Regulation of membrane-associated iPLA2 activity by a novel PKC isoform in ventricular myocytes

Sarah A. Steer1, Karin C. Wirsig2, Michael H. Creer2, David A. Ford1, and Jane McHowat2

Departments of 1 Biochemistry and 2 Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-epsilon 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-epsilon and PKC-delta are important for ischemic preconditioning, and PKC-alpha and PKC-beta 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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-beta 1, -eta , -theta , -zeta , and -lambda were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKC-alpha , -beta 2, -epsilon , and -gamma were from Sigma. PKC recombinant standards were from Oxford Biomedical Research (Oxford, MI). Anti-PKC-iota and -delta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Changes in membrane-associated and cytosolic calcium-independent phospholipase A2 (iPLA2) activity after thrombin stimulation (0.05 IU/ml, 1 min) and incubation with phorbol 12-myristate 13-acetate (PMA, 100 nm, 10 min). A and B: intact ventricular myocytes suspended in 1.2 mM Ca-HEPES buffer were incubated with thrombin or PMA, subcellular fractions were prepared, and iPLA2 activity was measured in each fraction. C: subcellular fractions were prepared from untreated ventricular myocytes, the membrane fraction was suspended in buffer 1 containing 200 µM ATP and incubated with thrombin or PMA where appropriate, and then iPLA2 activity was measured. Pretreatment of ventricular myocytes or isolated membrane fractions with the cell-permeable PKC inhibitor GF-109203X (10 nM, 10 min, open bars) had no effect on basal iPLA2 activity but completely inhibited activation of iPLA2 by thrombin or PMA (filled bars). iPLA2 activity was measured in subcellular fractions using 100 µM (16:0, [3H]18:1) plasmenylcholine substrate in the absence of Ca2+ (4 mM EGTA). Values are means + SE for independent results from 6 separate animals (A and B) or 6 separate membrane isolates (C). **P < 0.01 compared with control activity in the absence of GF-109203X.

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-alpha , -beta 1, -beta 2, -gamma , -delta , and -epsilon ; 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-alpha , -beta 1, -epsilon , -eta , and -iota ; the presence of PKC-beta 2, -gamma , -delta , -theta , and -lambda was not detected in several immunoblots.

PKC-alpha , -beta 1, -epsilon , -eta , and -iota were found to be present in the cytosolic fraction (Fig. 2). Both PKC-epsilon and PKC-iota 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-iota is involved; however, the PKC-epsilon 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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Fig. 2.   Immunoblot analysis of PKC isoforms in the cytosolic (Cyt) and membrane (Mem) subcellular fractions isolated from rabbit ventricular myocytes. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with PKC-alpha (1 in 10,000 dilution), PKC-beta 1 (1 in 3,000 dilution), PKC-epsilon (1 in 2,500 dilution), PKC-eta (1 in 2,000 dilution), or PKC-iota (1 in 5,000 dilution) antibodies and incubated with horseradish peroxidase (HRP)-linked secondary antibodies (1 in 50,000 dilution). Immunoblots were detected with enhanced chemiluminescence and multiple exposures to film. The corresponding PKC recombinant standards were used as positive controls on each blot (Pos Con).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta 1, beta 2, and gamma ) and the Ca2+-independent novel PKC isoforms (delta , epsilon , eta , and theta ) are activated by PMA, whereas the Ca2+-independent atypical PKC isoforms (zeta , iota , and lambda ) 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-gamma and -theta , 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-beta isoforms are present in human myocardium, neither is detected in rat myocytes (25), and only PKC-beta 1 was detected in rabbit myocytes. PKC-delta 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-zeta 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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Table 1.   Protein kinase C isoforms detected by immunoblot analysis in adult cardiac myocytes from human, rat, and rabbit

Of the novel PKC isoforms, only PKC-epsilon and PKC-eta were identified by immunoblot analysis in isolated rabbit ventricular myocytes. Immunoblot analysis of cytosolic and membrane subcellular fractions detected the presence of PKC-epsilon in the isolated membrane fraction that was not removed by repeated washing or by the removal of Ca2+. Thus PKC-epsilon 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-epsilon 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-epsilon 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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