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J. Biol. Chem., Vol. 276, Issue 45, 42050-42056, November 9, 2001
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From the Department of Pharmacology, Medical College of Ohio,
Toledo, Ohio 43614
Received for publication, August 16, 2001, and in revised form, September 18, 2001
We have shown before that
Na+/K+-ATPase acts as a signal
transducer, through protein-protein interactions, in addition to being an ion pump. Interaction of ouabain with the enzyme of the intact cells
causes activation of Src, transactivation of EGFR, and activation of
the Ras/ERK1/2 cascade. To determine the role of protein kinase C (PKC)
in this pathway, neonatal rat cardiac myocytes were exposed to ouabain
and assayed for translocation/activation of PKC from cytosolic to
particulate fractions. Ouabain caused rapid and sustained stimulation
of this translocation, evidenced by the assay of
Ca2+-dependent and Ca2+-independent
PKC activities and by the immunoblot analysis of the Na+/K+-ATPase is the intrinsic enzyme of
the plasma membrane that maintains the normal gradients of
Na+ and K+ across this membrane of most animal
cells (1, 2). In recent years, we have shown that
Na+/K+-ATPase also acts as a signal transducer;
i.e. it responds to extracellular stimuli such as ouabain or
low extracellular K+ to relay messages, through
protein-protein interactions and second messengers, to intracellular
signaling complexes, the mitochondria, and the nucleus (3-9). The
interaction of Na+/K+-ATPase with nontoxic
concentrations of the digitalis drug ouabain causes the activation of
multiple interrelated signal pathways that seem to begin with the
activation of Src kinase, followed by Src-induced transactivation of
EGFR,1 recruitment and
activation of Ras and activation of two Ras-dependent branched pathways: one communicating with the mitochondria to increase
the generation of mitochondrial reactive oxygen species and the other
being the Ras/Raf/MEK/ERK1/2 cascade (3-9). Although we have
demonstrated the ouabain-induced activation of these pathways in
several cell types (8, 9), to date the downstream consequences have
only been examined in cardiac myocytes (3-9). In these cells, where
the well established inhibitory effects of nontoxic ouabain concentrations on the ion-pumping function of
Na+/K+-ATPase lead to small increases in
[Na+]i and significant increases in
[Ca2+]i, this rise in
[Ca2+]i cooperates with the
Ca2+-independent activation of the mitochondrial reactive
oxygen species to regulate the transcription of growth-related genes
and cause myocyte hypertrophy (9). A question raised in the course of these studies on cardiac myocytes was on the possible role of PKC in
the proximal pathways that are activated by ouabain. Our early
experiments showed that inhibition of PKC blocked the ouabain-induced regulation of several growth-related genes (3, 4). Since PKC inhibition
also seemed to antagonize the rapid ouabain-induced activation of
ERK1/2 (6), we suggested that at least one locus of PKC involvement
must be upstream of ERK1/2 (6). This, coupled with the fact that direct
activation of PKC by PMA causes activation of ERK1/2 in myocytes (6,
10), led to the postulate that ouabain may indeed cause the rapid
activation of PKC in cardiac myocytes. The present studies were
initiated to test this hypothesis and to explore the mechanisms of any
ouabain-induced PKC activation and its linkage to activation of
ERK1/2.
Materials--
Chemicals of highest purity and culture media
were purchased from Sigma. Antibodies against PKC Cell Preparation and Culture--
The same protocols were used
to prepare and culture neonatal ventricular myocytes as described
before (3). In short, myocytes from 1-day-old Harlan Sprague-Dawley
rats were isolated and purified on Percoll gradients. In a medium
containing four parts of Dulbecco's modified Eagle's medium and one
part Medium 199 (Life Technologies, Inc.), penicillin (100 units/ml),
streptomycin (100 µg/ml), and 10% fetal bovine serum, myocytes were
cultured for 24 h at 37 °C in humidified air with 5%
CO2. After 24 h, the myocytes were serum-starved for
48 h, at which time all experiments were performed. Some of the
experiments were done in a nominally Ca2+-free medium,
which was prepared to contain the same components as Dulbecco's
modified Eagle's medium except that calcium salts were omitted and 0.1 mM EGTA was added to the medium. As we previously reported,
incubation of cardiac myocytes in this medium had no significant effect
on cell viability for the experimental durations used here (3).
Immunofluorescence staining with a myosin heavy chain antibody showed
that the cultures contained more than 95% myocytes.
Assay of PKC Activity and Translocation--
Myocytes were
washed with phosphate-buffered saline; suspended in a solution
containing 10 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 50 µg/ml leupeptin, 25 µg/ml aprotinin, and 20 mM Tris-HCl (pH 7.5); and homogenized in a Potter-Elvehjen homogenizer. The suspension was centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was removed (the
cytosolic fraction), and the pellet was suspended in the above
described solution to which Triton X-100 (1%) was also added. After 30 min on ice, the mixture was centrifuged at 25,000 × g
for 10 min, and the supernatant was collected (the particulate fraction).
PKC activities were determined by slight modifications of previously
described procedures (13-16). Briefly, the cytosolic and the
particulate fractions (above) were partially purified by passage through DE-52 cellulose columns (15, 16) and assayed either for
Ca2+-stimulated PKC activity using histone H1
as substrate (histone kinase) or for Ca2+-independent PKC
activity using the synthetic substrate of PKC
To assay the translocation of the various PKC isoforms, the cytosolic
and the particulate fractions were subjected to immunoblot analysis,
using the indicated isoform-specific antibodies and appropriate
secondary antibodies, as described before (15, 16). Quantitation of the
relative intensities of the immunoblots were done using
chemiluminescence and exposure to x-ray film. Images were scanned with
a Bio-Rad densitometer. When necessary, different dilutions of the
samples were subjected to immunoblotting, and multiple exposures of the
films were used to ensure that quantitative comparisons were made
within the linear range of the assay.
Measurement of Phosphorylation/Activation of ERK1/2--
After
the indicated treatments, cells were washed with phosphate-buffered
saline and lysed in an ice-cold solution containing 150 mM
NaCl, 1 mM NaF, 1 mM
Na3VO4, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 50 mM tetrasodium
pyrophosphate, 10 nM okadaic acid, 1% Triton X-100, 0.25%
sodium deoxycholate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 mM Tris-HCl (pH 7.4). Equal amounts of protein from various
lysates were subjected to SDS-polyacrylamide gel electrophoresis and
immunoblot analysis as described before (6) using antibodies that
detect only phosphorylated/active ERK1/2 and total ERK1/2. Activation
was measured as the ratio of the intensities of the appropriate bands
detected with the two antibodies. We have shown before that
ouabain-induced activation of ERK1/2 by this procedure correlates with
in-gel assay of ERK1/2 activities (6).
86Rb+ Uptake--
The initial rate of
ouabain-sensitive Rb+ uptake through the
Na+/K+-ATPase of intact myocytes was measured
as described before (17), using monensin in the assay medium to ensure
that the maximal capacity of the active uptake was being measured (18).
Briefly, myocytes were incubated in the culture medium at 37 °C for
10 min with different concentrations of ouabain, including the
maximally effective 1 mM ouabain; 25 µM
monensin and 86Rb+ as tracer for K+
were then added, and uptake was measured after 20 min. It was established that uptake was a linear function of time within this period.
Fluorescence Microscopic Assay of
[Ca2+]i--
This was done using fura-2, by
the procedures and calibrations described before (7, 17). Briefly,
fura-2 fluorescence was recorded using an Attofluor imaging system
(Atto Instruments, Rockville, MD) at excitation wavelengths of 340/380
nm and at an emission wavelength of 505 nm. Under each experimental
condition, time-averaged signals were obtained from about 20-40 single
cells. [Ca2+]i was calculated based on the
fluorescence ratio and the Ca2+ calibration curve (7,
17).
Data Analysis--
Means ± S.E. of the results of a
minimum of three experiments are presented. Student's t
test was used, and significance was accepted at p < 0.05.
Ouabain-induced Activation of PKC--
Stimulus-induced activation
of PKC involves the translocation of the enzyme from the soluble to the
particulate fractions of cells (19, 20). In experiments of Fig.
1, neonatal rat cardiac myocytes were
exposed to 100 µM ouabain for 10 min or 100 nM PMA for 5 min as positive control and were assayed for PKC activities in particulate and cytosolic fractions. In each fraction
Ca2+-stimulated PKC activity (histone kinase) and
Ca2+-independent PKC activity (Epep kinase) were assayed
(Fig. 1, A and B). As expected, in control
unstimulated cells, a small fraction of either histone kinase or Epep
kinase was in the particulate fraction (see legend to Fig. 1), and upon
PMA stimulation the greater portion of each activity was shifted from
the cytosolic to the particulate fraction (Fig. 1). Ouabain also caused
significant increases in the particulate fraction activities of histone
kinase (Fig. 1A) and Epep kinase (Fig. 1B);
however, these ouabain-induced increases were less than those induced
by PMA. The concomitant ouabain-induced decreases in cytosolic
activities, which were expected to be small, were not statistically
significant (Fig. 1).
To determine the effects of ouabain on individual PKC isoforms, we
focused on
The results of the experiments of Fig. 4A
on the dose dependence of the ouabain-induced translocation of PKC Role of PLC in Ouabain-induced Activation of PKC--
Because
activations of polypeptide growth factor receptors such as EGFR are
known to stimulate phosphoinositide turnover by activation of PLC- Relationship between Ouabain-induced Activations of PKC and
ERK1/2--
We showed before (6) that a rather nonspecific PKC
inhibitor, H-7, antagonized ouabain-induced activation of ERK1/2. This was confirmed with the use of more specific PKC inhibitors
chelerythrine and calphostin C (Fig. 7).
D609 also blocked ouabain-induced activation of ERK1/2 (Fig. 7),
indicating that sequential activations of PLC and PKC are required for
ouabain-induced activation of ERK1/2.
In previous studies, we showed that ouabain-induced activation of
ERK1/2 also required the presence of extracellular Ca2+
(6). The question arose as to whether this dependence on extracellular Ca2+ was exerted upstream or downstream of PKC activation.
In myocytes that were incubated in a nominally Ca2+-free
medium, exposure to ouabain did not activate PKC in contrast to the
activation noted in the Ca2+-containing medium (Fig.
8). Evidently, the well established
ouabain-induced net influx of Ca2+ that leads to a rise in
[Ca2+]i (Fig. 4) is required for ouabain-induced
activations of PKC and ERK1/2.
A main new finding reported here is that ouabain interaction with
intact cardiac myocytes leads to rapid translocation/activation of
several PKC isoforms from the soluble to the particulate pools of these
enzymes (Figs. 1-3). Before we discuss the implications of these and
related findings, it is important to emphasize that the data presented
here (Fig. 4), in conjunction with previous findings, clearly indicate
that the activating effect of ouabain on PKC is obtained at ouabain
concentrations that cause only partial inhibition of
Na+/K+-ATPase (Fig. 4B), small
changes in [Na+]i (9), and significant but
nontoxic increases in [Ca2+]i (Fig.
4B; Refs. 3 and 9). Such degrees of change in
Na+/K+-ATPase and intracellular cation
concentrations are indeed the requirements for obtaining the positive
inotropic effects of ouabain and related digitalis drugs (9, 35, 36).
In fact, it has been shown (35, 37) that the major portion of the
positive inotropic action of ouabain on rat ventricular strips is
obtained at ouabain concentrations (10-100 µM)
comparable with those used in the present studies. Thus, although
ouabain effect on the contractile strength of the cultured myocytes was
not measured here,2 it is
reasonable to conclude that the present findings suggest that
ouabain-induced PKC activation must accompany the classical effect of
ouabain on cardiac contractility, at least in the rat.
Activations of both PKC and Ras Are Required for the Linkage of
Na+/K+-ATPase to ERK1/2--
The present
findings provide strong support for the previous suggestion (6) that
ouabain-induced PKC activation precedes the activation of ERK1/2. To
date, the most proximal signaling events that have been identified to
be linked to Na+/K+-ATPase are the activation
of Src and the transactivation of EGFR (8). Evidently, these events
lead not only to the activation of Ras (6, 8) but also to the parallel
activation of PLC, stimulation of the phosphoinositide turnover, and
activation of PKC (Fig. 5). That this PKC activation is essential for
ouabain-induced activation of ERK1/2 (Fig. 7) must now be reconciled
with our previous data showing that activation of Ras is also essential for ouabain-induced activation of ERK1/2 (6). A reasonable hypothesis
is that Ras and PKC must cooperate to activate Raf. This is supported
by observations in cells other than cardiac myocytes (38-39),
indicating that Raf recruitment to the plasma membrane requires
activated Ras but that activation of the recruited Raf requires
additional steps that include phosphorylation of Raf, possibly by PKC.
Based on the present data and our previous findings, the status of the
signal pathway that links Na+/K+-ATPase to
ERK1/2 through PKC and Ras is summarized in Fig.
9. It is important to point out, however,
that the steps outlined in Fig. 9 may not be the only links between
Na+/K+-ATPase and PKC. In view of the recent
evidence suggesting the interaction of Src with RACK·PKC
complexes (40, 41), it is possible that ouabain-induced activated Src
may also be involved in directing the activated PKC isoforms to their
substrates, including Na+/K+-ATPase itself
(see below).
Necessity of Ouabain-induced Rise in [Ca2+]i
for Activations of PKC and ERK1/2--
We showed before (9) that
activation by ouabain of Src-induced tyrosine phosphorylation of a
number of proteins, and Ras-dependent increase in
mitochondrial generation of reactive oxygen species were independent of
the presence of extracellular Ca2+ and ouabain-induced rise
in [Ca2+]i. We also showed, however, that
ouabain-induced activation of ERK1/2 required the presence of
extracellular Ca2+ (6). This effect of Ca2+ is
now shown to be, at least in part, due to the dependence of ouabain-induced PKC activation on extracellular Ca2+ (Fig.
8), which is the source of the ouabain-induced rise in [Ca2+]i. Although the precise mechanism of this
Ca2+ effect (perhaps on PLC) remains to be determined, the
present findings reinforce the conclusion that there is cross-talk
between the ion-pumping function and the signal-transducing function of Na+/K+-ATPase (Fig. 9).
Further Implications of Ouabain-induced Activations of PKC
and ERK1/2--
If translocation/activation of some PKC isoforms does
indeed accompany the positive inotropic action and the therapeutic use of digitalis, as suggested by the present findings, several further implications are worthy of brief consideration. 1) In view of the above discussed necessity of ouabain-induced increase in
[Ca2+]i for the rapid and sequential activations
of PKC and ERK1/2, the question arises as whether the activation of
these protein kinases may in turn influence the ouabain-induced change in [Ca2+]i. Evidently, the answer is yes, since
Tian et al. (42) have shown recently that inhibition of
ERK1/2 in adult cardiac myocytes antagonizes ouabain-induced rise in
[Ca2+]i, perhaps through an effect of ERK1/2 on
Ca2+ channels and/or the Na+/Ca2+
exchanger. There is also a large body of evidence to indicate the role
of PKC in short term regulation of [Ca2+]i
through activating or inhibitory effects of PKC on voltage-gated
Ca2+ channels, the sarcoplasmic reticulum Ca2+
uptake/release system, and the sarcolemmal
Na+/Ca2+ exchanger (25, 43, 44). The
possibility that ouabain-induced activation of PKC may affect any of
these transporters, coupled with the demonstrated role of ERK1/2 in the
regulation of [Ca2+]i (42), suggests that PKC,
ERK1/2, Na+/K+-ATPase, and a number of
neighboring membrane receptors and transporters are within a signal
feedback cycle (Fig. 9). Thus, the mechanism of the positive inotropic
action of ouabain and related drugs may be more complex than previously
assumed. 2) Of special interest is the possibility that ouabain-induced
translocation/activation of PKC may cause the phosphorylation and
regulation of Na+/K+-ATPase. It has been known
for a long time that Na+/K+-ATPase is a
substrate for PKC (45, 46); however, establishment of the physiological
relevance of this has been difficult, at least in part due to isoform-
and species-specific structural differences among different
Na+/K+-ATPases that affect their interactions
with PKC (45-48). In the rat cardiac myocytes where the predominant
Na+/K+-ATPase isoforms are known to be good
substrates for PKC (47), it is not unlikely that PKC-induced changes in
the activity and/or endocytosis of
Na+/K+-ATPase (49, 50) may also be a part of
the ouabain-induced feedback cycle (Fig. 9). 3) Finally, we may note
that a number of previous studies, including those in cultured cells
and transgenic mice, have indicated the involvement of various PKC
isoforms and ERK1/2 in the development of cardiac hypertrophy and/or
failure (15, 16, 25, 51, 52). The present demonstration of the sequential ouabain-induced activations of PKC and ERK1/2, coupled with
the previously shown hypertrophic effects of ouabain in cultured myocytes (3, 4), emphasize the need for further studies on the roles of
PKC isoforms in the altered phenotype of the digitalis-treated heart.
*
This work was supported by NHLBI, National Institutes of
Health, Grants HL-36573 and HL-63238 and by institutional funds derived from The Ohio Board of Regents Research Challenge Program. Preliminary accounts of this work have been presented (11, 12).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.
Published, JBC Papers in Press, September 18, 2001, DOI 10.1074/jbc.M107892200
2
The low density, serum-starved myocyte cultures
used here consist of predominantly quiescent cells (i.e.
most cells do not contract spontaneously, and some contract at a low
rate). As indicated before (9), the easily noted ouabain effects in
these cultures are increases in the number of contracting cells and the
rate of contractions. Under these circumstances, it is difficult to quantitate the effect of ouabain on the contractile force (cell shortening) or Ca2+ transients. We have shown elsewhere
(42), however, that in electrically paced cultured myocytes, ouabain
increases systolic [Ca2+]i, diastolic
[Ca2+]i, and the time-averaged
[Ca2+]i, as expected.
The abbreviations used are:
EGFR, epidermal growth factor receptor;
D609, tricyclodecan-9-yl-xanthogenate;
Epep, PKC
Role of Protein Kinase C in the Signal Pathways That
Link Na+/K+-ATPase to ERK1/2*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 
, and 
isoforms of PKC. Dose-dependent stimulation of PKC
translocation by ouabain (1-100 µM) was accompanied by
no more than 50% inhibition of Na+/K+-ATPase
and doubling of [Ca2+]i, changes that do not
affect myocyte viability and are known to be associated with positive
inotropic, but not toxic, effects of ouabain in rat cardiac ventricles.
Ouabain-induced activation of ERK1/2 was blocked by PKC inhibitors
calphostin C and chelerythrine. An inhibitor of phosphoinositide
turnover in myocytes also antagonized ouabain-induced PKC translocation and ERK1/2 activation. These and previous findings indicate that ouabain-induced activation of PKC and Ras, each linked to
Na+/K+-ATPase through Src/EGFR, are both
required for the activation of ERK1/2. Ouabain-induced PKC
translocation and ERK1/2 activation were dependent on the presence of
Ca2+ in the medium, suggesting that the signal-transducing
and ion-pumping functions of Na+/K+-ATPase
cooperate in activation of these protein kinases and the resulting
regulation of contractility and growth of the cardiac myocyte.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(H-7), PKC
(C-15), PKC (C-17), ERK1/2 (K-23), and phosphorylated ERK1/2 (E-4)
were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Calphostin C and chelerythrine were purchased from Calbiochem, D609
from Sigma, phosphatidylserine and dioleyl-sn-glycerol from
Avanti Polar Lipids (Alabaster, AL), Epep from Quality Control
Biochemicals (Hopkinton, MA), and fura-2 and fura-2/AM from Molecular
Probes, Inc. (Eugene, OR).
, Epep
(Epep kinase). The reaction mixtures contained 20 mM
Tris-HCl (pH 7.5), 10 µM [
-32P]ATP, 10 mM magnesium acetate, 0.75 mM
CaCl2, 50 µg/ml leupeptin, either 100 µg/ml histone or
50 µg/ml Epep, with or without 30 µM
phosphatidylserine, and 0.5 µM
dioleyl-sn-glycerol. After incubation at 30 °C, reactions
were stopped by spotting aliquots on phosphocellulose paper (P81) and
washing each paper three times with 25 ml of 0.75% phosphoric acid.
The collected 32P-labeled substrate was counted by
conventional procedures. Reaction time and enzyme concentrations were
chosen to ensure the assay of initial velocities.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of ouabain and PMA on the
translocation of PKC from cytosolic to particulate fractions of
myocytes. A, Ca2+-stimulated PKC activity
(histone kinase). B, Ca2+-independent PKC
activity (Epep kinase). Cells were exposed to 100 µM
ouabain for 10 min or 100 nM PMA for 5 min, lysed, and
assayed for the indicated activities as described under "Experimental
Procedures." Control activities were as follows: particulate histone
kinase, 3.2 ± 1.6 pmol/µg/min; cytosolic histone kinase,
132 ± 22 pmol/µg/min; particulate Epep kinase, 14.7 ± 3.8 pmol/µg/min; cytosolic Epep kinase, 219 ± 33 pmol/µg/min.
Activity of each ouabain- or PMA-treated sample was compared with the
corresponding control. n 
6; *, p < 0.05.
, , and isoforms whose translocation from the
cytosolic to particulate fractions has been shown to be affected by a
number of other stimuli in the neonatal rat cardiac myocytes used here
(21-25). Exposure of myocytes to 100 µM ouabain for 10 min increased the amount of each isoform, assayed with isoform-specific antibodies, in the particulate fraction (Fig.
2). In the cytosolic fraction,
significant ouabain-induced decreases in the levels of immunoreactive
and isoforms, but not in that of isoform, were also
detected (Fig. 2). When the time course of the effects of 100 µM ouabain on the translocation of the three isoforms to the particulate fraction were examined, maximal increases were noted as
early as 1 min after exposure to ouabain, and the increases were
sustained for at least 20 min (Fig.
3).
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Fig. 2.
Effects of ouabain on the translocation of
PKC , PKC, and
PKC. Myocytes were exposed to ouabain as
in Fig. 1. The particulate and the cytosolic fractions were subjected
to Western blot analysis as indicated under "Experimental
Procedures." A, representative blots. B,
quantitative comparisons of blots from multiple experiments.
n 10; *, p < 0.05.
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Fig. 3.
Time course of the ouabain-induced increase
in immunoreactive PKC isoforms in the particulate fraction.
Myocytes were exposed to 100 µM ouabain, and the assays
were done as in Fig. 2. n 3; *, p < 0.05.
to the particulate fraction showed the effectiveness of ouabain
concentrations as low as 1 µM. To allow the direct
comparison of these effects with dose-dependent ouabain
effects on the ion transport function of the
Na+/K+-ATPase of these myocytes, the effects of
selected ouabain concentrations on active Rb+ uptake and on
[Ca2+]i of the intact myocytes were also
determined (Fig. 4, B and C). The highest
concentration of ouabain used in experiments on PKC (100 µM) caused about 50% inhibition of the maximal pumping capacity of Na+/K+-ATPase (Fig. 4B)
and about doubling of the [Ca2+]i (Fig.
4C). Clearly, ouabain-induced PKC activation/translocation is obtained in the absence of toxic Ca2+ overload. This is
in agreement with our previous data (3) showing no significant change
in the viability of these myocytes after 12 h of exposure to
100 µM ouabain.
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Fig. 4.
Comparison of the effects of varying ouabain
concentrations on PKC translocation to the
particulate fraction (A), active uptake of
Rb+ (B), and
[Ca2+]i (C). Immunoblots of
PKC were done as in Fig. 3 (n 3). Ouabain effects
on 86Rb+ uptake were assayed as described under
"Experimental Procedures." At each indicated ouabain concentration,
uptake is expressed as percentage of the maximal uptake inhibited by 1 mM ouabain (n 6). The
[Ca2+]i values, determined as indicated under
"Experimental Procedures," are the time-averaged means of values in
a minimum of 28 cells after 15-20 min of exposure to ouabain. *,
p < 0.05.
(26) and since ouabain transactivates EGFR in the myocytes used here
(8), it was reasonable to suspect that the above ouabain-induced
activation of PKC might be due to stimulation of phosphoinositide
turnover. In fact, ouabain-induced increases in inositol phosphates and
diacylglycerol in several cell types, including cardiac preparations,
have been noted before (27-30), albeit at ouabain concentrations that
are expected to be toxic to cardiac myocytes. To determine whether the
ouabain-induced activation/translocation of PKC noted here was indeed
linked to stimulation of phosphoinositide turnover, we used a PLC
inhibitor, D609. Although this compound is a more selective inhibitor
of phosphatidylcholine-specific PLC in several cell types (31, 32), it
has been shown to inhibit phosphatidylinositol-specific PLC in the
intact neonatal rat cardiac myocytes (24, 33, 34). Pretreatment of
myocytes with D609 blocked ouabain-induced translocation of PKC and
PKC to the particulate fraction (Fig.
5), but it had no significant effect on
PMA-induced translocation of the two isoforms (Fig.
6). This and the fact that D609 also had
no effect on PMA-induced activation of histone kinase and Epep kinase (data not shown) ruled out the possibility that D609 might have a
direct inhibitory effect on PKC. Taken together, the above data support
the proposition that ouabain-induced activation/translocation of PKC is
the consequence of PLC activation linked to the ouabain-induced transactivation of EGFR.
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Fig. 5.
Inhibition of ouabain-induced translocation
of PKC isoforms by D609. Effects of 100 µM ouabain
on control myocytes and those pretreated with 100 µM
D609, a PLC inhibitor, for 12 h were determined as indicated in
Fig. 2. A, representative blots; B, quantitative
comparison of blots from multiple experiments. Each value is expressed
relative to that of the corresponding control. n 5;
*, p < 0.05.
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Fig. 6.
Lack of effect of D609 on PMA-induced
translocation of the PKC isoforms. Experiments were done with 100 nM PMA as indicated in Fig. 5. n 3; *,
p < 0.05.
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Fig. 7.
Antagonism of the ouabain-induced activation
of ERK1/2 by calphostin C, chelerythrine, and D609. Control
myocytes and those pretreated with 0.1 µM calphostin C
(15 min), 5 µM chelerythrine (30 min), and 100 µM D609 (12 h) were treated with 100 µM
ouabain for 5 min and assayed for activations of ERK1/2 as indicated
under "Experimental Procedures." A, representative
blots. B, comparisons of blots from multiple experiments.
The value of each ouabain-treated sample is expressed as a percentage
of the respective control not treated with ouabain. n 4; *, p < 0.05.
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Fig. 8.
Effect of medium Ca2+ on
ouabain-induced translocation of PKC. Myocytes incubated in the
standard Ca2+-containing culture medium or the
Ca2+-free culture medium were exposed to 100 µM ouabain as in Fig. 1 and assayed for histone kinase
and Epep kinase activities of the particulate fractions.
n 4; *, p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Schematic representation of the pathways
involved in ouabain-induced activations of PKC and ERK1/2 in cardiac
myocytes. Ouabain induces an inhibited pool of
Na+/K+-ATPase to communicate with PKC and
ERK1/2 through protein-protein interactions (right).
Reduction of the ion pumping pool of
Na+/K+-ATPase by ouabain leads to rise in
[Ca2+]i (left), which mediates
cross-talk between the pathways linked to the two pools.
Solid arrows indicate events that are supported
by experimental evidence presented here or before. The broken arrows
are the postulated feedback loops; evidence for those involving PKC is
indirect, but there is direct evidence (42) for the feedback regulation
of [Ca2+]i by ERK1/2. See "Discussion" for
further details.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pharmacology,
Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-4182; Fax: 419-383-2871; E-mail: mheck@mco.edu.
ABBREVIATIONS
substrate peptide;
ERK, extracellular signal-regulated kinase/mitogen-activated protein kinase;
MEK, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase;
PKC, protein kinase C;
PLC, phospholipase C;
RACK·PKC, receptor for activated PKC;
PMA, phorbol
12-myristate 13-acetate;
Raf, Raf-1 kinase.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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