Ca2+-independent Phospholipase A2 Is Required for Agonist-induced Ca2+ Sensitization of Contraction in Vascular Smooth Muscle*

Zhenheng GuoDagger , Wen SuDagger , Zhongmin Ma§, George M. SmithDagger , and Ming C. GongDagger

From the Dagger  Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky 40536 and the § Division of Experimental Diabetes and Aging, Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, New York 1002

Received for publication, October 29, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Excitatory agonists can induce significant smooth muscle contraction under constant free Ca2+ through a mechanism called Ca2+ sensitization. Considerable evidence suggests that free arachidonic acid plays an important role in mediating agonist-induced Ca2+-sensitization; however, the molecular mechanisms responsible for maintaining and regulating free arachidonic acid level are not completely understood. In the current study, we demonstrated that Ca2+-independent phospholipase A2 (iPLA2) is expressed in vascular smooth muscle tissues. Inhibition of the endogenous iPLA2 activity by bromoenol lactone (BEL) decreases basal free arachidonic acid levels and reduces the final free arachidonic acid level after phenylephrine stimulation, without significant effect on the net increase in free arachidonic acid stimulated by phenylephrine. Importantly, BEL treatment diminishes agonist-induced Ca2+ sensitization of contraction from 49 ± 3.6 to 12 ± 1.0% (p < 0.01). In contrast, BEL does not affect agonist-induced diacylglycerol production or contraction induced by Ca2+, phorbol 12,13-dibutyrate (a protein kinase C activator), or exogenous arachidonic acid. Further, we demonstrate that adenovirus-mediated overexpression of exogenous iPLA2 in mouse portal vein tissue significantly potentiates serotonin-induced contraction. Our data provide the first evidence that iPLA2 is required for maintaining basal free arachidonic acid levels and thus is essential for agonist-induced Ca2+-sensitization of contraction in vascular smooth muscle.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 GTPgamma S1 (15). Second, the time course and the mass of arachidonic acid released by GTPgamma 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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. alpha -Toxin was purchased from List Biological Laboratories (Campbell, CA). GTPgamma 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 alpha -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 alpha -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 iPLA2beta ). Shown in the figure are representative blots of at least three independent experiments.

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.

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 alpha -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. alpha -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.

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.

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. alpha -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.

To exclude the possibility that BEL selectively blocks the alpha 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 alpha -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.

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 beta -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.

    ACKNOWLEDGEMENT

We thank Dr. Eric J. Smart for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health (NIH) Grant HL67284 (to M. C. G.) and by NINDS, NIH, Grant NS38126 (to G. M. S.).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.

To whom correspondence should be addressed. Tel.: 859-323-6996; Fax: 859-323-1070; E-mail: mcgong2@uky.edu.

Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M211075200

    ABBREVIATIONS

The abbreviations used are: GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; BEL, bromoenol lactone; DAG, diacylglycerol; PDBu, phorbol-12,13-dibutyrate; PE, phenylephrine; PLA2, phospholipase A2; cPLA2, cytosol PLA2; iPLA2, Ca2+-independent PLA2; sPLA2, secretory PLA2; PAP, phosphatidate phosphohydrolase; GFP, green fluorescent protein; DPPC, 1-palmitoyl-2-palmitoyl-L-3-phosphatidylcholine.

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