Departments of 1 Biochemistry and 2 Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
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Thrombin stimulation of rabbit ventricular
myocytes increases membrane-associated, Ca2+-independent
phospholipase A2 (iPLA2) activity, resulting in
accelerated hydrolysis of membrane plasmalogen phospholipids and
increased production of arachidonic acid and lysoplasmenylcholine. This study was designed to investigate the signal transduction pathways involved in activation of membrane-associated iPLA2.
Incubation of isolated membrane fractions suspended in
Ca2+-free buffer with thrombin or phorbol 12-myristate
13-acetate resulted in a two- to threefold increase in
iPLA2 activity. Prior treatment with the PKC inhibitor
GF-109203X blocked iPLA2 activation by thrombin. These data
suggest that a novel PKC isoform present in the membrane fraction
modulates iPLA2 activity. Immunoblot analysis revealed a
significant portion of PKC- present in the membrane fraction, but no
other membrane-associated novel PKC isoform was detected by this
method. These data indicate that activation of membrane-associated
iPLA2 is mediated by a membrane-associated novel PKC
isoform in thrombin-stimulated rabbit ventricular myocytes.
signal transduction; ventricular myocytes; calcium-independent phospholipase A2; protein kinase C
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INTRODUCTION |
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IN PREVIOUS STUDIES, we have demonstrated that thrombin stimulation of isolated rabbit ventricular myocytes results in activation of a membrane-associated, calcium-independent phospholipase A2 (iPLA2) that preferentially hydrolyzes membrane plasmalogen phospholipids (21). Activation of membrane-associated iPLA2 is also observed when isolated ventricular myocytes are incubated for short intervals with phorbol 12-myristate 13-acetate (PMA), suggesting that the increase in iPLA2 activity is mediated by protein kinase C (PKC) (20). In addition, downregulation of PKC activity by prolonged incubation with PMA results in complete inhibition of the thrombin-stimulated iPLA2 activity (20).
Proteins that participate in signal transduction are generally subject to strict regulation. For PLA2, the enzyme has to be brought into contact with its substrate and its catalytic activity modulated via posttranslational mechanisms such as increased intracellular Ca2+ concentration or protein phosphorylation. In isolated ventricular myocytes, the majority of thrombin-stimulated PLA2 activity does not exhibit a catalytic requirement for Ca2+ and is membrane associated (20, 21), where it is presumably in direct contact with its endogenous phospholipid substrate. Accordingly, activation of iPLA2 by phosphorylation may account for thrombin stimulation of iPLA2 activity. This is supported by the observation that analysis of the sequences of known iPLA2 isoforms demonstrates the presence of several potential PKC phosphorylation sites (12, 17).
Several PKC isoforms have been identified in the myocardium that have
different intracellular locations and are activated preferentially in
response to different stimuli. Alterations in specific myocardial PKC
isoform activity have been reported previously, particularly in
ischemic preconditioning (6, 15, 27),
ischemia-reperfusion (1, 28), heart failure
resulting from cardiomyopathy (3), and diabetes
(11). The exact PKC isoforms that are preferentially activated in these conditions have been difficult to determine; however, PKC- and PKC-
are important for ischemic
preconditioning, and PKC-
and PKC-
1/2 are activated
in heart failure associated with diabetes or nonviral cardiomyopathy.
The PKC isoform(s) that may activate iPLA2 have not been identified to date. Recently, we have treated the isolated membrane fraction prepared from ventricular myocytes with thrombin and observed a significant increase in iPLA2 activity. This suggests that any kinases involved in a signal transduction pathway between the thrombin receptor and membrane-associated iPLA2 would have to be present in this isolated membrane fraction. This study was designed to investigate which kinases may be responsible for membrane-associated iPLA2 activation in ventricular myocytes in response to thrombin stimulation.
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MATERIALS AND METHODS |
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Isolation and incubation of rabbit ventricular myocytes. Adult rabbits of either sex weighing 2-3 kg were anesthetized with intravenous pentobarbitone sodium (50 mg/kg), and the heart was removed rapidly. The heart was mounted on a Langendorff perfusion apparatus and perfused for 5 min with a Tyrode solution containing (in mmol/l) 118 NaCl, 4.8 KCl, 1.2 CaCl2, 1.2 MgCl2, 24 NaHCO3, 1.2 KH2PO4, and 11 glucose (all Sigma Chemical, St. Louis, MO); the Tyrode solution was saturated with 95% O2-5% CO2 to yield a pH of 7.4. This was followed by a 4-min perfusion with a Ca2+-free Tyrode solution containing EGTA (100 µM) and a final perfusion for 20 min with the Tyrode solution containing 100 µM Ca2+ and 0.033% collagenase (type II; Worthington Biochemical). The ventricles were cut into small pieces and shaken in fresh enzyme solution. Individual myocytes were washed with a HEPES buffer containing (in mmol/l): 133.5 NaCl, 4.8 KCl, 1.2 MgCl2, 0.3 CaCl2, 1.2 KH2PO4, 10 glucose, and 10 HEPES (pH = 7.4). Extracellular Ca2+ was increased to 1.2 mM in three stages at intervals of 20 min. Myocytes were incubated overnight in medium 199 (Sigma) with 10% FCS (GIBCO) at 37°C and then washed three times with 1.2 mM Ca2+-HEPES solution.
Phospholipase A2 activity. Myocytes were suspended in 1 ml buffer containing (in mmol/l) 250 sucrose, 10 KCl, 10 imidazole, 5 EDTA, and 2 dithiothreitol (DTT) with 10% glycerol, pH = 7.8 (buffer 1). The suspension was sonicated on ice six times for 10 s, and the sonicate was centrifuged at 20,000 g for 20 min to remove cellular debris and nuclei. The supernatant was then centrifuged at 100,000 g for 60 min to separate the membrane fraction (pellet) from the cytosolic fraction (supernatant). The pellet was washed two times to minimize contamination of the membrane fraction with cytosolic protein by resuspension in buffer 1 and centrifugation at 100,000 g for 60 min. The final pellet was resuspended in buffer 1. In experiments where membrane fractions were isolated and then incubated with thrombin or PKC activity modulators, 200 µM ATP were added to buffer 1 for control and stimulated samples. Phospholipase A2 activity in cytosolic and membrane fractions was assessed by incubating enzyme (8 µg membrane protein or 200 µg cytosolic protein) with 100 µM (16:0, [3H]18:1) plasmenylcholine substrate in assay buffer containing (in mmol/l) 10 Tris, 4 EGTA, and 10% glycerol, pH = 7.0 at 37°C for 5 min in a total volume of 200 µl. Reactions were terminated by the addition of 100 µl butanol, and released radiolabeled fatty acid was isolated by application of 25 µl of the butanol phase to channeled Silica Gel G plates, development in petroleum ether-diethyl ether-acetic acid (70:30:1, vol/vol/vol), and subsequent quantification by liquid scintillation spectrometry. Protein content of each sample was determined by the Lowry method using freeze-dried BSA (Bio-Rad, Richmond, CA) as the protein standard, as described previously (18).
Immunoblot analysis.
Myocytes were suspended in lysis buffer containing (in mmol/l) 10 HEPES
(pH 7.6), 250 sucrose, 2 DTT, 2 EDTA, 2 EGTA, 10 -glycerophosphate, 1 sodium orthovanadate, 2 phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 10 µg/ml aprotinin, and 5 µg/ml pepstatin A
(buffer 2). Cells were sonicated on ice for six bursts of
10 s and centrifuged at 20,000 g at 4°C for 20 min to
remove cellular debris and nuclei. Cytosolic and membrane fractions
were separated by centrifuging the supernatant at 100,000 g
for 60 min. The pellet was resuspended in buffer 2, and the
suspension was centrifuged at 100,000 g for 60 min two times
to minimize contamination of the membrane fraction with cytosolic
protein. The final pellet was resuspended in buffer 2 containing 0.1% Triton X-100. Protein (cytosol or membrane) was mixed
with an equal volume of SDS sample buffer and heated at 95°C for 5 min before loading on a 10% polyacrylamide gel. Protein was separated
by SDS-PAGE at 200 volts for 35 min and electrophoretically transferred
to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) at 100 volts
for 1 h. Nonspecific sites were blocked by incubating the
membranes with Tris buffer solution containing 0.05% (vol/vol) Tween
20 (TBST) and 5% (wt/vol) nonfat milk for 1 h at room
temperature. The blocked PVDF membrane was incubated with primary
antibodies to iPLA2 or PKC isoforms for 1 h at room
temperature. Unbound antibodies were removed with three washes with
TBST solution, and membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies. After six washes with TBST,
regions of antibody binding were detected using enhanced
chemiluminescence (Amersham, Arlington Heights, IL) after exposure to
film (Hyperfilm; Amersham). Multiple exposures of film to the blots
were developed.
Statistics. Statistical comparison of values was performed by the Student's t-test or ANOVA with the Fisher's multiple-comparison test as appropriate. All results are expressed as means ± SE. Statistical significance was considered to be P < 0.05.
Materials.
Anti-iPLA2 was from Cayman Chemical (Ann Arbor, MI).
Anti-PKC-1, -
, -
, -
, and -
were from Santa
Cruz Biotechnology (Santa Cruz, CA). Anti-PKC-
, -
2,
-
, and -
were from Sigma. PKC recombinant standards were from
Oxford Biomedical Research (Oxford, MI). Anti-PKC-
and -
and rat
brain lysate were from Transduction Laboratories (Lexington, KY).
Bisindolylmaleimide (GF-109203X) was from Calbiochem (Santa Cruz, CA).
Bromoenol lactone was a generous gift from Hoffmann La Roche.
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RESULTS |
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In a previous study, we demonstrated that thrombin stimulation of
isolated rabbit ventricular myocytes results in increased membrane-associated iPLA2 activity that is maximal at
30 s and remains elevated over 10 min of stimulation
(19). In this study, we stimulated isolated ventricular
myocytes with thrombin (0.05 IU/ml, 1 min) and measured
iPLA2 activity in cytosolic and membrane subcellular
fractions in the absence of Ca2+ (4 mM EGTA) and using
(16:0, [3H]18:1) plasmenylcholine substrate (Fig. 1,
A and B). Thrombin stimulation resulted in a significant increase in membrane-associated iPLA2 activity (Fig. 1A) with no change in
cytosolic iPLA2 activity (Fig. 1B), suggesting
that thrombin stimulation was not a result of translocation of
cytosolic iPLA2 to the membrane fraction and may be the
result of activation of a latent membrane-associated iPLA2,
as has been proposed previously (13-15). A similar
increase in membrane-associated iPLA2 activity was observed
when isolated ventricular myocytes were incubated with PMA (100 nM, 10 min) before subcellular fractionation, indicating that activation of PKC results in increased membrane-associated iPLA2 activity
(Fig. 1A). Inhibition of PKC activity by pretreatment of
ventricular myocytes with the cell-permeable PKC inhibitor GF-109203X
(10 nM, 10 min) before thrombin stimulation completely inhibited the thrombin-induced increase in iPLA2 activity (Fig.
1A). No significant change in cytosolic iPLA2
activity was observed by modulation of PKC activity (Fig.
1B).
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In further studies, subcellular fractions were prepared from untreated
ventricular myocytes, and the membrane fraction was resuspended in
buffer 1 containing 200 µM ATP. Thrombin stimulation of
the isolated membrane fraction resulted in a significant increase in
membrane-associated iPLA2 activity (Fig. 1C).
Incubation of the isolated membrane fraction with PMA also resulted in
a significant increase in membrane-associated iPLA2
activity that was similar to the increase in iPLA2 activity
observed with thrombin (Fig. 1C). Pretreatment of the
isolated membrane fraction with GF-109203X (an inhibitor of PKC-,
-
1, -
2, -
, -
, and -
; see Ref. 29) before
incubation with thrombin or PMA resulted in complete inhibition of
iPLA2 activation (Fig. 1C).
If thrombin treatment of membrane vesicles isolated from cardiac
myocytes under basal conditions in the absence of Ca2+ can
activate iPLA2 by a mechanism involving PKC, then it is
reasonable to expect that the PKC isozyme involved in the process must
be membrane associated in resting cells. To determine the identity of
PKC isoforms in the membrane fraction that may regulate
thrombin-stimulated iPLA2 activity, subcellular fractions
from untreated ventricular myocytes were prepared in buffer
2, and the cytosolic and membrane fractions were submitted to
immunoblot analysis. Cytosolic and membrane fractions isolated from
rabbit ventricular myocytes were subjected to immunoblot analysis for
each of the PKC isoforms identified to date. Isolated rabbit
ventricular myocytes were found to contain PKC-, -
1,
-
, -
, and -
; the presence of PKC-
2, -
, -
,
-
, and -
was not detected in several immunoblots.
PKC-, -
1, -
, -
, and -
were found to be
present in the cytosolic fraction (Fig.
2). Both PKC-
and PKC-
were also
detected in the isolated membrane fraction from ventricular myocytes,
even after repeated washing and sonication of the membrane fraction under basal conditions, suggesting that a portion of these isoforms is
present in the membrane fraction, although the majority still remains
in the cytosol (Fig. 2). Because incubation of the isolated membrane
fraction in the absence of Ca2+ with PMA results in
iPLA2 activation, it is unlikely that the atypical,
PMA-independent PKC-
is involved; however, the PKC-
isozyme is
Ca2+ independent and stimulated by PMA and is thus an
excellent candidate to mediate the activation of membrane-associated
iPLA2 activity.
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DISCUSSION |
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The sarcolemma of cardiac myocytes is composed of a phospholipid bilayer containing integral membrane proteins that regulate cellular responses through a variety of signal transduction mechanisms and a host of proteins that perform active and passive transport functions (22). Receptor-mediated activation of PLA2 and accompanying hydrolysis of membrane phospholipids results in the stoichiometric production of a free fatty acid and a lysophospholipid; both can affect membrane properties directly or serve as precursors for biologically active metabolites such as eicosanoids and platelet-activating factor. Thus activation of PLA2 and the consequent production of phospholipid metabolites has a vitally important role in the regulation of cardiac myocyte function. Several isoforms of PLA2, differing in substrate preference and calcium dependency, have been identified in the heart. Secretory PLA2 has been identified in the myocardium and isolated cardiac myocytes, and both iPLA2 and cytosolic PLA2 have been identified in either the cytosol or membrane fractions of the heart or in isolated ventricular myocytes (4, 5, 10, 14, 19-21). We have demonstrated that the majority of cardiac myocyte PLA2 activity is iPLA2 (19-21) and that membrane-associated iPLA2 is activated in response to thrombin stimulation in isolated ventricular myocytes, resulting in the preferential hydrolysis of membrane plasmalogen phospholipids and the production of free arachidonic acid and lysoplasmenylcholine (21). In this study, we have demonstrated that direct thrombin stimulation of the isolated membrane fraction from ventricular myocytes results in increased iPLA2 activity, suggesting that this fraction contains the entire signaling pathway between the thrombin receptor and iPLA2. Accordingly, iPLA2 activation after thrombin treatment does not appear to be dependent on cytosolic kinases. Very little is known about the regulation of iPLA2 in the heart, although previous studies have demonstrated that myocardial iPLA2 activity may be regulated by PKC (20), phosphofructokinase (9), ATP (8), and calcium/calmodulin (7, 30).
Incubation of the isolated membrane fraction with PMA results in
activation of iPLA2, and pretreatment with GF-109203X
completely inhibits thrombin-stimulated iPLA2 activity;
thus, a role for regulation of iPLA2 by PKC is likely. A
role for PKC in signal transduction has been demonstrated previously in
several studies (2, 16, 23). PKC isoforms are divided into
three groups based on structural features and cofactor requirements.
Both the Ca2+-dependent conventional PKC isoforms (,
1,
2, and
) and the Ca2+-independent novel PKC isoforms (
,
,
, and
) are activated by PMA, whereas the Ca2+-independent
atypical PKC isoforms (
,
, and
) are PMA independent. The
presence of conventional PKC isoforms in the membrane fraction was not
detected by immunoblot. Additionally, the membrane fraction was
resuspended in a Ca2+-free buffer and, since conventional
PKC isoforms are Ca2+ dependent, it is unlikely they are
involved in membrane-associated iPLA2 activation.
Similarly, because activation of iPLA2 is observed in the
presence of PMA, it is unlikely that the PMA-independent atypical PKC
isoforms are involved.
Multiple PKC isoforms have been demonstrated previously to be present
in the myocardium and cardiac myocytes (Table
1). All PKC isoforms, apart from PKC-
and -
, have been detected in human ventricular myocytes
(26). Cardiac myocytes isolated from other species
demonstrate a different pattern of expression of PKC isoforms. For
example, although PKC-
isoforms are present in human myocardium, neither is detected in rat myocytes (25), and only
PKC-
1 was detected in rabbit myocytes. PKC-
is
present in human and rat myocytes but is not detected in rabbit
myocytes (Fig. 2). Additionally, there are age-dependent differences in
expression of PKC isoforms (24, 25). For example, although
PKC-
is expressed in abundance in the rat fetal heart, its
expression declines markedly by the second postnatal day, and the faint
detection in the adult heart is the result of the presence of the
isoform in nonmyocytic cells. Thus there are both species- and
age-specific differences in myocardial expression of PKC isoforms.
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Of the novel PKC isoforms, only PKC- and PKC-
were identified by
immunoblot analysis in isolated rabbit ventricular myocytes. Immunoblot
analysis of cytosolic and membrane subcellular fractions detected the
presence of PKC-
in the isolated membrane fraction that was not
removed by repeated washing or by the removal of Ca2+. Thus
PKC-
is a likely candidate as the isoform that regulates membrane-associated iPLA2. However, it is possible that the
other novel PKC isoforms may be present in the membrane fraction in a
sufficient amount to activate iPLA2 but not be detectable
by immunoblot analysis.
Although several PKC phosphorylation sites can be identified from the
amino acid analysis of known iPLA2 isoform sequences (12, 17), it is not known whether PKC- is activating
membrane-associated iPLA2 via phosphorylation of the
enzyme. Stimulation of iPLA2 activity in isolated membrane
fractions from ventricular myocytes was performed in the presence of
200 µM ATP (Fig. 1C). Repeated sonication and washing of
the isolated membrane fraction to remove endogenous ATP resulted in an
almost complete loss of iPLA2 activation by thrombin or PMA
(data not shown). Because ATP was present in both the control and
stimulated membrane fractions, the thrombin- or PMA-induced increase in
membrane-associated iPLA2 activity is due to the presence
of thrombin or PMA and does not involve activation of iPLA2
by ATP itself. Together, these data suggest that PKC modulation of
membrane-associated iPLA2 activity requires the presence of
ATP and thus likely occurs via phosphorylation of the enzyme.
In conclusion, from iPLA2 activity measurements, activation
of ventricular myocyte membrane-associated iPLA2 by
thrombin is mediated by a novel PKC isoform. Immunoblot analysis
suggests that PKC- is a likely candidate for this activation since
its presence in the membrane fraction is apparent; however, other novel
PKC isoforms may be present in sufficient amounts to activate iPLA2 but not enough to detect by immunoblot analysis.
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ACKNOWLEDGEMENTS |
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This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-68588 (to J. McHowat) and HL-42665 (to D. A. Ford) and the American Heart Association (National Center to M. H. Creer and Missouri Affiliate to J. McHowat).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. McHowat, Dept. of Pathology, St. Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: mchowatj{at}slucare1.sluh.edu).
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.
10.1152/ajpcell.00109.2002
Received 11 March 2002; accepted in final form 6 August 2002.
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