into a 1 × 1-cm fluorescence cuvette at
37 °C and bathed in the ischemia and reperfusion (washout) buffers
as stated above (KRH buffer at pH 6.2 or 7.4, respectively).
Steady-state pHi was measured for 1 min after the specified
ischemia or ischemia-reperfusion protocol (see above). In some
experiments, the cells were pretreated with the MEK1 inhibitor PD98059
(50 µM) during the last 30 min of ischemia and during the
reperfusion period. PD98059 was added during the reperfusion phase and
at all subsequent steps. For other experiments, SB202190 (1 µM) was added during the ischemic phase as well as at the
reperfusion phase.
Rates of recovery from an acid load in control, ischemic, and
ischemia-reperfused neonatal cardiac myocytes (with and without PD98059
or SB202190) were also measured following an acid challenge using the
ammonium chloride prepulse method as previously described (9, 32).
Cells were treated either with an ischemia induced by using KCN only
(I1) or with a harsher form of ischemia induced by using KCN
and 2-deoxy-D-glucose (I2). Briefly, ammonium
chloride (20 mM) was added for 4 min followed by a pH recovery that was obtained by transferring the cells to buffer containing 130 mM sodium chloride after a brief exposure to
a Na+-free buffer. For measuring initial rate of recovery
from an acid load we examined the initial changes in pHi during
the first 10-20 s after sodium chloride addition.
We calculated proton flux for all experiments involving rate of
recovery from an acid load. Buffering capacity (B, millimole/liter/pH unit) was estimated as the amount of acid loaded divided by the observed change of cell pHi produced by this load. Buffering capacity was determined at various pHi by varying amounts of
ammonium chloride. Proton flux (JH+) produced due to the Na+/H+ exchanger after acid
loading was calculated from the buffer capacity at mid point pH × Data Analysis--
Autoradiographs were quantified using a model
BAS1000 PhosphorImager (Fuji Photo Film Co., Ltd) to examine
radioactivity incorporated into protein (in-gel kinase assay). Analysis
of these results was by Mann-Whitney U test and/or
Student's unpaired t test on direct numerical values
obtained from the image analysis. Quantification and analysis of
results of pHi measurements was done using a Mann-Whitney
U test. Values presented are mean ± S.E. with at least
four and up to eight experiments being done in every case; p
values < 0.05 were considered statistically significant.
A MAPK (ERK)-dependent Pathway Facilitates Steady-state
pHi Regulation by the Na+/H+
Exchanger in Response to Ischemia Reperfusion--
To observe the
regulation of the endogenous Na+/H+ exchanger,
the effect of ischemia and ischemia reperfusion on steady-state pHi of neonatal cardiac myocytes was examined. Isolated neonatal cardiac myocytes were grown on coverslips and serum-starved overnight prior to stimulation. We utilized methods that
"chemically" induce ischemia and ischemia-reperfusion events
similar to that observed in perfused hearts. This "chemical"
ischemia acts by inhibition of oxidative phosphorylation
(I1), or by inhibiting both oxidative phosphorylation and
glycolysis (I2) (30). Fig. 1
summarizes the effects of ischemia (I1) and ischemia reperfusion on steady-state pHi in the presence or absence of
the MEK1 inhibitor 50 µM PD98059. Ischemia reduced
steady-state pHi dramatically while 5 and 30 min of reperfusion
brought about a complete recovery of normal pHi (to ~7.2).
Treatment of the myocytes with 50 µM PD98059
significantly reduced the ability of the cells to return pHi to
the values observed without PD98059. This indicated that a MAPK (ERK)
pathway is partially responsible for facilitating
Na+/H+ exchanger activity during ischemia
reperfusion in neonatal ventricular myocytes. PD98059 alone had no
effect on resting pHi of isolated myocytes that had not
undergone ischemia or reperfusion (not shown).
A MAPK (ERK)-dependent Pathway Facilitates the Rate of
Recovery from Acid Load Induced by Ischemia Reperfusion--
To
observe changes in proton efflux during ischemia and ischemia
reperfusion, an ammonium chloride prepulse was used to induce acute
acidosis in control myocytes and in various groups of myocytes treated
with ischemia (I1 or I2) as described for Fig. 1.
Three groups were examined. Control cells were treated with KRH buffer
for 30 min to 4 h and then treated with ammonium chloride. This
was followed by a brief period in Na+-free KRH buffer and
then recovery in KRH buffer with normal Na+. These cells
recovered rapidly from the acid load (Fig.
2A). A second group of cells
(Isch) was given the same kind of treatment except that at all stages
the KRH buffer was replaced either with KCN containing buffer
(I1) or buffer used to induce chemical ischemia. These cells
retained a much more acidic pHi at all stages and did not
recover from an acid load (Fig. 2, A and B). The
third group of cells (I/R) was subject to 3-4 h of chemical ischemia
(either I1 or I2), followed by varying
reperfusion periods (4.5-30 min) in normal KRH buffer (with or without
PD98059 (I1) or SB202190 (I1 and I2)).
They were then subjected to ammonium chloride-induced acid load and
recovery in normal KRH buffer (with or without PD98059 or SB202190).
Cells treated with PD98059 recovered much more slowly from the acid
load in comparison to untreated cells and reached a lower steady state
pHi (Fig. 2, A and B). There was
no difference in the rate of recovery from an acid load when comparing
PD98059-treated control cells and PD98059-treated cells subjected to
ischemia and reperfusion (Fig. 2B). Ischemia (I1)
followed by reperfusion resulted in a large (up to 50%) reproducible,
time-dependent decrease in the ability of cells to recover
from an acid load. At both 12 and 17 min of reperfusion there was a
decrease in the ability of cells to recover from acid load (Fig. 2,
A-C). This effect largely disappeared after 30 min of
reperfusion.
The effect of SB202190 on rate of recovery from an acute acid load is
shown in Fig. 2C. Control cells treated with SB202190 showed
only a slight, variable reduction in the rate of recovery from acid
load. Isolated myocytes were treated with ischemia (I1) followed by reperfusion for 17 min. SB202190 had no effect on the
ability of these I1-treated cells to recover from an acid
load. When isolated myocytes were treated with the harsher form of
ischemia (I2) followed by reperfusion for 17 min, SB202190
slightly (but significantly) reduced the ability of these cells to
recover from an acid load. SB201290 had no effect on the steady state
resting pHi of isolated myocytes (not shown).
Protein Kinases Phosphorylating the
Na+/H+ Exchanger Are Activated by
Ischemia and Reperfusion in Isolated Neonatal Ventricular
Myocytes--
We then examined the effect of ischemia and ischemia
reperfusion on phosphorylation of the Na+/H+
exchanger COOH terminus in isolated, neonatal cardiac myocytes. Cells
were maintained either under control conditions (Krebs-Ringer-HEPES (KRH) buffer, pH 7.4, Fig. 3, lanes
1-3), subjected to ischemia (I1) for 4 h (KRH
buffer + 2.5 mM KCN, pH 6.2, lanes 4-6) or
subjected to ischemia followed by reperfusion or washout for 30 min with the control KRH buffer (lanes 7-9). Samples of
myocyte extracts were used in an in-gel kinase assay with the
Na+/H+ exchanger as a substrate as described
earlier (10). Four major kinases of approximate molecular masses
90, 55, 44, and 40 kDa were activated during both ischemia and ischemia
reperfusion of neonatal myocytes. In-gel kinase analysis with only GST
as a substrate did not give any of these major bands and usually gave
only weak activity at sizes of ~70 and 37 kDa. Based on the size of
the protein kinases phosphorylating the Na+/H+
exchanger on our previous study (10) and on Western blot analysis of
the same samples (Fig. 3, C and D), we
tentatively identified ERK1/2 (44 and 42-kDa) and p90rsk
(90-kDa) as the 44- and 90-kDa protein kinases that were present in the
cell extracts. The two remaining kinases of molecular masses 40 and 55 kDa remained to be identified. Increasing amounts of phosphorylation of
the Na+/H+ exchanger was observed by all four
of the major protein kinases activated by ischemia and ischemia
reperfusion (Fig. 3, A and E). Western blot
analysis with antibodies to MF-20 (Fig. 3B) and ERK1/2 (Fig.
3D) confirmed that equal amounts of protein were present in
control versus treated myocytes. Immunoblotting with anti-phospho ERK1/2 (Fig. 3C, lanes 7-9) show increased
activation of both these kinases during ischemia followed by
reperfusion. Qualitatively similar results were obtained with extracts
of cells from myocytes treated with the more severe ischemia
(I2).
In another set of experiments (Fig. 4) we
used a similar approach to examine the effects of more severe ischemia
(I2) and reperfusion on phosphorylation of the
Na+/H+ exchanger COOH terminus in isolated,
neonatal cardiac myocytes. Using a similar protocol we found the same
proteins phosphorylated in control isolated myocytes (Fig. 4,
lanes 1-3). Ischemia and I/R resulted in large increases in
the level of phosphorylation of the 90-kDa band. There was no increase
in the level of the 55-, 44-, or 40-kDa band in ischemia alone,
however, for all these kinases, the activity was significantly
increased by reperfusion following ischemia. The 90-kDa kinase was most
strongly activated by the severe ischemia and reperfusion treatment.
The level of phosphorylation by the 44-kDa kinase was decreased from
the control levels by ischemia alone.
Ischemia and Ischemia Reperfusion of the Intact Rat Myocardium
Activate Multiple Protein Kinases That Phosphorylate the
Carboxyl-terminal of the Na+/H+
Exchanger--
In-gel kinase assay and Western analysis were used to
identify potential protein kinases that phosphorylated the
carboxyl-terminal of the Na+/H+ exchanger
during ischemia and ischemia reperfusion of the intact adult rat
myocardium. Samples of heart cell extracts (Fig.
5A, controls, lanes
1-3; ischemic, lanes 4-6; ischemia-reperfused, lanes 7-9) were
used in in-gel kinase assays as described under "Experimental
Procedures." Four major kinases of approximate molecular masses 90, 55, 44, and 40 kDa were able to significantly phosphorylate the
carboxyl-terminal region of the Na+/H+
exchanger. Control experiments with GST alone (not shown) as a
substrate did not show phosphorylation of the kinases of approximate molecular masses 90, 55, 44, and 40 kDa. The four kinases were all
activated during both ischemia and ischemia reperfusion of the rat
myocardium in comparison to control hearts. For the 90- and 55-kDa
protein kinases the phosphorylation was increased by reperfusion of the
hearts in comparison to the effect of ischemia alone. With the 40- and
90-kDa protein kinases reperfusion caused further activation of the
kinases (Fig. 5E).
Immunoprecipitated ERK1/2 and p90rsk Phosphorylate the
Na+/H+ Exchanger during
Ischemia and Ischemia Reperfusion of the Rat Myocardium--
To more
conclusively identify ERK1/2 and p90rsk (RSK1) as activated
protein kinases that phosphorylate the carboxyl-terminal of the
Na+/H+ exchanger, these kinases were
immunoprecipitated with antibodies specific to ERK1/2 and
p90rsk (RSK1). Specific kinases were immunoprecipitated from
cell extracts of control (Fig. 6,
lanes 1-3), ischemia (lanes 4-6), and ischemic-reperfused (lanes 7-9) hearts and the immunoprecipitates were used in
an in-gel kinase analysis (Fig. 6, A and E). The
first two lanes for each group are before and after
immunoprecipitation while the third lane is the
immunoprecipitate. Western blots of the samples confirmed the presence
of equal amounts of immunoprecipitated protein for each of the
different groups and confirmed the identity of the immunoprecipitates
(Fig. 6, B, D, and F). Fig. 6A shows that during ischemia and ischemic reperfusion, p90rsk was
activated and phosphorylated the Na+/H+
exchanger more than controls. p90rsk was highly activated
during ischemia reperfusion in comparison to either control hearts or
hearts treated with ischemia alone. A similar result occurred with
immunoprecipitated ERK1 (Fig. 6, E and F). ERK1
was successfully immunoprecipitated from control, ischemic, and
ischemia-reperfused tissue (lanes 3, 6, and 9, respectively). Western analysis with anti-ERK1 antibody confirmed the
identity of the immunoprecipitates (Fig. 6F). The in-gel
kinase assay showed that ERK1 was highly activated during ischemia
reperfusion and phosphorylated the Na+/H+
exchanger fusion protein more than both control and ERK1
immunoprecipitates from hearts treated with ischemia. Western blot
analysis with anti-phospho-ERK1 antibody confirmed that ERK1 was highly
activated by ischemia followed by reperfusion (Fig. 6C).
ERK2 was also weakly activated during ischemia reperfusion and
phosphorylated PCRA, but to a much lesser degree (data not shown). It
appears that both ERK1 and -2 are phosphorylated during ischemia
followed by reperfusion. However, immunoprecipitation of ERK2 with
anti-ERK2 antibodies suggested that ERK2 does not appear to
phosphorylate the Na+/H+ exchanger as strongly
as ERK1 (data not shown).
Activity of p38 and JNK2 in IGKAs Directed Toward the
Na+/H+ Exchanger--
To
identify p38 (38-kDa) and/or JNK2 (54-kDa) (stress kinases) as
activated protein kinases that phosphorylate the
Na+/H+ exchanger, these kinases were
immunoprecipitated with specific antibodies. (Antibody to JNK2
recognizes both JNK1/2 in Western analysis, but only immunoprecipitates
JNK 2.) p38 (Fig. 7, A and B) and JNK2 (Fig. 7, D and E) were
immunoprecipitated from cell extracts from control (Fig. 7, lanes
1-3), ischemic (lanes 4-6), and ischemia-reperfused
(lanes 7-9) hearts. The immunoprecipitates were used in an
in-gel kinase analysis (p38, Fig. 7A; JNK, Fig. 7D). Western blot analysis of the samples confirmed the
presence of immunoprecipitated protein for each type of cell extract
(Fig. 7, B and E). Fig. 7, C and
F, also confirm the presence of p38 and JNK1/2 in control
(lanes 1-3), ischemia (lanes 4-6), and ischemic reperfused (lanes 7-9) hearts. Fig. 7A shows
that there was no phosphorylation of the Na+/H+
exchanger by immunoprecipitated p38 from heart extracts. Similarly, Fig. 7D shows no phosphorylation of the
Na+/H+ exchanger by immunoprecipitated JNK2.
The results demonstrated that neither our immunoprecipitate of p38 nor
that of JNK2, phosphorylate the carboxyl-terminal of the
Na+/H+ exchanger. Fig. 7G confirms
that p38 from heart extracts phosphorylates the substrate ATF-2 in an
in-gel kinase assay (Fig. 7G). Despite repeated attempts at
improvement, the immunoprecipitation of p38 from the isolated myocytes
was relatively inefficient, while that from other cells was greatly
improved and phosphorylated the Na+/H+
exchanger very readily (not shown). The reason for this difference is
not clear at this time but could be due to differences in isoforms of
the p38 kinase that occur in various tissues (34). Our results suggest
that either p38 and JNK2 from the myocardium do not phosphorylate the
Na+/H+ exchanger or that their phosphorylation
was below the level of detection of this assay.
In another experiment, we examined the effect of SB202190 on the
protein kinase activity of heart extracts in an in-gel kinase assay.
Prior to incubation with ATP, the gel containing the heart extract was
incubated with 10 µM SB202190. There was no reduction of
any specific molecular weight protein kinase activity directed toward
the Na+/H+ exchanger, despite a general overall
reduction in all protein kinases activity (not shown).
Inhibition of MEK1 by PD98059 Reduces
Na+/H+ Exchanger Kinase
Activation during Ischemia Reperfusion of the Myocardium--
In-gel
kinase assay and Western analysis were used to identify differences in
protein kinase activation during ischemia reperfusion in the absence
(Fig. 8, A and B, lanes
1-3) or presence (lanes 4-6) of 40 µM
PD98059, a MEK1 inhibitor. The use of PD98059 reduced the
phosphorylation activity of the 90-kDa kinases toward the Na+/H+ exchanger (Fig. 8, A and
C). The use of antibodies to the phosphorylated form of
ERK1/2 (Fig. 8B) confirmed that there was a large reduction in activation of ERK1/2 by treatment with PD98059. There was no reduction in the level of phosphorylation by the 40-kDa kinase and
surprisingly, there was also no significant reduction in the amount of
phosphorylation by the 44-kDa kinase.
In another series of experiments we postulated that some of the kinase
activity we observed might be due to MEK1 (43-kDa) and/or MEK2
(56-kDa). However, in a series of experiments we immunoprecipitated MEK1/2 and examined their ability to phosphorylate the antiporter in
in-gel kinase assays. The results showed that phosphorylation by the
55- and 40-kDa protein kinases were not due to MEK1/2 (not shown). A
similar experiment with anti-CaM kinase II antibody also showed that
the 55-kDa protein kinase was not CaM kinase II (not shown).
It is well established that the activity of the
Na+/H+ exchanger exacerbates the tissue injury
that occurs during ischemia and reperfusion of the myocardium (4, 12).
However, regulation of the activity of the
Na+/H+ exchanger during this time period has
not been studied. To investigate this phenomenon further, we examined
protein kinase-mediated regulation of the
Na+/H+ exchanger during ischemia and
reperfusion of the myocardium. We initially demonstrate that
ERK-dependent pathways are involved in activity of the
Na+/H+ exchanger when recovering from ischemia
and reperfusion. When isolated myocytes were exposed to ischemia
followed by reperfusion, PD98059 inhibited their ability to return to
resting pH. PD98059 also inhibited the ability to recover from an acute
acid load, although there was no difference between control hearts
treated with PD98059 and hearts that had undergone ischemia and
reperfusion. These results suggest that ERK-dependent
pathways are important in maintenance of resting pH in the ischemic
myocardium and that they are normally important in the ability of the
Na+/H+ exchanger to recover from an acute acid
load. Because of the time required for inducing acute acid loading, it
was not possible to measure the effects of reperfusion on
Na+/H+ exchanger very early after ischemia.
Therefore it was not possible to examine if very early reperfusion
elevates Na+/H+ exchanger activity beyond the
control levels. Nevertheless, it is clear that
ERK-dependent phosphorylation was important in NHE1 activity and is important in resting pHi.
We also found that ischemia followed by reperfusion caused a large,
transient, inhibitory effect on Na+/H+
exchanger activity. The rate of recovery from an acid load was decreased as was the resting pHi (Fig. 2). This was unlikely to
be due to nonspecific or proteolytic effects since it occurred reproducibly between 12 and 17 min after reperfusion, and then later
was reversed. These results leave open the possibility that protein
kinase mediated activation of the Na+/H+
exchanger through ischemia reperfusion, may have an inhibitory effect
on Na+/H+ exchanger activity. Protein
kinase-mediated inhibition of the Na+/H+
exchanger has been suggested earlier in the heart (35) and smooth
muscle (36). While it is not yet certain whether the physiological
effects we observed are protein kinase mediated, they nevertheless
demonstrate that ischemia followed by reperfusion has a significant
physiological effect on Na+/H+ exchanger activity.
We found that the p38 kinase was not a major regulator of
Na+/H+ exchanger activity in isolated myocytes.
The inhibitor SB202190 caused only a slight, variable effect on the
rate of recovery from an acute acid load. In addition, there was only a
minor effect on the rate of recovery from an acute acid load in one
group of isolated myocytes treated with severe ischemia followed by
reperfusion. Our results agree with others that have suggested that p38
does not have significant activity toward the antiporter in the
myocardium (37). However, they disagree with those that suggest that
p38 activity is important in smooth muscle (36). This difference could
reflect dissimilar regulation of the Na+/H+
exchanger which is known to be dependent on cell type (3, 38).
Different isoforms of p38 could be responsible for the differential
regulation of activity in different tissues (34).
To elucidate the specific kinases involved in the regulatory effects we
examined the protein kinases that phosphorylated the antiporter during
ischemia and reperfusion. Our initial experiments clearly demonstrate
that both ischemia and ischemia reperfusion activate a number of
protein kinases that phosphorylate the carboxyl-terminal region of the
Na+/H+ exchanger (Figs. 3-5). Similar to
previous results (10), there were four major protein kinases that
phosphorylated the antiporter. We found that ischemia reperfusion
induced large increases (over 10-fold in some cases) in the activity of
these kinases directed toward the Na+/H+
exchanger. We used three models of ischemia reperfusion, the isolated
perfused heart and two models of ischemia-treated isolated myocytes.
One treatment was with cyanide alone to inhibit oxidative phosphorylation; the other more severe treatment also contained 2-deoxyglucose to also inhibit glycolysis. Ischemia alone increased most kinases' activity in the less severe ischemia model while in the
more severe model, ischemia alone only increased phosphorylation by the
90-kDa protein. In contrast, severe ischemia, followed by reperfusion,
increased most protein kinase activity toward the
Na+/H+ exchanger more than the less severe
ischemia reperfusion. The reason for these differences is not known but
it can be hypothesized that severe ischemia results in more ATP
depletion that reduces protein kinase activity. Greater activation of
the kinases by the severe ischemia was similar to the results obtained
with the intact hearts, where ischemia and reperfusion caused up to
8-fold increases in kinase activity. These very large increases were surprising and extremely interesting observations. They suggest that
that in the clinical situation of ischemia and reperfusion, Na+/H+ exchanger-directed protein kinases may
be greatly activated.
We attempted to identify the different protein kinases phosphorylating
the Na+/H+ exchanger. Immunoprecipitation
experiments with anti-p90rsk and anti-ERK antibodies confirm
the identity of these Na+/H+ exchanger-directed
protein kinases (10). In addition, they demonstrate that ischemia and
reperfusion (Fig. 5) activate p90rsk and
ERK-dependent phosphorylation of the
Na+/H+ exchanger. Our laboratory (9, 10) and
others (5, 28, 36, 39, 40) have shown that MAP
kinase-dependent pathways are important in regulation of
the Na+/H+ exchanger, although the details of
the specific pathway involved appear to vary from one tissue to
another. While one report (40) has suggested that ERK does not directly
phosphorylate the Na+/H+ exchanger, we have
earlier shown that ERK directly phosphorylates the protein in
stoichiometric amounts (9). In addition, we (10) and others (36) have
more recently demonstrated ERK-mediated phosphorylation of the
Na+/H+ exchanger in muscle tissue. The reason
for the discrepancy between some of these studies (40) is not known at
this time but for now, the preponderance of evidence suggests that ERK
directly phosphorylates the Na+/H+ exchanger
and is critical for regulation of activity (9).
The identification of the 90-kDa kinase as p90rsk is similar to
a report in smooth muscle (41). The identification of the 44-kDa kinase
as ERK1/2 is also similar to other reports in assays of myocytes and
other tissues (5, 9, 28). However, there was an important discrepancy
between the amount of phosphorylation observed in in-gel kinase assays
of the 44-kDa protein and the amount of phospho-ERK detected in some
samples. With isolated myocytes, the amount of 44-kDa kinase
phosphorylating the Na+/H+ exchanger was
increased by ischemia (Fig. 3A), however, anti-phospho ERK
antibodies detected a decrease in phospho-ERK1 and -ERK2 (Fig. 3C, lanes 4-6). Similar results occurred with isolated
perfused hearts (Fig. 5C) when ischemia increased the amount
of phosphorylation by 44-kDa kinase but the amount of phospho-ERK was
reduced. The most likely explanation is that there is another protein
kinase at the same molecular weight as the ERK isoforms and that this kinase accounts for a large part of signal we see at this molecular weight. Immunoprecipitation showed that ERK1 is at least partly responsible for the phosphorylation observed at this molecular weight
(Fig. 6E). However, after treatment of hearts with PD98059 there was an almost total absence of phosphorylated ERK1 and ERK2 protein (Fig. 8B), but no significant decrease in the level
of 44-kDa protein kinase directed toward the
Na+/H+ exchanger. These results suggest that
there is second protein kinase of ~44 kDa that phosphorylates the
Na+/H+ exchanger. It is likely a kinase in a
pathway that is independent of the MAP kinase pathway since its
activity remained in the presence of PD98059. In addition, its activity
is stimulated by ischemia and reperfusion in both isolated myocytes and
in the perfused intact heart.
It is known that many MAP kinase-dependent pathways are
activated in the heart during ischemia. This includes the ERK kinases, p38 MAPK, and JNK kinases (22-26). Treatment of isolated perfused hearts increased the level of 44-kDa protein kinase that phosphorylated the carboxyl-terminal of the Na+/H+ exchanger.
While it is clear that this is not all due to ERK, immunoprecipitation
experiments demonstrated that ERK1 was more active in tissues from
hearts subjected to ischemia and reperfusion (Fig. 6E).
Experiments with anti-ERK2 antibodies suggest that ERK2 was not
phosphorylating the Na+/H+ exchanger COOH
terminus as strongly as ERK1 (not shown). The stimulation of ERK by
ischemia and reperfusion is in agreement with previous studies (22, 22,
27, 28). These results demonstrated that ischemia and reperfusion
activate ERK1 activity toward the Na+/H+ exchanger.
p90rsk is a protein kinase downstream of ERKs. Similar to
results with ERKs, it was possible to immunoprecipitate p90rsk
and demonstrate that the immunoprecipitated protein could phosphorylate the carboxyl-terminal of the Na+/H+ exchanger
(Fig. 6A). Both ischemia and ischemia reperfusion increased the level of phosphorylation by this protein kinase. Our results are in
agreement with our earlier study (10) and that of others (37, 41) which
suggest that p90rsk is an important regulator of the
Na+/H+ exchanger. Hypoxia and reoxygenation
have been shown to activate MAP kinase-dependent pathways
earlier, including activation of p90rsk (42). Our results
demonstrate that ischemia and reperfusion of isolated hearts and
isolated myocytes also increase p90rsk activity toward the
Na+/H+ exchanger. Because of the significant
role this kinase is known to play in Na+/H+
exchanger regulation (10, 41), it is clear that this may be an
important event in the activity of the antiporter during ischemia and
reperfusion. It has recently been shown that reactive oxygen species
can activate p90rsk in pathways that are both
MEK1/2-dependent and MEK1/2-independent (28, 43, 44). We
found that PD98059 blocked ~60% of p90rsk activity toward
the Na+/H+ exchanger (Fig. 8A),
suggesting that both MEK1/2-dependent and MEK1/2-independent pathways could be involved in the present series of experiments.
Aside from the 44- and 90-kDa protein kinases that phosphorylate the
Na+/H+ exchanger, in-gel kinase assays
demonstrated that two other protein kinases of 40 and 55 kDa also
phosphorylated the Na+/H+ exchanger. Both were
highly stimulated markedly by ischemia and reperfusion. The identity of
these protein kinases is not yet certain, however, their size and
activation by ischemia suggest that they could be p38, JNK (stress
kinases), MEK1/2 (23-26), or CaM kinase II. Immunoprecipitation of p38
kinase showed that this kinase might not account for the 40-kDa
Na+/H+ exchanger-directed protein kinase. It
should be noted, however, that immunoprecipitation of p38 from other
cells types led to strong phosphorylation of the
Na+/H+ exchanger in IGKAs (not shown). It may
be that the isoform of p38 present in the myocardium was not as
reactive toward the Na+/H+ exchanger as in
other tissues, or that its abundance and specificity was below the
level of detection of our IGKA. Future experiments will examine this in
more detail. At present our IGKA results largely agree with our
physiological experiments that have shown that the p38 pathway plays a
minor role in activity of the Na+/H+ exchanger
in the myocardium.
The identity of the 55-kDa protein kinase is not known at this time.
Immunoprecipitation of JNK2 and an examination of its activity in
in-gel kinase assays suggested that it likely does not account for this
Na+/H+ exchanger-directed kinase we see in our
assays. A similar result was found for CaM kinase II and MEK. The lack
of activity of JNK toward the Na+/H+ exchanger
agrees with the results of Kusuhara et al. (36) in smooth
muscle. It should be noted, however, that it is possible that JNK1
phosphorylates the Na+/H+ exchanger since our
immunoprecipitates only contain the JNK2 isoform of the protein.
Further studies will be necessary to identify the 55-kDa protein kinase
in the myocardium.
Regulation of the Na+/H+ exchanger is a balance
between phosphorylation of the protein and dephosphorylation of the
many potential sites available on the carboxyl-terminal region. We
found that ischemia and reperfusion resulted in a transient decrease in
the activity of the protein. Recently, two reports have suggested that
protein kinase-mediated phosphorylation can be inhibitory to the
Na+/H+ exchanger (35, 36). It is well known
that the activity of the Na+/H+ exchanger is
detrimental to the myocardium during ischemia and reperfusion (12, 13).
It is therefore tempting to suggest that a protein kinase-mediated
inhibitory mechanism exists for reducing Na+/H+
exchanger activity during reperfusion. Such a mechanism could help
reduce the damage the Na+/H+ exchanger activity
normally mediates (12, 13). Our study demonstrates that several unknown
protein kinases of size 55, 44, and 40 kDa are activated during
ischemia followed by reperfusion. Further experiments are necessary to
examine if these kinases mediate the inhibitory effects we found on the
Na+/H+ exchanger.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-Adrenergic receptor stimulation regulates
the antiporter in the myocardium causing both an alkalization of steady
state intracellular pH (pHi) and an enhanced rate of
Na+/H+ exchanger mediated recovery from an acid
load (14-16).
1-Adrenergic stimulation is known to
exacerbate reperfusion induced arrhythmias and increased
Na+/H+ exchanger activity may play an important
role in this phenomenon (17). In addition, the 21-amino acid vasoactive
peptide endothelin (ET-1) has also been shown to stimulate
Na+/H+ exchange in cardiac myocytes (18, 19).
Under some circumstances endothelin may also aggravate ischemic
reperfusion injury, possibly through Na+/H+
exchanger activation (20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32]ATP was purchased
from Amersham Pharmacia Biotech (Oakville, Ontario, Canada).
Collagenase Type 2 was obtained from Worthington Biochemical Corp.
(Lakewood, NJ) and BCECF-AM was from Molecular Probes (Eugene, OR). All
other chemicals were of analytical grade and were purchased from Fisher
Scientific (Ottawa, ON), Sigma, or BDH (Toronto, ON).
-D-galactoside. GST-Na+/H+ exchanger fusion protein was
purified via glutathione-Sepharose 4B affinity chromatography as
described earlier (9, 10).
pH/
time essentially as described earlier (33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of ischemia and ischemia reperfusion
conditions on steady-state pHi of isolated neonatal ventricular
myocytes. Myocytes were grown on coverslips and serum-starved
overnight prior to stimulation in the presence or absence of 50 µM PD98059. Ischemia (I1) and reperfusion were
for the indicated times. Results are mean ± S.E. of at least four
experiments. Where error bars are absent they
were too small to be displayed. Asterisk (*) indicates that
the value in the presence of PD98059 is significantly different from
the untreated value at p < 0.05.
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Fig. 2.
Recovery of pHi from acute acid load
by isolated neonatal ventricular myocytes after ischemia and ischemia
reperfusion in the presence or absence of 50 µM PD98059 or 1 µM SB202190). Ischemia was induced
by the presence of KCN (2.5 mM) or KCN plus
2-deoxy-D-glucose (10 mM) where indicated
(I2). Ammonium chloride prepulse was used to induce acute
acidosis. Control cells were treated 30 min to 4 h with KRH,
followed by ammonium chloride prepulse, and a brief period in
Na+-free KRH buffer and recovery in KRH buffer with
Na+. I/R (ischemia-reperfusion) cells
were treated 3-4 h with chemical ischemia buffer (as described under
"Experimental Procedures") followed by various incubations in
normal KRH buffer (with or without PD98059 (A and
B) or SB202190 (A and C)). Ammonium
chloride prepulse and recovery measurement followed as with controls.
The inhibitors PD98059 and SB202190 were present in all solutions used
for pH recovery measurements. A, illustrates representative
tracings of pHi recovery of myocytes. B and
C, bar graphs summarize the results of initial
rate of pHi recovery from an acute acid load. Results are
mean ± S.E. of at least four experiments. Plus and
asterisk (+, *) indicate significant difference from control
values at p < 0.01 and p < 0.05, respectively. indicates significant difference from no SB202190
treatment at p < 0.01.
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Fig. 3.
Na+/H+
exchanger-directed protein kinase activity of cell extracts from
ischemic-reperfused isolated neonatal ventricular myocytes. Cells
were maintained either under control conditions (KRH buffer pH 7.4, lanes 1-3), subjected to ischemia (I1) for
4 h in KRH buffer + 2.5 mM KCN, pH 6.2 (lanes
4-6), or subjected to ischemia followed by reperfusion or washout
for 30 min with the control KRH buffer (lanes 7-9). Myocyte
extracts were used for: A, in-gel kinase assay as described
under "Experimental Procedures." Lane 10 is a myocyte
extract run in a gel using GST alone as a substrate (1 mg/ml).
B-D, Western blots of simultaneously run samples (1-9) that
were used in A. The Western blots were probed with
antibodies to MF-20, phospho-ERK1/2, and ERK1/2 for B-D,
respectively. E, bar graph summarizing results in
A. Asterisk (*) indicates significant difference
from control values at p < 0.05.
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Fig. 4.
Na+/H+
exchanger-directed protein kinase activity of cell extracts from
ischemic-reperfused isolated neonatal ventricular myocytes. Cells
were maintained either under control conditions (KRH buffer pH 7.4, lanes 1-3), subjected to severe ischemia (I2)
for 4 h in KRH buffer + 2.5 mM KCN, pH 6.2, and 10 mM 2-deoxyglucose (lanes 4-6), or subjected to
ischemia followed by reperfusion or washout for 30 min with the control
KRH buffer (lanes 7-9). Myocyte extracts were prepared as
described in the legend to Fig. 3. Panel B is a Western blot
of a simultaneously run sample, probed with an antibody to MF-20.
C, bar graph summarizing results in A. Asterisk (*) indicates significant difference from control
values at p < 0.05.
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Fig. 5.
Characterization of
Na+/H+ exchanger-directed protein kinase
activity of cell extracts from isolated perfused rat hearts. Heart
extracts were prepared from control isolated perfused hearts
(lanes 1-3), from hearts subjected to 30 min ischemia
(lanes 4-6), and from hearts subjected to 30 min ischemia
followed by 30 min reperfusion (lanes 7-9). A,
autoradiogram showing in-gel kinase assay using
Na+/H+ exchanger-GST as substrate (1 mg/ml,
lanes 1-9) and a ischemia-reperfused sample using GST
protein as substrate (1 mg/ml, lane 10). Right of
lane 9 indicates the 90-, 55-, 44-, and 40-kDa protein
kinases. B, Coomassie Blue-stained SDS-PAGE gel of identical
samples (lanes 1-9) noting the position of cardiac
sarcomeric myosin heavy chain protein. C, Western blot
analysis of the same samples using anti-ERK1 (rabbit polyclonal,
1:4000) antibody to detect ERK1/2 (44 and 42 kDa) in cell extracts.
D, Western blot using anti-RSK (p90rsk) (mouse
monoclonal, 1:500) to detect the presence of this kinase in the heart
cell extracts. E, bar graph summarizing the
effects of ischemia and ischemia reperfusion on exchanger-directed
protein kinase activity of 4 protein kinases (90, 55, 44, and 40 kDa)
present in heart cell extracts. Results are mean ± S.E. of three
experiments. Asterisk (*) indicates significant difference
from control value at p 0.05. Plus (+)
indicates significant difference from ischemic value at
p < 0.05.
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Fig. 6.
In-gel kinase assays demonstrating activated
p90rsk (RSK1) and ERK1-mediated phosphorylation of the
Na+/H+ exchanger. Cell extracts were
prepared from the various isolated perfused rat hearts as described
under "Experimental Procedures." p90rsk (RSK1)
(A) and ERK1 (E) antibodies were used to
immunoprecipitate these protein kinases. Samples of immunoprecipitates
and cell extracts before and after immunoprecipitation were then used
for in-gel kinase assays with Na+/H+ exchanger
protein as substrate. Lanes 1-3 are from control
(C) hearts: lane 1, cell extracts before; and
lane 2, after immunoprecipitation; lane 3 is the
immunoprecipitate. Lanes 4-6 are ischemic (I)
samples and lanes 7-9 are ischemic-reperfused
(I/R) samples run on the gel using the same
loading pattern. B and F are Western blots of
simultaneously run samples that were used in A and
E, respectively. They were probed with antibodies to
p90rsk (RSK1) and ERK1, respectively. C and
D are Western blots performed with antibodies to the
phosphorylated form of ERK1 (mouse monoclonal, 1:1000) and ERK1,
respectively (lanes 1 and 2 are control cell
extracts; 3 and 4, ischemic cell extracts; and
5 and 6 ischemic-reperfused cell extracts).
HC indicates the position of the heavy chain of IgG used for
immunoprecipitation and is evident when probing the immunoprecipitate
with secondary antibody.
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Fig. 7.
In-gel kinase assays demonstrating lack of
ability of MAPK family members, p38 (38-kDa) and JNK2 (54-kDa) (stress
kinases), to phosphorylate the Na+/H+
exchanger. Cell extracts were prepared from the various isolated
perfused rat hearts as described for Fig. 1. p38 (A) and
JNK2 (D) antibodies were used to immunoprecipitate these
protein kinases. Immunoprecipitates were used in in-gel kinase assays
with Na+/H+ exchanger protein as substrate
(lanes 1-3 are control (C) cell extract; before
immunoprecipitation (lane 1), after immunoprecipitation
(lane 2), and the immunoprecipitate is in lane
3. Lanes 4-6 are ischemic (I) samples and
lanes 7-9 are ischemic-reperfused
(I/R) samples using the same loading pattern as
the controls. B and E are Western blots of
simultaneously run samples that were used in A and
D. They were probed with antibodies to p38 (rabbit
polyclonal, 1:1000) and JNK2 (rabbit polyclonal, 1:1000), respectively.
C and F are Western blots with antibodies to p38
and JNK2, respectively. They demonstrate the presence of these protein
kinases in the three types of treated hearts (lanes 1-3 are
control heart extracts; lanes 4-6, ischemic heart extracts;
and lanes 7-9, ischemic-reperfused heart extracts).
HC indicates the position of the heavy chain of IgG used for
immunoprecipitation. G, in-gel kinase assay showing p38
protein kinase from control (lane 1), ischemic (lane
2), and ischemic-reperfused (lane 3) heart extracts
phosphorylating its ATF-2 substrate.
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Fig. 8.
In-gel kinase assay and Western blot analysis
of isolated perfused rat hearts subjected to ischemia reperfusion in
the presence or absence of 40 µM of
the MEK1 inhibitor PD98059. A, samples of
ischemia-reperfused heart extracts were used in in-gel kinase analysis
with the Na+/H+ exchanger protein as a
substrate. Lanes 1-3 are ischemic-reperfused hearts without
PD98059; lanes 4-6 are ischemic-reperfused hearts treated
with PD98059. B, Western blot analysis with
anti-phospho-ERK1 antibody indicating the phosphorylated forms of
ERK1/2. C, bar graph summarizing the in-gel
kinase assay data and Western analysis data of five experiments.
Asterisk (*) indicates significant difference from untreated
values at p < 0.5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by a grant from the Heart and Stroke Foundation of Canada (to L. F.) and by grants from the Canadian Institute of Health Research (to L. F. and M. K.).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.
¶ Career Investigator of the Heart and Stroke Foundation of Ontario.
Scientist of the Alberta Heritage Foundation for Medical
Research. To whom correspondence should be addressed Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Tel.: 780-492-1848; Fax: 780-492-0886; E-mail:
lfliegel@gpu.srv.ualberta.ca.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M100519200
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ABBREVIATIONS |
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The abbreviations used are: NHE1, Na+/H+ exchanger isoform 1; CaM kinase II, calmodulin dependent protein kinase II; ERK1/2, extracellular signal-regulated kinase 1 and 2; JNK1/2, c-Jun NH2-terminal kinase1 and 2; GST, glutathione S-transferase; MAPK, mitogen activated protein kinase; MEK1/2, MAPK kinase 1 and 2; pHi, intracellular pH; IGKA, in-gel kinase assay; KRH, Krebs-Ringer-HEPES; PAGE, polyacrylamide gel electrophoresis.
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