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(Received for publication, May 1, 1996, and in revised form, June 13, 1996)
From the Division of Bioorganic Chemistry and Molecular
Pharmacology, Departments of Medicine, Chemistry, Pharmacology, and
Molecular Biology, Washington University School of Medicine,
St. Louis, Missouri 63110
ABSTRACT Herein we demonstrate the
calcium-dependent regulation of myocardial phospholipase
A2 activity, which is mediated by a cytosolic protein
constituent that can be chromatographically resolved from, and
subsequently reconstituted with, purified myocardial phospholipase
A2. Purification of this protein by sequential column
chromatographies revealed an 18-kDa doublet, which was identified as
calmodulin by Western blotting, calcium-dependent precipitation with
W-7 agarose beads, and reconstitution of calcium-mediated phospholipase
A2 inhibition with authentic homogeneous calmodulin.
Calcium-induced calmodulin-mediated inhibition of myocardial
phospholipase A2 was titrated by physiologic increments of
calcium ion (Kd ~200 n). Moreover,
ternary complex affinity chromatography with calmodulin-Sepharose
demonstrated that inhibition of myocardial phospholipase A2
activity by calmodulin resulted from the direct interaction of
calmodulin with the myocardial phospholipase A2 catalytic
complex. Exposure of cultured A-10 muscle cells to three structurally
disparate calmodulin antagonists (W-7, trifluoperazine, and
calmidazolium) resulted in the robust release of arachidonic acid,
which was entirely ablated by pretreatment of cells with
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran-2-one.
Collectively, this study identifies a novel mechanism whereby latent
phospholipase A2 activity can be released from tonic
inhibition by alterations in the interactions between the phospholipase
A2 catalytic complex, calcium ion, and the intracellular
calcium transducer, calmodulin.
INTRODUCTION In myocardium the majority of phospholipase A2
activity is catalyzed by a calcium-independent phospholipase
A2, which is selective for plasmalogen substrate containing
arachidonic acid (1, 2, 3). Since this enzyme neither requires calcium as
an obligatory cofactor in catalysis nor employs calcium for membrane
association, it has traditionally been assumed that calcium ion does
not directly regulate the activity of this enzyme (1, 2, 3, 4, 5, 6). However,
alterations in calcium homeostasis play prominent roles in cardiac
physiology, the predominant phospholipid constituents in the
electrically active membrane of myocytes are plasmalogens containing
arachidonic acid (7, 8), and both reaction products of phospholipase
A2 catalysis are potent modulators of ion channel function
(9, 10, 11, 12). Since prior results demonstrated that calcium ion could
inhibit the activity of crude (i.e. cytosolic) myocardial
phospholipase A2 activity (1), we sought to identify a
pathway that could integrate alterations in myocardial phospholipase
A2 activity with changes in myocytic calcium homeostasis
and electrophysiologic function. Herein we describe a novel mechanism
through which calcium ion regulates nominally ``calcium-independent''
myocardial phospholipase A2 through its physical
association with, and functional coupling to, the intracellular calcium
transducer, calmodulin. We now report that myocardial phospholipase
A2 specifically and tightly binds to calmodulin in a
calcium-dependent fashion, that this interaction is
titrated over physiologic increments of calcium ion, that the
interaction between calmodulin and the phospholipase A2
catalytic complex regulates phospholipase A2 activity
in vitro, and that pharmacologic ablation of this
interaction by three structurally disparate calmodulin antagonists
results in the release of arachidonic acid by nominally
``calcium-independent'' phospholipase A2 in intact muscle
cells.
EXPERIMENTAL PROCEDURES The preparation of rabbit myocardial cytosolic
calcium-independent phospholipase A2 and the synthesis of
1-O-(Z)-hexadec-1 Ventricular myocardium
from New Zealand White rabbits was placed in ice-cold buffer (250 m sucrose, 10 m imidazole, 10 m
KCl, 5 m EDTA, pH 7.5) and homogenized utilizing a
Potter-Elvehjem homogenizer. Homogenates were centrifuged at
20,000 × gmax for 20 min, and the
resultant supernatant was heat-treated (90 °C for 3 min) and rapidly
cooled, and precipitated protein was pelleted by centrifugation
(20,000 × gmax for 20 min). The resultant
cytosol was dialyzed against 20 m imidazole, 1 m magnesium acetate, pH 7.5 (Buffer A) at 4 °C and
loaded onto a DEAE-Sephacel column (2.5 × 8.0 cm), and adsorbed
protein was eluted by a linear gradient of NaCl (100 m-1
NaCl). The calcium-dependent inhibitor was
concentrated (Amicon Centriplus-10) and diluted 3-fold with buffer A,
and 500 µg of protein was loaded onto a PC 1.6/5 Mono-Q column
(Pharmacia Biotech Inc.) prior to elution with a linear 1
NaCl gradient in buffer A.
Calcium-dependent inhibition of
myocardial phospholipase A2 was assessed by incubating
partially purified phospholipase A2 (150 µg) with column
fractions (25 µl) or bovine brain calmodulin in a final volume of 200 µl in 100 m Tris-HCl, pH 7.0, containing either 10 m CaCl2 or 4 m EGTA for 2 min at
25 °C. Phospholipase A2 activity was quantified by the
release of [3H]oleic acid from
1-O-(Z)-hexadec-1 Protein
samples (500 µ CaCl2 final concentration)
were loaded onto calmodulin-Sepharose columns pre-equilibrated with 50 m Tris-HCl (pH 7.0) (buffer B) containing 500 µ CaCl2. After application of 10 column
volumes of equilibration buffer, columns were washed with buffer B
containing 4 m EGTA. For experiments involving W-7
agarose, 75 µl of Mono-Q-purified calcium-dependent
inhibitor was adjusted to either 1 m CaCl2 or
4 m EGTA prior the addition of W-7 agarose equilibrated in
20 m imidazole, pH 7.5. After a 60-min incubation at
4 °C, the W-7 agarose was pelleted by centrifugation, and the pellet
was washed (3 times) with 20 m imidazole, pH 7.5, containing either 1 m CaCl2 or 4 m EGTA prior to resuspension.
RESULTS AND DISCUSSION Previously, we have demonstrated that calcium ion inhibits over
80% of crude myocardial cytosolic phospholipase A2
activity (1) and have repeatedly observed this effect in multiple
independent preparations (
To identify the protein constituent mediating these effects,
heat-treated cytosol was subjected to anion exchange chromatography. A
single, well resolved peak of calcium-dependent inhibition
was identified (Fig. 1B), pooled, and subjected to Mono-Q
chromatography (Fig. 1C). The calcium-dependent
inhibitor of myocardial phospholipase A2 eluted as a
single, well resolved UV peak at ~300 m NaCl, which
contained an 18-kDa doublet that precisely cochromatographed with the
observed calcium-dependent inhibition of myocardial
phospholipase A2 (Fig. 1D). SDS-PAGE in the
presence of EGTA demonstrated the collapse of the doublet to a single
band.
Since calmodulin is a heat-stable, acidic 18-kDa protein that migrates
as a doublet on SDS-PAGE in the presence of calcium ion and a single
band in the presence of EGTA (18, 19), the active fractions from Mono-Q
chromatography were further analyzed by Western blotting utilizing
antibodies directed against calmodulin. The 18-kDa protein doublet,
which cochromatographed with calcium-dependent inhibition,
was recognized by anti-calmodulin monoclonal antibody (Fig. 1).
Moreover, authentic bovine brain calmodulin both cochromatographed
with the homogeneous calcium-dependent inhibitor of
myocardial phospholipase A2 (utilizing identical conditions
for Mono-Q chromatography) and inhibited myocardial phospholipase
A2 activity in a calcium-dependent fashion.
To further substantiate the identity of the
calcium-dependent inhibitor of myocardial phospholipase
A2 as calmodulin, W-7 agarose affinity resin was employed
(20). W-7 agarose beads bound the Mono-Q-purified
calcium-dependent inhibitor in the presence of calcium
ion, and the inhibitor was completely released by
subsequent incubation with 4 m EGTA (Fig. 2A).
No binding was manifest in the presence of buffer containing 4 m EGTA. The partitioning of the protein mediating
calcium-dependent inhibition onto W-7 agarose in the
presence of calcium ion and its subsequent release with EGTA correlated
with the amount of calcium-dependent inhibition of
phospholipase A2 (Fig. 2, B and C).
Since the 18-kDa polypeptide, which cochromatographed with
calcium-dependent inhibition of myocardial phospholipase
A2, was: 1) recognized by antibodies directed against
calmodulin; 2) cochromatographed with authentic calmodulin; 3) bound to
W-7 agarose beads in a calcium-dependent fashion; 4)
possessed identical electrophoretic characteristics as authentic
calmodulin; and 5) calmodulin entirely reproduced the
calcium-dependent inhibition of myocardial phospholipase
A2, we conclude that the cytosolic protein constituent
mediating calcium-dependent inhibition of myocardial
phospholipase A2 was calmodulin.
To determine the calcium dependence of calmodulin-mediated inhibition
of myocardial phospholipase A2 activity, measurements of
phospholipase A2 activity were conducted with highly
purified myocardial phospholipase A2 (50,000-fold purified;
10 ng of protein) and authentic calmodulin. Half-maximal inhibition was
manifest at 200 n calcium ion (Fig. 2D), which
closely parallels the Kd of calcium ion for
calmodulin (21, 22, 23). The calcium-induced calmodulin-mediated inhibition
was reversible by subsequent chelation of calcium ion.
To distinguish between the potential mechanisms that were responsible
for the calmodulin-induced alterations in myocardial phospholipase
A2 activity (i.e. phospholipid substrate
sequestration versus a direct interaction between
calcium-activated calmodulin and myocardial phospholipase
A2) calmodulin-Sepharose chromatography was employed (24,
25). The application of myocardial cytosol (in buffer containing 500 µ CaCl2) to a calmodulin-Sepharose affinity
column resulted in the adsorption of less than 5% of the applied
protein while myocardial phospholipase A2 activity was
completely adsorbed. Phospholipase A2 activity was
quantitatively desorbed by application of buffer containing 4 m EGTA (Fig. 3A). The load and
the void contained an identical protein banding pattern and protein
masses (39 versus 38.5 mg for load and void, respectively),
while the EGTA eluent contained only 0.5 mg of protein, which possessed
a completely different banding pattern (Fig. 3B). Myocardial
cytosolic phospholipase A2 activity applied to a
calmodulin-Sepharose column in the presence of EGTA was not adsorbed,
demonstrating that activated calmodulin was required for association
with the myocardial phospholipase A2 catalytic complex.
Moreover, 100-fold increases in substrate concentration (from 2 to 200 µ) did not attenuate calmodulin-mediated phospholipase
A2 inhibition.
To determine whether the interaction between activated
calmodulin and myocardial phospholipase A2 present in
myocardial cytosol was an inherent component of the myocardial
phospholipase A2 complex, highly purified myocardial
phospholipase A2 (>50,000-fold purified from the ATP
affinity chromatography eluent in Ref. 2) was utilized. Similar to
results with crude fractions, activity was quantitatively absorbed in
the presence of calcium ion and desorbed by washing with 4 m EGTA. For comparison, highly purified recombinant 85-kDa
calcium-dependent phospholipase A2 was not
adsorbed onto calmodulin-Sepharose.
To determine the biologic significance of calmodulin-mediated
regulation of phospholipase A2 in intact cells, we examined
the effects of three structurally disparate calmodulin antagonists on
arachidonic acid release in cultures of A-10 muscle cells, a cell line
that predominantly contains ``calcium-independent'' phospholipase
A2 (14). Exposure of prelabeled A-10 muscle cells to W-7
resulted in over an 8-fold increase in [3H]arachidonic
acid release (Fig. 4). Furthermore, half-maximal effects
were manifest at a concentration of W-7, which parallels the
Kd for binding of W-7 by calmodulin (26, 27, 28).
Moreover, preincubation of A-10 muscle cells with BEL prior to the
addition of W-7 ablated the release of [3H]arachidonic
acid (Fig. 4). Finally, utilization of two structurally disparate
calmodulin antagonists, calmidazolium and trifluoperazine, resulted in
similar increases in [3H]arachidonic acid release from
A-10 cells with effective concentrations that approximated the
Kd for the binding of calmidazolium and
trifluoperazine to calmodulin (<1 µ and ~1
µ, respectively) (29, 30, 31, 32, 33). No significant differences in
[3H]arachidonic acid incorporation into phospholipids
were manifest in the presence of W-7.
The results of this study identify the physical association of
calmodulin with the myocardial phospholipase A2 catalytic
complex, the role of calcium ion in facilitating this association, and
the importance of this interaction in modulating phospholipase
A2 activity in intact muscle cells. Since calcium ion is
neither an obligatory cofactor in catalysis nor necessary for the
membrane association of this enzyme (in contrast to secretory
phospholipase A2 (34) and calcium-dependent
phospholipase A2 (35, 36, 37)), it has traditionally been
assumed that calcium ion does not play an important role in modulating
the activity of this enzyme in intact cells (1, 2, 3, 4, 5, 6). These results
clearly demonstrate the importance of calcium ion and the intracellular
calcium transducer, calmodulin, in regulating the activity of nominally
``calcium-independent'' myocardial phospholipase A2. The
calmodulin antagonists resulted in release of
[3H]arachidonic acid from cellular phospholipids even at
ambient cytosolic calcium concentrations ( FOOTNOTES Acknowledgment We gratefully acknowledge the technical
expertise of Jian Wang in the cell culture experiments.
REFERENCES
Volume 271, Number 35,
Issue of August 30, 1996
pp. 20989-20992
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
IMPLICATIONS FOR CARDIAC CYCLE-DEPENDENT ALTERATIONS IN
PHOSPHOLIPOLYSIS*
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
-enyl-2-[9,10-3H]octadec-9-enoyl-sn-glycero-3-phosphocholine
and
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran-2-one
(BEL)1 were performed as described
previously (13, 14). Recombinant 85-kDa calcium-dependent
phospholipase A2 was purified from a baculovirus expression
system as described previously (15). A-10 muscle cells (ATTC CRL 1476)
were cultured and labeled according to established methods (16).
-enyl-2-[9,10-3H]octadec-9-enoyl-sn-glycero-3-phosphocholine
(plasmenylcholine) (2 µ) as described previously (2).
The indicated free calcium ion concentrations employed in the assays
were prepared using buffered CaCl2/EGTA solutions (17).
= ~96 pmol/mg·min in
the presence of EGTA and ~ 18 pmol/mg·min in the presence of
10 m calcium ion). During the course of investigating this
phenomenon, we observed that highly purified preparations of myocardial
phospholipase A2 did not manifest the calcium-mediated
inhibition of enzymic activity, which was present in the crude
cytosolic fraction (Fig. 1). The calcium-mediated
inhibition of purified phospholipase A2 activity could be
reconstituted by the addition of heat-treated cytosol (which did not
contain measurable phospholipase A2 activity) to highly
purified preparations of myocardial phospholipase A2 (Fig.
1A). Furthermore, incubation of heat-treated cytosol with
trypsin completely ablated its ability to reconstitute calcium-mediated
inhibition of purified myocardial phospholipase A2
activity. Collectively, these experiments demonstrate that a
heat-stable protein constituent present in myocardial cytosol was
responsible for the calcium-mediated inhibition of myocardial
phospholipase A2 activity.
Fig. 1.
Characterization and purification of the
calcium-dependent inhibitor of myocardial phospholipase
A2. A, the presence and trypsin sensitivity of
the cytosolic calcium-dependent inhibitor of myocardial
phospholipase A2 were assessed in the absence or presence
of calcium after the addition of heat-treated cytosol to highly
purified myocardial phospholipase A2 as described under
``Experimental Procedures.'' B, the supernatant from
heat-treated myocardial cytosol was chromatographed on a DEAE-Sephacel
column, and individual column fractions were assayed for the
calcium-dependent inhibition of myocardial phospholipase
A2 as described under ``Experimental Procedures.'' 
,
10 m CaCl2; 
, 4 m EGTA.
C, the calcium-dependent inhibitor from
DEAE-Sephacel chromatography was loaded onto a Mono-Q column prior to
elution utilizing a linear gradient of 1 NaCl as
described under ``Experimental Procedures.'' , 10 m
CaCl2; , 4 m EGTA. D, proteins
from Mono-Q chromatography were resolved by SDS-PAGE and subjected to
either silver staining (top) or Western blotting utilizing
mouse monoclonal anti-calmodulin-IgG1 (bottom)
as described under ``Experimental Procedures.''
Fig. 2.
N-(6-Aminohexyl)-5-chloro-1-naphthalene
sulfonamide (W-7) precipitation and calcium dependence of the inhibitor
of myocardial phospholipase A2. The active fraction
(fraction 13) from Mono-Q chromatography (MQ) was incubated
with W-7 agarose beads in the presence of 1 m
CaCl2 as described under ``Experimental Procedures.'' The
mixture was centrifuged, the supernatant was reserved
(SW7), and the pellet was washed and resuspended
in 4 m EGTA (PW7). The resultant
fractions were assessed for inhibition of phospholipase A2
activity (A), protein mass by Coomassie staining
(B), or immunoreactive calmodulin (C) as
described under ``Experimental Procedures.'' D, the
calcium and calmodulin sensitivity of highly purified myocardial
phospholipase A2 was assessed by incubating mixtures
containing selected concentrations of calmodulin (
, 0.2 µg; 
,
0.5 µg; , 1 µg; 
, 2.0 µg; 
, 5.0 µg) with the indicated
amounts of calcium ion in the presence of myocardial phospholipase
A2 and [3H]plasmenylcholine as described
under ``Experimental Procedures.''
Fig. 3.
Calmodulin-Sepharose chromatography of
phospholipase A2 from myocardial cytosol. A,
dialyzed rabbit heart cytosol (39 mg of protein) was loaded onto a 1-ml
column of calmodulin-Sepharose, myocardial phospholipase A2
activity was eluted by the application of buffer containing 4 m EGTA, and phospholipase A2 activity was
quantified as described under ``Experimental Procedures.'' The void
volume contained 38.5 mg of total protein, and the EGTA 1 fraction
contained 0.5 mg of total protein. B, calmodulin-Sepharose
column fractions were individually diluted to a final concentration of
100 µg/ml protein with 10% SDS and 0.05% 
-mercaptoethanol, and
the proteins were resolved on a 10% polyacrylamide gel and
subsequently stained with silver. Calmodulin-Sepharose column fractions
were assayed for calcium-dependent phospholipase
A2 activity as described under ``Experimental
Procedures.''
Fig. 4.
Calmodulin antagonists stimulate the
calcium-independent phospholipase A2-catalyzed release of
arachidonic acid release from A-10 muscle cells. A-10 muscle cells
were prelabeled in media containing 50 µCi of
[3H]arachidonic acid for 16 h as described under
``Experimental Procedures.'' Next, the cells were preincubated with
10 µ BEL or vehicle alone for 15 min prior to exposure
to W-7 (0, 10, 25, or 50 µ) for 60 min. The results are
expressed as the disintegrations/min/plate of released radiolabeled
fatty acid. Forty thousand dpm represents the release of 
40% of
[3H]arachidonate incorporated into cellular phospholipids
during the 16-h prelabeling interval.
100 n)
implying that at least a portion of the calmodulin·phospholipase
A2 catalytic complex is likely present in subcellular loci
containing calcium ion in concentrations greater than that present in
the cytosolic compartment (e.g. membranous intracellular
calcium pools, subsarcolemmal locations at or near calcium channels).
During the cardiac cycle, concentrations of calcium vary from 150 n during diastole to 600 n during systole
(38, 39, 40). The electrophysiologically active membrane of myocytes is
highly enriched in plasmalogen molecular species (7, 8) containing
arachidonic acid (the preferred substrate of this enzyme), and
nonesterified arachidonic acid is a potent modulator of ion channel
function (9, 10, 11, 12). Thus, these results constitute the first description
of a biochemical mechanism that can mediate the cardiac
cycle-dependent alterations in phospholipase
A2-catalyzed liberation of arachidonic acid, thereby
modulating the function of critical sarcolemmal proteins in a cardiac
cycle-dependent fashion.
To whom correspondence should be addressed: Division of Bioorganic
Chemistry and Molecular Pharmacology, Washington University School of
Medicine, 660 South Euclid, Box 8020, St. Louis, MO 63110. Tel.:
314-362-2690; Fax: 314-362-1402.
1
The abbreviations used are: BEL,
(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran-2-one;
PAGE, polyacrylamide gel electrophoresis.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. J. Wolf, J. Wang, J. Turk, and R. W. Gross Depletion of Intracellular Calcium Stores Activates Smooth Muscle Cell Calcium-independent Phospholipase A2. A NOVEL MECHANISM UNDERLYING ARACHIDONIC ACID MOBILIZATION J. Biol. Chem., January 17, 1997; 272(3): 1522 - 1526. [Abstract] [Full Text] [PDF]
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M. J. Wolf and R. W. Gross Expression, Purification, and Kinetic Characterization of a Recombinant 80-kDa Intracellular Calcium-independent Phospholipase A2 J. Biol. Chem., November 29, 1996; 271(48): 30879 - 30885. [Abstract] [Full Text] [PDF]
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G.-i. Atsumi, M. Murakami, K. Kojima, A. Hadano, M. Tajima, and I. Kudo Distinct Roles of Two Intracellular Phospholipase A2s in Fatty Acid Release in the Cell Death Pathway. PROTEOLYTIC FRAGMENT OF TYPE IVA CYTOSOLIC PHOSPHOLIPASE A2alpha INHIBITS STIMULUS-INDUCED ARACHIDONATE RELEASE, WHEREAS THAT OF TYPE VI Ca2+-INDEPENDENT PHOSPHOLIPASE A2 AUGMENTS SPONTANEOUS FATTY ACID RELEASE J. Biol. Chem., June 9, 2000; 275(24): 18248 - 18258. [Abstract] [Full Text] [PDF]
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C. M. Jenkins, M. J. Wolf, D. J. Mancuso, and R. W. Gross Identification of the Calmodulin-binding Domain of Recombinant Calcium-independent Phospholipase A2beta . IMPLICATIONS FOR STRUCTURE AND FUNCTION J. Biol. Chem., March 2, 2001; 276(10): 7129 - 7135. [Abstract] [Full Text] [PDF]
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