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INTRODUCTION |
Whereas the increase of intracellular free Ca2+ plays
a pivotal role in the regulation of smooth muscle contraction,
excitatory agonists can induce significant smooth muscle contraction
under constant free Ca2+ by increasing the sensitivity of
the contractile apparatus to Ca2+. The latter is called the
Ca2+ sensitization mechanism. Ca2+
sensitization plays an important physiological role in the regulation of the tonic phase of contraction induced by various agonists (1).
Abnormalities of Ca2+ sensitization and its machinery have
been implicated in the pathophysiology of several cardiovascular
diseases including hypertension, coronary artery spasms, and restenosis
(2-6). Therefore, the molecular basis underlying Ca2+
sensitization has been extensively studied, and many signaling molecules have been identified that regulate Ca2+
sensitization, such as small G-protein RhoA (7, 8) and its effector
Rho kinase (2, 9), myosin phosphatase (10-12), the
heterotrimeric G protein G12/13 (13), arachidonic acid (14, 15), ZIP-like kinase (16), ZIP kinase (17), and phosphatase inhibitory
protein CPI-17 (18-20).
Several lines of evidence suggest that arachidonic acid contributes to
agonist-induced Ca2+ sensitization in vascular smooth
muscle. First, many Ca2+-sensitizing agonists increase
arachidonic acid in vascular smooth muscle, including norepinephrine
(67), angiotensin II (21), endothelin (22), vasopressin (23), and
GTP
S1 (15). Second, the
time course and the mass of arachidonic acid released by GTP
S are
consistent with the role of arachidonic acid as a messenger in the
G-protein-coupled inhibition of phosphatase (15). Third, exogenous
arachidonic acid, not its metabolic products, increases 20-kDa myosin
light chain phosphorylation and causes the contraction of smooth muscle
at constant Ca2+ by dissociating and reducing myosin
phosphatase activity (14, 15). Fourth, arachidonic acid can stimulate
Rho kinase (24, 25) in solution, and arachidonic acid-induced
Ca2+ sensitization of contraction is partially inhibited by
a Rho kinase inhibitor, Y-27632 (25, 68). Finally, a PLA2
inhibitor, ONO-RS-082, concomitantly blocks agonist-induced arachidonic
acid release and Ca2+ sensitization of force (26). However,
the physiological significance of arachidonic acid in agonist-induced
Ca2+ sensitization remains to be established. One major
obstacle in demonstrating the physiological importance of arachidonic
acid in the regulation of smooth muscle contraction is that, to date, the enzyme responsible for agonist-induced arachidonic acid release and
Ca2+ sensitization has not been identified.
Phospholipase A2 (PLA2) is the key enzyme
responsible for the release of arachidonic acid, although the
sequential action of phospholipase C and mono- or diglycerol lipase or
of phospholipase D and phosphatidic acid phosphohydrolase and
diglycerol lipase can also release arachidonic acid. PLA2
comprises a large superfamily of enzymes that hydrolyze the
sn-2 ester bond of phospholipids with the concomitant
production of free fatty acid and lysophosphlipids. Based upon their
cellular location and Ca2+ requirement for enzymatic
activity, PLA2 can be classified into three groups:
secretory PLA2 (sPLA2), cytosolic
PLA2 (cPLA2), and calcium-independent
PLA2 (iPLA2) (27). sPLA2 is an
extracellular enzyme and requires millimolar Ca2+
for enzymatic activity. Both cPLA2 and iPLA2
are intracellular enzymes, but cPLA2 requires
micromolar Ca2+ for its enzymatic activity, whereas
iPLA2 is Ca2+ independent (28). A wide variety
of Ca2+-independent phospholipase A2 activities
have been found in many different tissues (29), but until recently they
were not characterized at the molecular level (30). The cDNA
encoding iPLA2 was cloned and identified in several species
and cell types (31-37). It is now clear that iPLA2
represents a diverse group of enzymes that have distinct and different
sequences, molecular weights, subcellular localizations, and tissue
distributions (30, 38). Consistent with these diversities,
iPLA2 has been implicated in divergent cellular functions,
including membrane phospholipid remodeling (39, 40), glucose-induced
insulin secretion (33, 41), Fas-induced apoptosis (42, 43),
phosphatidylcholine homeostasis (44, 45), cell proliferation (46-48),
eicosanoid synthesis (49, 50), Ca2+ influx (51, 52),
preadipocyte spreading (53), and membrane traffic (54). In smooth
muscle tissue, multiple PLA2 activities, including both
Ca2+-dependent and
Ca2+-independent, have been reported (23, 55, 56).
In the current work, we test the hypothesis that the
iPLA2 mediates, at least in part, free arachidonic
acid release and Ca2+ sensitization of contraction in
vascular smooth muscle. Our results show that iPLA2 is
expressed in vascular smooth muscle tissue, and inhibition of the
endogenous iPLA2 activity by BEL decreases basal free
arachidonic acid levels and diminishes phenylephrine-induced Ca2+ sensitization of contraction. Moreover,
adenovirus-mediated overexpression of exogenous iPLA2 in
mouse portal vein tissue significantly potentiates agonist-induced
contraction. We conclude that iPLA2 is required for
agonist-induced Ca2+ sensitization of contraction in
vascular smooth muscle.
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EXPERIMENTAL PROCEDURES |
Materials--
New Zealand White Rabbits (male, 2-3 kg) were
purchased from Myrtle's Rabbitry, Inc. (Thompson Station, TN).
C57BL/6J mice (male, 7 weeks old) were purchased from Jackson
Laboratories (Bar Harbor, ME). HELSS (BEL), an iPLA2
inhibitor, was purchased from Biomol (Plymouth Meeting, PA).
3H-Labeled arachidonic acid
([5,6,8,9,11,12,14,15-3H]arachidonic acid) was
purchased from ARC (St. Louis, MO). Unlabeled arachidonic acid and a
rabbit polyclonal anti-iPLA2 antibody
(PRFNQNINLKPPTQPADQLV) were purchased from Cayman (Ann Arbor, MI).
Another rabbit polyclonal anti-iPLA2 antibody that
recognizes a different epitope (CTDPDGRAVDR) was purchased from Upstate
(Lake Placid, NY).
1-Palmitoyl-2-[1-14C]palmitoyl-sn-glycero-3-phosphorylcholine
(14C-DPPC) was purchased from Amersham Biosciences. The
unlabeled DPPC was purchased from Avanti Polar Lipids (Alabaster, AL).
1,2-Dioleoyl-sn-glycerol (DAG) and a mouse monoclonal
anti-FLAG antibody M2 was purchased from Sigma. The channeled silica
gel G thin layer chromatography (TLC) plates (20 × 20 cm) with a
preabsorbent zone were purchased from Alltech (Newark, DE). The BCA
protein assay kit was purchased from Pierce.
-Toxin was purchased
from List Biological Laboratories (Campbell, CA). GTP
S was purchased
from Roche Molecular Biochemicals. Phorbol-12,13-dibutyrate (PDBu) was
purchased from Calbiochem. Other chemicals and reagents were purchased
from Sigma or Fisher.
Tissue Preparation--
Rabbit or mouse portal veins were
prepared as previously described (15). Briefly, the adventitia was
removed carefully, and under a light microscope, the endothelium cells
were denuded by gentle rubbing of the inner surface with a razor
blade. The denudation of endothelium was verified by the lose of
acetylcholine-induced relaxation in early experiments.
Isometric Tension Measurement--
Isometric tension
of small strips (3 mm long, 150-200 µm wide, and 75 µm thick) was
measured with a force transducer (AE801; AME, Horten, Norway) in a well
on a "bubble" plate at 24 °C. Details of the solution used for
studies on intact or
-toxin permeabilized were described previously
(57). After the steady responses to high [K+] were
observed, the strips were incubated in a Ca2+-free solution
and permeabilized with 17.5 µg/ml
-toxin for 60 min. To deplete
the sarcoplasmic reticulum of calcium, all permeabilized strips were
treated with A23187 (10 µM; Calbiochem) as described (57).
Western Blot Analysis--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred to a
polyvinylidene difluoride membrane (Fisher). Nonspecific binding sites
on the polyvinylidene difluoride membrane were blocked by 4% nonfat
milk in PBST buffer (PBS plus 0.1% Tween 20). iPLA2 was
detected by using anti-iPLA2 antibodies from Upstate or
from Cayman (Ann Arbor, MI). The immunoreactive bands were blotted with
horseradish peroxidase-conjugated goat-anti-rabbit antibodies (1:7500
dilution; Jackson ImmunoResearch Laboratories, Inc.) and detected by
enhanced chemiluminescence.
Phospholipase A2 Assays--
The iPLA2
activity was assayed using a well established method (58). The reaction
was carried out in a Ca2+-free buffer that virtually
abolishes sPLA2 and cPLA2 activity. 1 mM ATP and 2 mM dithiothreitol were included in
order to stabilize the iPLA2 and inactive sPLA2
activities, respectively. In preliminary experiments, we found that the
majority of the iPLA2 activity was present in the membrane
fraction. Therefore, the membrane fraction was used in the subsequent
iPLA2 assay experiments. The tissue homogenate was
separated to the cytosol and membrane fraction by a 100,000 × g centrifugation at 4 °C. ~100 µg of membrane
proteins were incubated with mixture of 14C-labeled and
unlabeled DPPC for 90 min at 40 °C. iPLA2 activity was
linear with an incubation time of up to 2 h (data not shown). The
free fatty acid released was extracted by the modified Dole reagents,
and the radiolabeled free fatty acid was quantified by liquid
scintillation counting. The iPLA2 specific activity was
expressed as pmol of free fatty acid released/mg of protein in 1 min
(pmol/min/mg).
[3H]Arachidonic Acid Release and DAG
Production--
Rabbit portal vein strips were labeled with
[3H]arachidonic acid (2 µCi/ml) in HEPES-buffered Krebs
solution overnight at 37 °C (15). The strips were washed three times
(20 min each time) in Krebs solution containing 0.2% fatty acid-free
bovine serum albumin (Sigma) and 10 µM BEL or vehicle
(Me2SO, as controls). The strips were then
stimulated with 10 µM phenylephrine for 5 min at
24 °C. The medium containing released [3H]arachidonic
acid was removed and counted by liquid scintillation to determine the
amount of arachidonic acid released. The tissue was used to determine
the tissue DAG level as previously described (15). Briefly, tissue
lipids were extracted twice with chloroform and separated by thin layer
chromatography. The bands corresponding to free fatty acid and DAG were
scrapped off and counted by liquid scintillation.
[3H]Arachidonic acid release and DAG production were
normalized as a percentage of the total 3H counts
incorporated into lipids.
Recombinant Adenovirus Construction--
Rat iPLA2
cDNA was cloned as previously described (33). The
SpeI/StuI-MluI restriction fragment of
pBK-CMV/iPLA2 (33) was generated by PCR. The
SpeI/StuI-MluI fragment of PCR
products was ligated into a modified version of pBluescript KS
(pFLAGmluI), which contains the FLAG epitope (DYKDDDDK). The
SpeI-StuI fragment of pBK-CMV/iPLA2
and the StuI-NotI fragment of
pFLAGmluI/iPLA2 were ligated into a modified version of an
adenoviral shuttle vector (pAdtracgfptre) containing two expression
cassettes, one that uses the cytomegalovirus promoter to drive green
fluorescent protein (GFP) expression and one that uses the tetracycline
response element promoter to drive iPLA2 expression (59).
To generate the pAd-iPLA2 adenovirus, an adenoviral
backbone vector (pAdEasy-1) and the PmeI-linearized
iPLA2 shuttle vector (pAdtracgfptre/iPLA2) were
co-transformed into electrocompetent Escherichia coli BJ5183 cells (60). The successful recombination of these two vectors was
screened by restriction enzyme analyses. To generate the adenovirus, the identified recombinants were linearized with PacI and
transfected into a mammalian packaging cell line (HEK293) by using
LipofectAMINE-plus according to the manufacturer's protocol
(Invitrogen). Expression of GFP and lysis of the HEK 293 cells
were taken as an indication of successful viral production. Moreover,
the expression of iPLA2 was confirmed by Western blot using
anti-iPLA2 and anti-FLAG antibodies. Large quantities of
adenovirus were produced by infecting HEK293 cells in
100-mm2 dishes and purified by cesium chloride gradient
ultracentrifugation (61). The physical number of viral particles was
determined by optical absorbency.
Adenoviral Infection--
The Ad-iPLA2 and Ad/Tet-on
(encoding a tetracycline-regulatory transcription factor) viruses were
co-transfected into A10 smooth muscle cells or mouse portal vein
tissue. The expression of iPLA2 was achieved by adding
doxycycline to the cell culture medium (10% fetal bovine
serum/Dulbecco's modified Eagle's medium; Invitrogen). The adenoviral
transfection condition was optimized to obtain maximal expression of
GFP, FLAG, and iPLA2 and to minimize the cytopathic effect
of the adenovirus. For cultured A10 smooth muscle cells, a multiplicity
of infection of ~200 was found to be sufficient to achieve over 90%
transfection. For mouse portal vein tissue, however, higher doses of
adenovirus (1.77 × 109 viral particles in a total of
100 µl/one-half mouse portal vein) and a longer incubation time (24 h
at 37 °C) were applied.
Statistical Analysis--
Each experiment was repeated a minimum
of three times. Data were expressed as mean ± S.E. Statistical
analysis was performed by using an unpaired t test.
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RESULTS |
iPLA2 Protein Is Expressed in Vascular Smooth Muscle
Tissues--
iPLA2 protein is expressed in many cell
types, including cultured rat vascular smooth muscle cells (62);
however, it is not clear whether the iPLA2 protein is
expressed in fully differentiated vascular smooth muscle tissue. To
address this question, homogenate from the medium of rabbit aorta,
portal vein, or femoral artery were examined by Western blot analysis
using two iPLA2 antibodies that recognize distinct epitopes
of iPLA2. Both antibodies recognized one major band at the
expected molecular mass of about 85 kDa, strongly suggesting
that iPLA2 is expressed in the tissue (Fig. 1). In addition, since iPLA2
has been shown to be the predominant phospholipase A2 in
the brain, rabbit brain was included as a positive control.

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Fig. 1.
Expression of iPLA2 protein in
vascular smooth muscle tissue. Rabbit aorta, femoral arteries
(FA), portal veins (PV), skeletal muscle
(SM), and brain were homogenized in radioimmune
precipitation buffer. 10 µg of cell lysates from each tissue were
analyzed by Western blot using anti-iPLA2 antibodies from
Cayman and Upstate. Note that one major band was identified using
either of the two antibodies that recognizes different epitopes on the
iPLA2 protein (Cayman antibody, amino acids 561-575;
Upstate antibody, amino acids 681-690 of rat iPLA2 ).
Shown in the figure are representative blots of at least three
independent experiments.
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Inhibition of iPLA2 Activity Diminishes PE-induced
Ca2+ Sensitization of Contraction in Vascular Smooth Muscle
Tissues--
To investigate whether iPLA2 plays a role in
the regulation of smooth muscle contraction, the effect of inhibiting
iPLA2 activity on agonist-induced contractions was
determined. BEL was used to selectively inhibit iPLA2
activity, since it has been shown to be over 1,000-fold more selective
for iPLA2 over cPLA2 and sPLA2 (63,
64). We determined and compared PE-induced contractions in a single
tissue strip in the presence or absence of BEL. In preliminary
experiments, the muscle strips were incubated with BEL for 15, 30, and
60 min. We found that 60-min preincubation induced the largest
inhibition of PE-induced contractions; therefore, it was used in all of
the subsequent experiments. We found that BEL not only significantly
inhibits the amplitude of the sustained phase of PE-induced contraction
from 46.8 ± 5.38 mg (n = 4) to 26.4 ± 2.74 mg (n = 4, p < 0.05) but also potently
slows down the rate of force development (the t1/2 from 3.0 ± 0.86 min (n = 4) to 9.0 ± 2.18 min (n = 4, p < 0.05)) (Fig.
2A). In contrast, BEL did not
significantly affect high K+ depolarization-induced
contractions (Fig. 2B). The fact that BEL selectively
inhibits PE-induced but not K+ depolarization-induced
contractions suggests that BEL selectively acts on an agonist-activated
process. Interestingly, BEL does not completely abolish the PE-induced
contraction (Fig. 2A). In the presence of BEL, the
sustained/slow phase of PE-induced contractions may be mediated by
Ca2+. It may also be mediated by BEL-insensitive
phospholipase A2 or due to incomplete inhibition of
iPLA2 activity by BEL.

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Fig. 2.
Inhibition of iPLA2 by BEL
diminishes PE-induced contractions in intact rabbit portal veins.
A, a representative isometric force recording showing that
BEL (10 µM, 60 min) selectively inhibits PE-induced, but
not high K+ (143 mM)-induced contractions, in
intact and endothelium-denuded rabbit portal vein strips. B,
summary of the experiments shown in A. n = 4 each. *, p < 0.05.
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Various agonists including PE cause smooth muscle contractions by both
Ca2+-dependent and Ca2+-independent
mechanisms. To determine specifically whether iPLA2 is
involved in the Ca2+-independent Ca2+
sensitization process, we used a well established
-toxin
permeabilized smooth muscle system (1). In this system, the cytosolic
free Ca2+ can be clamped, and the Ca2+
sensitization can be selectively determined. BEL
dose-dependently inhibited PE-induced
Ca2+-sensitization of contraction. The PE-induced
Ca2+ sensitization of contraction (percentage of maximal
Ca2+-induced contraction) was 49.0 ± 3.63%
(n = 8) in controls and was inhibited to 21.6 ± 3.20% (n = 5, p < 0.01) by 3 µM BEL and to 12.0 ± 1.01% (n = 3, p < 0.01) by 10 µM BEL (Fig.
3A). In contrast, BEL did not
significantly affect the Ca2+-induced contraction (Fig.
3B). The maximal concentration of Ca2+-induced
contraction (absolute force) was 58.9 ± 4.83 mg
(n = 12) in the control and 57.3 ± 4.27 mg
(n = 12, p > 0.05) in the 10 µM BEL-treated groups (Fig. 3B). Contraction
induced by submaximal concentration of Ca2+ (pCa6.3,
expressed as percentage of maximal Ca2+-induced
contraction) was 6.8 ± 3.23% (n = 5) in the
control and was 7.4 ± 1.67% (n = 5, p > 0.05) in the presence of 10 µM
BEL.

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Fig. 3.
Inhibition of iPLA2 by BEL
diminishes PE-induced Ca2+ sensitization of force in
permeabilized rabbit portal veins. -Toxin-permeabilized and
endothelium-denuded rabbit portal vein strips were incubated with
different concentrations of BEL (0, 3, and 10 µM) for 60 min. The strips were then partially contracted with submaximal
concentrations of Ca2+ and stimulated with PE (10 µM) plus GTP (10 µM). Finally, maximal
contractions were obtained by 10 µM Ca2+.
A, PE-induced Ca2+-sensitization of force was
expressed as a percentage of maximal Ca2+-induced
contractions (n = 8 for vehicle, n = 5 for 3 µM BEL, and n = 3 for 10 µM BEL). **, p < 0.01. B,
contractions induced by maximal concentrations of Ca2+ (10 µM) expressed as absolute force (mg). n = 12 for each.
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BEL Inhibits iPLA2 Activity and Decreases Basal Free
Arachidonic Acid Levels in Portal Vein Smooth Muscle Tissue--
To
ensure that the inhibition of Ca2+ sensitization of smooth
muscle contraction by BEL results from its inhibition of
iPLA2 activity, we assayed iPLA2 activity using
an exogenous 14C-DPPC as a substrate in the presence and
absence of BEL and/or PE. As shown in Fig.
4A, BEL (10 µM,
60 min preincubation) significantly inhibited the iPLA2
activity from 5.3 ± 0.30 pmol/mg/min (n = 12) to
2.8 ± 0.2 pmol/mg/min (n = 3, p < 0.01) (Fig. 4A). The iPLA2 activity was not
significantly increased by PE stimulation (10 µM, 5 min),
although there was a trend of increase (from basal 5.3 ± 0.30 pmol/mg/min (n = 12) to 6.6 ± 0.56 pmol/mg/min
(n = 13), p = 0.06). This trend of
iPLA2 activity increase by PE stimulation was abolished by
BEL: 2.7 ± 0.19 pmol/mg/min (n = 4) in the
absence of PE and 2.8 ± 0.20 pmol/mg/min (n = 3)
in the presence of PE.

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Fig. 4.
BEL inhibits iPLA2 activity
(A) and decreases basal free arachidonic acid
(AA) level (B) in vascular smooth
muscle tissue. A, rabbit portal vein strips were
incubated with BEL (10 µM, 60 min) or vehicle
(Me2SO) and then stimulated with PE (10 µM, 5 min). The iPLA2-specific activity was assayed as described
under "Experimental Procedures." n = 4-12 for each
bar. **, p < 0.01. B, rabbit
portal vein strips were labeled with [3H]arachidonic acid
for 24 h and incubated with BEL (10 µM) or vehicle
(Me2SO) for 60 min prior to PE stimulation (10 µM, 5 min). The [3H]arachidonic acid
released to the medium was determined by liquid scintillation counting
and normalized to the total radioactivity incorporated. The arachidonic
acid release was expressed as the means ± S.E. from four
independent experiments. *, p < 0.05; **,
p < 0.01.
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We then investigated the role of BEL-sensitive iPLA2
in maintaining free arachidonic acid levels under basal conditions and with agonist (PE)-stimulation. As shown in Fig. 4B, PE (10 µM, 5 min) stimulated arachidonic acid release in rabbit
portal vein tissues (from 0.07 ± 0.009% (n = 5)
to 0.14 ± 0.013% (n = 5), p < 0.01). Concomitant with the inhibition of basal iPLA2
activity, BEL (10 µM, 60 min preincubation) significantly
inhibited the basal free arachidonic acid level from 0.07 ± 0.009% (n = 5) to 0.03 ± 0.006 (n = 5), p < 0.01). Importantly, in
the presence of BEL, PE stimulation still caused a significant free
arachidonic acid increase (from 0.03 ± 0.006% (n = 5) to 0.08 ± 0.007% (n = 5), p < 0.01). However, free arachidonic acid levels with PE stimulation
were only able to reach a level that is comparable with the resting
level in the absence of BEL (Fig. 4B).
The Selectivity of BEL's Effect on iPLA2 and
Ca2+ Sensitization of Contraction--
In addition to
potently and selectively inhibiting iPLA2 among the classes
of phospholipase A2, BEL also inhibits
Mg2+-dependent phosphatidic acid
phosphohydrolase (PAP-1) (63). PAP-1 can dephosphorylate phosphatidic
acid and yield DAG. PE has been shown to increase the cellular DAG
level in vascular smooth muscle (15), although the DAG and protein
kinase C pathway plays only a minor role in PE-induced Ca2+
sensitization of contraction (19, 64). However, to vigorously test our
hypothesis, we determined whether BEL diminished DAG levels in rabbit
portal vein smooth muscle tissue under our experimental conditions. BEL
(10 µM) did not significantly affect the DAG level in the
presence of PE (3.7 ± 0.14% (n = 4)
versus 4.6 ± 0.62% (n = 4),
p > 0.05) although this concentration of BEL potently inhibited PE-induced Ca2+ sensitization of contraction
(Figs. 2 and 3). This suggests that, under our experimental conditions,
10 µM BEL selectively inhibits iPLA2
activity, or PAP-1 does not contribute significantly to DAG level in
portal vein smooth muscle tissues.
To further investigate the selectivity and the action site of BEL on
the signaling pathways that regulate Ca2+ sensitization of
contraction, we examined the effect of BEL on exogenous arachidonic
acid- and PDBu-induced contractions. Free arachidonic acid is a product
of PLA2 action; therefore, BEL is not expected to affect
exogenous free arachidonic acid-induced contraction. Indeed, BEL (10 µM) had no effect on arachidonic acid (50 µM)-induced contractions (Fig.
5A). On the other hand, PDBu
directly activates protein kinase C to induce Ca2+
sensitization of contraction, and iPLA2 activity is not
required in the pathway downstream of protein kinase C. Therefore, BEL is not expected to affect PDBu-induced Ca2+-sensitization
of contraction. Indeed, BEL (10 µM) did not affect PDBu
(1 µM)-induced Ca2+ sensitization of
contraction (Fig. 5B).

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Fig. 5.
BEL selectively inhibits agonist-induced but
not PDBu- or arachidonic acid-induced Ca2+ sensitization of
force. -Toxin-permeabilized endothelium-denuded rabbit portal
vein strips were incubated with BEL (10 µM) 60 min prior
to stimulation with either arachidonic acid (A, 50 µM), PDBu (B, 1 µM), or
endothelin (ET) (C, 100 nM
endothelin). Contractions are expressed as a percentage of the maximal
concentration of Ca2+ (10 µM
Ca2+)-induced contraction. Data are expressed as the
mean ± S.E. for n = 3 each (A),
n = 3 each (B), and n = 6 each (C). **, p < 0.01.
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To exclude the possibility that BEL selectively blocks the
1 receptor and thus selectively inhibits PE-induced
contractions, we also investigated the effect of BEL on other
agonist-induced contractions. We found that BEL (10 µM)
also significantly inhibited endothelin (100 nM)-induced
Ca2+ sensitization of contraction (percentage of maximal
Ca2+-induced contraction) in
-toxin permeabilized rabbit
portal vein tissue from 11.2 ± 2.09% (n = 3) to
0.6 ± 0.34% (n = 3, p < 0.01) (Fig. 5C). BEL (3 µM) also significantly
inhibited serotonin (1 µM)-induced contractions in intact
(nonpermeabilized) mouse portal vein tissue to 33.1 ± 2.84% of
the control level (n = 5, p < 0.01).
Overexpression of iPLA2 in Vascular Smooth Muscle
Tissues Potentiates Agonist-induced Contractions--
To further
establish the role of iPLA2 in agonist regulation of smooth
muscle contractions, we determined whether overexpression of
iPLA2 in vascular smooth muscle tissue potentiates
agonist-induced contractions. A tetracycline-inducible,
replication-deficient recombinant adenovirus encoding rat
iPLA2 (33) was constructed and used to infect vascular
smooth muscle tissue and overexpress iPLA2 (Fig. 6 and Fig.
7).

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Fig. 6.
Ad-iPLA2 transfection expresses
active iPLA2 protein in A10 smooth muscle cells. The
cultured A10 smooth muscle cells were incubated with a recombinant
adenovirus (multiplicity of infection of 200) encoding
iPLA2 and doxycycline (at concentrations indicated in the
figure) for 12 h. Forty-eight hours later, the cells
were lysed. Expression of iPLA2 protein was determined by
Western blot, and iPLA2 activity was assayed using the
methods described in detail under "Experimental Procedures."
A, schematic representation of the adenoviral construct.
B, iPLA2 immunoblot. 10 µg of protein was
loaded in each lane. Shown is a representative blot from three
independent experiments. C, induction of iPLA2
expression is associated with over a 100-fold increase in
iPLA2 specific activity. n = 3 for each
bar. *, p < 0.05.
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Fig. 7.
Overexpression of iPLA2 in
vascular smooth muscle tissue potentiates serotonin-induced
contractions. The portal vein from each mouse was cut into two
parts of equal size and incubated with Ad-iPLA2 for
24 h. One part was incubated in the presence of doxycycline (2 µg/ml) to induce the expression of iPLA2, whereas the
other part was incubated in the absence of doxycycline to serve as a
control. After incubation with the adenovirus, the portal vein tissues
were incubated at 37 °C for another 40 h. A,
expression of iPLA2 was analyzed by Western blot using an
anti-iPLA2 antibody that recognizes both endogenous and
exogenous iPLA2 and an anti-FLAG antibody that only
recognizes exogenous iPLA2. Six mice portal veins were used
for Western blotting of iPLA2 in three independent blots.
B, transfection efficiency was determined by confocal
microscopy that detects the expression of GFP (left
panel). GFP is expressed regardless of the presence or
absence of doxycycline. Right, a Nomarski image showing all
of the smooth muscle cells at the same cross-layer. Three mice were
used for confocal microscopy detection of GFP expression. C,
expression of iPLA2 shifts the serotonin dose-response
curve to the left and increases the maximal contraction to 153% of
that of the control group. Six mice portal veins were used to determine
the serotonin dose-response curve. **, p < 0.01.
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To demonstrate that the tetracycline-inducible recombinant adenoviral
system produces functional iPLA2 expression, we used an A10
cell line derived from rat aortic smooth muscle. As shown in Fig.
6B, lane 1, FLAG-tagged
iPLA2 (recombinant iPLA2) was not detectable by
Western blot in the absence of doxycycline, indicating that the
expression of recombinant iPLA2 in the smooth muscle cell
line was tightly controlled by tetracycline. The addition of different
concentrations of doxycycline caused a dose-dependent increase in iPLA2 expression (Fig. 6B,
lanes 2-6). Importantly, overexpression of
iPLA2 increased iPLA2 activity up to 100-fold when compared with endogenous iPLA2 activity (Fig.
6C), indicating that the expressed iPLA2 is
enzymatically active.
Adenoviruses have been successfully used to transfer specific genes
into cultured vascular cells to enable gene expression and functional
activity, but application of this technique to the vascular wall is
currently limited by its relatively low efficiency of transfection
(65). In order to monitor the transfection efficiency, a GFP whose
expression was driven by a cytomegalovirus promoter (not an
inducible promoter) was included in the recombinant adenoviral construct (Fig. 6A) (59). Confocal microscopy was used to
monitor the expression of GFP as an indication of the efficiency of
adenoviral transfection. In addition, in order to maximize the
possibility of obtaining a significant functional effect of
overexpressing iPLA2, we sought to use a tissue with a low
level of endogenous iPLA2. Through combined analyses of GFP
expression and the endogenous iPLA2 protein expression
levels in various vascular tissues (portal veins and femoral arteries
from rabbits, rats, and mice), we found that a higher smooth muscle
cell transfection rate and a higher recombinant
iPLA2/endogenous iPLA2 ratio could be obtained
with mouse portal vein tissue. However, PE only induces minimal
contractions in mouse portal vein tissue, whereas serotonin initiates a
good contractile response. Accordingly, we sought to examine the effect of overexpressing iPLA2 on serotonin-induced contractions.
First, we tested whether serotonin-induced contractions were sensitive to BEL. We found that 3 µM BEL very significantly
inhibited serotonin-induced contractions to 33.1 ± 2.84% of the
control level (n = 5, p < 0.01).
Therefore, mouse portal veins were used in subsequent adenovirus experiments. As shown in Fig. 7A, recombinant
iPLA2 protein is readily detected by Western blot using an
anti-iPLA2 antibody and confirmed by an anti-FLAG antibody.
The iPLA2 protein reached about 2.3-fold of the control
level with the adenoviral transfection. In the absence of doxycycline,
only endogenous iPLA2 protein is detected. Moreover,
expression of GFP was detected by confocal microscopy in about 50% of
the smooth muscle cells (Fig. 7B). What is responsible for
the mobility shift between the endogenous mouse iPLA2 and
the overexpressed recombinant iPLA2 (Fig. 7A) remains to be identified; it may relate to the splice variants of
iPLA2.
Finally, we determined the effect of iPLA2 overexpression
on the dose-response curve of serotonin-induced contractions. The portal vein tissue from each mouse was cut into two equal pieces and
randomly divided into the two groups. A total of six mice were used.
Both groups were infected with the same concentration of recombinant
adenovirus encoding iPLA2. One group of portal vein tissue
was treated with doxycycline to induce iPLA2
overexpression, and the other group of portal vein tissue served as the
control (without doxycycline). As shown in Fig. 7C,
expression of iPLA2 in mouse portal vein smooth muscle
significantly potentiated serotonin-induced contractions in comparison
with the controls (0.1 µM serotonin, from 12 ± 1.44 (n = 6) to 20 ± 2.74 mg (n = 6, p < 0.05); 10 µM serotonin, from 19 ± 2.11 (n = 6) to 29 ± 3.9 mg (n = 6, p < 0.05); 100 µM serotonin, from
19 ± 2.07 (n = 6) to 30 ± 3.89 mg
(n = 6, p < 0.05)). The
EC50 was not significantly different between the control
and doxycycline-treated group (log EC50 was
7.3 ± 0.058 versus
7.4 ± 0.039, n = 6 each, p > 0.05). However, the high potassium
depolarization-induced contractions were not significantly different
between the two groups (9 ± 0.66 mg versus 10 ± 1.57 mg, p > 0.05, n = 6 each).
Importantly, doxycycline alone did not significantly affect
serotonin-induced contractions. The maximal contraction induced by
serotonin was 20 ± 2.45 mg (n = 6) in the absence
of doxycycline and 14 ± 4.05 mg (n = 6, p > 0.05) in the presence of doxycycline. The
EC50 of serotonin-induced contractions was not affected by
doxycycline (log EC50 was
7.4 ± 0.11 in the absence
of doxycycline and
7.3 ± 0.09 in the presence of doxycycline, n = 6 each, p > 0.05).
 |
DISCUSSION |
Ca2+ sensitization plays a physiological role in
mediating agonist-induced smooth muscle contractions (1, 66). Multiple lines of evidence suggest that arachidonic acid plays a role in mediating agonist-induced Ca2+ sensitization of contraction
in smooth muscle; however, the molecular mechanisms responsible for
maintaining and regulating free arachidonic acid level are not
completely understood. The major finding of the present study is that
an iPLA2 is required for Ca2+ sensitization of
smooth muscle contraction, and such a requirement results
from the essential role of iPLA2 in maintaining the basal free arachidonic acid levels. Several lines of evidence support this
conclusion. First, immunodetectable iPLA2 protein is
present in portal vein smooth muscle (Fig. 1). Second, inhibition of
iPLA2 activity with BEL diminishes basal free arachidonic
acid levels (Fig. 4), contractions in intact (nonpermeabilized) portal
veins (Fig. 2), and Ca2+ sensitization of contraction in
-toxin-permeabilized tissue preparations (Figs. 3 and 5).
Importantly, BEL does not affect agonist-induced generation of DAG or
high K+-, Ca2+-, PDBu-, or arachidonic
acid-induced contractions (Figs. 2 and 5), indicating the selectivity
of BEL under our experimental conditions. Third, overexpression of
iPLA2 in mouse portal veins by adenovirus-mediated gene
transfer leads to a significant increase in serotonin-induced smooth
muscle contractions (Figs. 6 and 7).
iPLA2 has been implicated in multiple cellular functions in
a variety of cell types and tissues. First, iPLA2 has been
proposed to be responsible for membrane phospholipid remodeling by
providing lysophospholipids as an acceptor for free fatty acid in
murine P388D1 macrophage-like cell line (28, 39). Second,
iPLA2 has been proposed to play a signaling role in
mediating agonist-induced net free arachidonic acid release in
pancreatic islet
-cell and submandibular gland ductal cell (30, 33,
41). Third, proteocleavage by caspase-3-induced
iPLA2 activation has been proposed to be required for the
execution of apoptosis in U937 cells (42, 43). Fourth,
iPLA2 has also been suggested to play a role in cell
proliferation and cell spreading (47, 53). With regard to the function
of iPLA2 in smooth muscle, Gross and co-workers (23)
demonstrated that iPLA2 activity is largely responsible for
[Arg8]vasopressin-induced net arachidonic acid release in
a cultured A10 rat aortic vascular smooth muscle cell line. However,
little is known about the physiological role of iPLA2 in
the regulation of mature vascular smooth muscle function. In
particular, it is unknown whether iPLA2 is involved in the
regulation of mature smooth muscle contractions. In the present study,
we provide several lines of evidence strongly suggesting that
iPLA2 is required for Ca2+ sensitization of
smooth muscle contractions. Our evidence suggests an essential role of
iPLA2 in maintaining basal free arachidonic acid levels
(Fig. 4). Interestingly, agonist stimulation only induces a small and
statistically insignificant increase of iPLA2 activity that
is not proportional to the significant net increase in free arachidonic
acid level. In addition, the phenylephrine-induced net increase in free
arachidonic acid is not significantly different in the presence
versus in the absence of BEL. These findings suggest that
iPLA2 plays only a minor role in the phenylephrine-induced net increase of free arachidonic acid, and a phospholipase
A2 other than the BEL-sensitive iPLA2 is mainly
responsible for the agonist-induced net increase in free arachidonic
acid. This is consistent with the notion that multiple phospholipase
A2 activities, including
Ca2+-dependent and
Ca2+-independent, are present in vascular smooth muscle
tissues (25).
Free arachidonic acid released by iPLA2 may mediate
agonist-induced Ca2+-sensitization of contraction by
multiple molecular mechanisms. Inhibition of the myosin phosphatase
that dephosphorylates the 20-kDa myosin light chain is the downstream
mechanism mediating Ca2+ sensitization of smooth muscle
contractions (12). There are at least two pathways that can couple
agonist stimulation to inhibition of myosin phosphatase. One is the
Rho/Rho kinase pathway, and the other is the protein kinase C/CPI-17
pathway. Arachidonic acid may interact with both pathways or either
pathway to contribute to Ca2+ sensitization of contraction.
For example, arachidonic acid may interact with the Rho/Rho kinase
pathway by activating Rho kinase. Arachidonic acid has been shown to
activate Rho kinase in solution (24), and exogenous arachidonic
acid-induced Ca2+ sensitization of contraction is partially
reversed by inhibiting Rho kinase (68). Arachidonic acid may interact
with the protein kinase C/CPI-17 pathway by activating certain isoforms
of protein kinase C (26). In addition, arachidonic acid can directly
inhibit phosphatase by dissociating the phosphatase subunits (12).
Interestingly, the dissociation of the phosphatase subunits was
reported to occur in isolated smooth muscle cells in an
agonist-specific manner (69). In addition, multiple physiological
agonists can, with different potency and time course, induce vascular
smooth muscle contractions and Ca2+ sensitization (1).
These agonists couple to divergent pathways and, therefore, may
initiate Ca2+ sensitization by turning on different
signals. Arachidonic acid may act through different mechanisms when
stimulated by different agonists. These various possibilities by which
arachidonic acid induces Ca2+ sensitization of contraction
are currently under active investigation in our laboratory.
In summary, we used pharmacological and adenovirus-mediated gene
transfer approaches to either inhibit or promote iPLA2
activity in vascular smooth muscle cell tissues. Our results establish the importance of iPLA2 in maintaining basal free
arachidonic acid levels, contractions, and Ca2+
sensitization in vascular smooth muscle tissues. Our findings may
provide a molecular basis for developing new therapeutic agents for
cardiovascular diseases associated with Ca2+ sensitization.