Eicosanoid Signaling and Vascular Dysfunction: Methylmercury-Induced Phospholipase D Activation in...

ORIGINAL PAPER

Eicosanoid Signaling and Vascular Dysfunction: Methylmercury-Induced Phospholipase D Activation in Vascular Endothelial Cells

Shariq I. Sherwani • Sheila Pabon • Rishi B. Patel • Muzzammil M. Sayyid •

Thomas Hagele • Sainath R. Kotha • Ulysses J. Magalang •

Krishna R. Maddipati • Narasimham L. Parinandi

Published online: 22 October 2011

� Springer Science+Business Media, LLC 2011

Abstract Mercury, especially methylmercury (MeHg), is

implicated in the etiology of cardiovascular diseases. Ear-

lier, we have reported that MeHg induces phospholipase D

(PLD) activation through oxidative stress and thiol-redox

alteration. Hence, we investigated the mechanism of the

MeHg-induced PLD activation through the upstream reg-

ulation by phospholipase A2 (PLA2) and lipid oxygenases

such as cyclooxygenase (COX) and lipoxygenase (LOX) in

the bovine pulmonary artery endothelial cells (BPAECs).

Our results showed that MeHg significantly activated both

PLA2 (release of [3H]arachidonic acid, AA) and PLD

(formation of [32P]phosphatidylbutanol) in BPAECs in

dose- (0–10 lM) and time-dependent (0–60 min) fashion.

The cPLA2-specific inhibitor, arachidonyl trifluoromethyl

ketone (AACOCF3), significantly attenuated the MeHg-

induced [3H]AA release in ECs. MeHg-induced PLD

activation was also inhibited by AACOCF3 and the COX-

and LOX-specific inhibitors. MeHg also induced the for-

mation of COX- and LOX-catalyzed eicosanoids in ECs.

MeHg-induced cytotoxicity (based on lactate dehydroge-

nase release) was protected by PLA2-, COX-, and LOX-

specific inhibitors and 1-butanol, the PLD-generated PA

quencher. For the first time, our studies showed that MeHg

activated PLD in vascular ECs through the upstream action

of cPLA2 and the COX- and LOX-generated eicosanoids.

These results offered insights into the mechanism(s) of the

MeHg-mediated vascular endothelial cell lipid signaling as

an underlying cause of mercury-induced cardiovascular

diseases.

Keywords Cyclooxygenase � Lipoxygenase �Phospholipase D � Eicosanoid signaling �Phospholipase A2 � Vascular endothelial cells � PLD

Introduction

Mercury (Hg), a highly toxic heavy metal, is a potent

environmental pollutant and has been established to cause

neurotoxicity, cytotoxicity, and immunotoxicity in humans,

but appears to play no known physiological role [1–3].

Mercury-containing dental amalgam fillings have been in

use in dental care worldwide and mercury leaching from

the dental implants in the mouth has been attributed to the

adverse health effects in humans [4]. Mercury also arises as

a toxicant in the industrial environments, contributing to

the occupational disorders/diseases such as respiratory and

lung diseases, cardiovascular diseases, musculoskeletal

disorders, and nervous system diseases among transport

workers [5]. Inorganic form of mercury, which is toxic to

many organisms, undergoes biomethlyation into the highly

toxic form, methylmercury (MeHg) [6]. Dietary con-

sumption of fish has been shown as a major source of

environmental mercury (especially MeHg) in humans that

could lead to suppression of the beneficial effects of

omega-3 fatty acids on the coronary artery disease [7, 8].

Overall, humans are exposed to all these forms of mercury

S. I. Sherwani � S. Pabon � R. B. Patel �M. M. Sayyid � T. Hagele � S. R. Kotha �U. J. Magalang � N. L. Parinandi (&)

Lipidomics, Lipid Signaling, and Vasculotoxicity Laboratory,

Division of Pulmonary, Allergy, Critical Care, and Sleep

Medicine, Department of Internal Medicine, Dorothy M. Davis

Heart and Lung Research Institute, The Ohio State University

College of Medicine, 473 W. 12th Avenue, Columbus,

OH 43210, USA

e-mail: [email protected]

K. R. Maddipati

Department of Pathology, Wayne State University

School of Medicine, Detroit, MI 48202, USA

123

Cell Biochem Biophys (2013) 67:317–329

DOI 10.1007/s12013-011-9304-3

through accidents, environmental pollution, food contami-

nation, dental care, preventive medical practices, industrial

and agricultural operations, and occupational exposures

[7].

Increased exposure to mercury has been correlated to

risk of cardiovascular disease in humans [9]. The role of

mercury toxicity as a possible risk factor in cardiovascular

disease has been discussed [10]. Reports have been made

on the toxic effects of metals in several diseases among

humans including the vascular diseases [9]. Elevated body

levels of mercury, due to fish consumption by humans,

have been hypothesized as a risk factor in coronary heart

disease [11]. Increased levels of urinary mercury have been

shown to be associated with elevated cholesterol levels in

humans and mercury has been suggested as a risk factor of

myocardial infarction, coronary disease, and cardiovascular

disease [12]. Levels of mercury in toenail and urine sam-

ples have been found to be directly correlated with the

increased risk of myocardial infarction, and coronary heart

disease [13]. MeHg has been reported to cause hyperten-

sion in rats [13]. MeHg has also been shown to generate

reactive oxygen species (ROS) in several systems including

the vascular ECs, which can lead to cellular oxidative

stress [14, 15]. Despite the existence of a correlation

between mercury and cardiovascular diseases, there

remains a void on the role of vascular endothelial cells

(ECs) in the mechanism of mercury-induced cardiovascu-

lar diseases. Vascular endothelium is known to play a

pivotal role in the structure and function of the blood vessel

and maintains the homeostasis of the circulatory system

and the body in general [16]. Endothelial dysfunction has

been associated with vascular leak and the breakdown of

the cardiovascular system [17]. Hence, the mercury-

induced cardiovascular diseases may conceivably be a

result of the toxic effects of mercury on the vascular

endothelium.

Mammalian phospholipases are enzymes which specifi-

cally hydrolyze the membrane phospholipids and generate

bioactive lipid second messengers, which play a vital role in

cell signaling and regulation of the cellular functions [16,

18, 19]. One such enzyme, PLA2, is crucial in regulating the

cellular signaling cascades involving the formation and

repair of the phospholipid membrane and generation of the

inflammatory lipid metabolites [19, 20]. In addition to

functioning as a housekeeping enzyme [21], PLA2 hydro-

lyzes the membrane phospholipid at the sn-2 position,

releasing the free unsaturated fatty acid (arachidonic acid,

AA) from the membrane phospholipid and forming the

lysophospholipid [19, 20, 22]. The PLA2-released AA is a

preferred substrate for the lipid oxygenases including the

cyclooxygenases (COXs) and lipoxygenases (LOXs),

which catalyze the formation of bioactive AA metabolites

(eicosanoids) such as prostaglandins and leukotrienes [20].

Generation of the eicosanoids is tightly regulated by PLA2

action and they have been identified as crucial players in the

inflammatory cascades [22]. Another important member of

the phospholipase family, phospholipase D (PLD), is a

ubiquitous lipid signaling enzyme present in all mammalian

cells, and acts exclusively on the substrate phosphatidyl-

choline (PC) [19]. PLD hydrolyzes PC to generate choline

and phosphatidic acid (PA), which is subsequently metab-

olized to either 1,2-diacylglycerol (DAG) by phosphatidate

phosphohydrolase or lysophosphatidic acid (LPA) by

PLA1/PLA2 [18, 19, 23]. These PLD-derived lipid media-

tors have been shown to play vital roles in cell signaling and

signal transduction [16, 19, 22–25].

Earlier, we have reported that MeHg induces activation

of both PLA2 and PLD in the vascular ECs [15, 26–28].

However, the interdependent regulation of these two

phospholipases in the vascular ECs under mercury expo-

sure is yet to be shown. In order to define the underlying

bioactive lipid-mediated mechanism of mercury-induced

vascular dysfunction(s), we hypothesized that MeHg would

induce cytotoxicity in the vascular ECs through the inter-

active signaling of PLA2, COX, LOX, and PLD. Our

results demonstrated that MeHg induced the activation of

PLD and generation of the bioactive lipid, PA, through the

upstream activation of PLA2 and formation of COX- and

LOX-catalyzed eicosanoids, which led to the MeHg-

induced cytotoxicity in the well-established bovine pul-

monary artery ECs (BPAECs).

Materials and Methods

Materials

Bovine pulmonary artery endothelial cells (passage 2)

were purchased from Cell Applications, Inc. (San Diego,

CA). Antibiotic–antimycotic (10,000 units/ml penicillin,

10,000 lg/ml streptomycin, 25 lg/ml amphoteracin B),

fetal bovine serum (FBS), trypsin, and nonessential amino

acids were obtained from Gibco Invitrogen Co. (Grand

Island, NY). Phosphate-free Dulbecco’s modified Eagle’s

medium (DMEM), minimum essential medium (MEM),

MeHg chloride, and lactate dehydrogenase (LDH) cyto-

toxicity assay kits were obtained from Sigma Chemical Co.

(St. Louis, MO). [32P]orthophosphate (carrier-free) was

obtained from New England Nuclear (Wilmington, DE).

Phosphatidylbutanol (PBt) was purchased from Avanti

Polar Lipids (Alabaster, AL). Endothelial cell growth fac-

tor was obtained from Upstate Biotechnology (Lake Placid,

NY). [3H]arachidonic acid (AA) was acquired from

American Radiolabeled Chemicals, Inc. (St. Louis, MO).

Arachidonyl trifluoromethyl ketone (AACOCF3), BEL,

ibuprofen, cinnamyl-3,4-dihydroxy-a-cynanocinnamate

318 Cell Biochem Biophys (2013) 67:317–329

123

(CDC), 5,8,11-eicosatriynoic acid (ETI), 5,8,11,14-eico-

satetraynoic acid (ETYA), baicalein, caffeic acid, aspirin,

and indomethacin were obtained from Cayman Chemical

Co. (Ann Arbor, MI). Primary antibodies for cPLA2 and

phosphoserine-cPLA2, raised in rabbit, were obtained from

Cell Signaling Technology, Inc (Danvers, MA). Secondary

antibody (Anti-Rabbit IgG) and anti-b-actin antibody were

obtained from Amersham Biosciences (Piscataway, NJ).

Cell Culture

Bovine pulmonary artery endothelial cells were cultured in

MEM supplemented with 10% FBS, 1% nonessential

amino acids, 100 units/ml antibiotics (penicillin and

streptomycin) and 5 lg/ml endothelial growth factor

according to our previously published procedures [15, 16].

Cells in culture were maintained at 37�C in a humidified

environment of 95% air–5% CO2 and grown to contact-

inhibited monolayers with typical cobblestone morphol-

ogy. When confluence was reached, cells were trypsinized

and sub-cultured in sterile 35 or 17.5-mm tissue culture

dishes. Confluent cells were observed for cobblestone

morphology under light microscope and stained positive

for factor VIII. All experiments were conducted between 2

and 10 passages (75–80% confluence).

LDH Assay of Cytotoxicity

Cytotoxicity in BPAECs was determined by spectropho-

tometric determination of the extent of release of lactate

dehydrogenase (LDH) from cells according to our previ-

ously published methods [17, 26, 27]. BPAECs grown in

17.5-mm dishes were pretreated with MEM alone or MEM

containing the selected pharmacological inhibitors for 1 h

and then treated with MEM alone or MEM containing

MeHg for the desired lengths of time. At the end of

treatment, the medium was collected and the LDH released

into the medium was determined according to the manu-

facturer’s recommendations (Sigma Chemical Co.,

St. Louis, MO).

Ecosanoid Determination by Liquid Chromatography-

Mass Spectrometry (LC-MS)

Samples (media) were spiked with 10 ng of 8-iso prosta-

glandin F2a-d4 and 15(S)-HETE-d8 as internal standards and

mixed well. SEP-Pak C18 cartridges (100 mg adsorbent;

Waters Corporation, MA) were equilibrated with 1 ml each

of methanol followed by water. The internal standard sup-

plemented samples were applied to the conditioned C18

cartridges and the cartridges were washed with 5 ml of water

followed by 5 ml of hexane. Eicosanoids were eluted with

500 ll of ethyl acetate. The eluate was dried under nitrogen

and reconstituted in methanol:25 mM aqueous ammonium

acetate (1:1). The extracted and reconstituted sample was

subjected to HPLC on a Luna C18 column (2 9 150 mm,

3 l, Phenomenex) eluted with methanol:13 mM aqueous

ammonium acetate:acetonitrile gradient at a flow rate of

0.2 ml/min. Initial composition of the solvent gradient

50:45:5 (CH3OH:aq.NH4OCOCH3:CH3CN) was changed to

90:5:5 in 30 min and the final conditions were maintained for

5 min. The eluent was monitored for the eicosanoids by mass

spectrometer (QuattroLC; Micromass, UK) in the negative

ion mode under the following conditions: Source block:

120�C, Desolvation: 350�C, and Collision gas pressure:

3.2 9 10-4 mbar. Multiple Reaction Monitoring (MRM)

was used to detect and quantify the entire range of eicosa-

noids using this method and the complete list of transitions

are published elsewhere [29]. MRM transitions for the

detected eicosanoids were as follows (m/z): 8-iso PGF2a-d4:

357 ? 197, 8-iso PGF2a: 353 ? 193, PGE2: 351 ? 271,

15(S)-HETE-d8: 327 ? 226, 15(S)-HETE: 319 ? 219,

12(S)-HETE: 391 ? 179, and 5(S)-HETE: 319 ? 115.

Cone voltage (CV) and collision energies (CE) used for

MRM of the detected eicosanoids and the corresponding

deuterated standards were as follows: 8-iso PGF2a—40 V,

25 eV; prostaglandin E2—25 V, 15 eV; and for all HE-

TEs—25 V, 14 eV. Under these conditions, the retention

times for the detected eicosanoids were as follows:

8-iso prostaglandin F2a—9.8 min, PGE2—8.4 min, 15(S)-

HETE—26.3 min, 12(S)-HETE—27.5 min, and 5(S)-HETE—

29.2 min.

Phospholipase D activation in intact ECs: PLD activity in

BPAECs was determined according to our previously pub-

lished procedures [15, 16, 19]. BPAECs in 35-mm dishes

(5 9 105 cells/dish) were labeled with [32P]orthophosphate

(5 lCi/ml) in DMEM phosphate-free medium containing

2% (v/v) fetal bovine serum for 6–14 h. Cells were washed

with MEM and incubated at 37�C in 1 ml of MEM con-

taining 0.05% (v/v) 1-butanol in absence and presence of the

desired concentrations of MeHg for different lengths of time

under a humidified 95% air–5% CO2 atmosphere. In some

experiments, wherever required, ECs were pre-treated for

1 h with the selected pharmacological inhibitors prior to

exposure to MeHg for 30 min. The incubations were termi-

nated by addition of 1 ml methanol:HCl (100:1 v/v). Lipids

were extracted essentially according to the method of Bligh

and Dyer as described previously [15, 28]. [32P]-labeled

phosphatidylbutanol ([32P]PBt), formed from PLD activa-

tion and transphosphatidylation reaction as an index of PLD

activity in intact cells, was separated by thin-layer chroma-

tography (TLC). Radioactivity associated with the [32P]PBt

was quantified by liquid scintillation counting and data were

expressed as DPM normalized to 106 counts in the total

cellular lipid extract.

Cell Biochem Biophys (2013) 67:317–329 319

123

[3H]AA Labeling and Assay of PLA2 Activity

The activity of PLA2 was assayed according to our previ-

ously published procedure [19, 26, 27]. BPAECs in 35-mm

dishes (70% confluence) were labeled for 12 h with 1 ml of

the medium containing 0.5 lCi/ml of [3H]AA following

which they were washed and treated with MEM alone or

MEM-containing MeHg (1–10 lM) for the desired lengths

of time. Following the treatment of cells, [3H]AA released

into the medium was measured in the Packard Tricarb 2900

TR liquid scintillation counter. PLA2 activity was expres-

sed as DPM of [3H]AA released/dish.

SDS-PAGE and Western Blotting

Preparation of cell lysates, separation of proteins by

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS-PAGE), and Western blotting were done according

to our previously published procedures [16]. Cell lysates

containing equal amounts of proteins (40 lg) were sub-

jected to SDS-PAGE on 12% gels, transferred on to

polyvinylidene difluoride (PVDF) membranes, and then

subjected to overnight immunoblotting with either anti-

cPLA2 or anti-phosphoserine-cPLA2 (1:1000 dilution)

rabbit polyclonal antibodies at 4�C. The membranes were

then washed three times with TBST and incubated for

1–2 h at room temperature with horseradish peroxidase-

conjugated goat anti-rabbit secondary antibody (1:2000

dilution). The immunoblots were then developed on the

film with the enhanced chemiluminescence (ECL) reagents

according to the manufacturer’s recommendations. The

intensities of protein bands developed on the film were

quantified by digital densitometric analysis.

Statistical Analysis of Data

All experiments were done in triplicates and data were

expressed as mean ± standard deviation (SD). One-way

analysis of variance (ANOVA) and pair wise multiple

statistical comparisons were done by Dunnett’s method

with P \ 0.05 indicating significance.

Results

MeHg Activates PLD in Dose- and Time-Dependent

Manner in BPAECs

We conducted studies to determine whether MeHg would

cause activation of PLD in BPAECs. MeHg (1, 5, and

10 lM) at 30 and 60 min of incubation, induced significant

activation of PLD ([32P]PBt formation) as compared with

the same in the control untreated cells (6-, 12-, and 13-fold

for 30 min and 2-, 9-, and 17-fold for 60 min of treatment

at concentrations of 1, 5, and 10 lM, respectively)

(Fig. 1a). Treatment of ECs with MeHg (5 lM) for 15, 30,

45, and 60 min induced significant enhancement (6-, 4-,

13-, and 19-fold) of the activation of PLD as compared

with the same in the control untreated cells (Fig. 1b). These

results revealed that MeHg induced PLD activation in

dose- and time-dependent manner in BPAECs.

MeHg Activates PLA2 in Dose- and Time-Dependent

Manner in BPAECs

We conducted studies to determine whether MeHg would

cause activation of PLA2 in BPAECs. MeHg (1, 5, and

10 lM) at 30 and 60 min of incubation, induced significant

activation of PLA2 ([3H]AA release) as compared with the

same in the control untreated cells (3-, 10-, and 24-fold for

30 min and 5-, 22-, and 59-fold for 60 min of treatment at

concentrations of 1, 5, and 10 lM, respectively) (Fig. 2a).

Treatment of ECs with MeHg (5 lM) for 15, 30, 45, and

60 min induced significant enhancement (4-, 2-, 11-, and

12-fold) of the activation of PLA2 as compared with the

same in the control untreated cells (Fig. 2b). These results

Fig. 1 MeHg activates PLD in dose- and time-dependent manner in

BPAECs. BPAEC monolayers (5 9 105 cells/35-mm dish) were

labeled with [32P]orthophosphate (5 lCi/ml) in phosphate-free MEM

for 6-14 h. Following [32P]orthophosphate labeling, the cells were

treated with MEM alone or MEM containing different concentrations

(1, 5, and 10 lM) of MeHg for 30 and 60 min (a) or for different time

periods (0–60 min; b) in presence of 0.05% (v/v) 1-butanol. At the

end of incubation period, [32P]PBt formed was determined as

described under Materials and Methods. Data represent mean ± SD

calculated from three independent experiments. *Significantly differ-

ent at P \ 0.05 as compared to cells treated with MEM alone


123

revealed that MeHg-induced PLA2 activation in dose- and

time-dependent manner in BPAECs.

PLA2-Specific Inhibitors Attenuate MeHg-Induced

PLD Activation in BPAECs

Earlier experiments of the current study showed that MeHg

caused the activation of both PLA2 and PLD. Therefore,

we investigated whether PLA2 activation would be

upstream of PLD activation in ECs under MeHg exposure.

In order to establish the role of PLA2 activation in the

MeHg-induced PLD activation in BPAECs, the well-

established PLA2-specific inhibitors (AACOCF3 for cPLA2

and BEL for iPLA2) were used. Cells were pre-treated for

1 h with MEM alone or MEM containing the chosen PLA2-

specific inhibitor (1, 3, and 5 lM) and then treated for

30 min with MeHg (5 lM). AACOCF3 caused effective

and significant attenuation of the MeHg-induced PLD

activation in BPAECs (58, 78, and 76% of inhibition at

concentrations of 1, 3, and 5 lM, respectively) (Fig. 3a).

BEL also offered effective and significant attenuation of

the MeHg-induced PLD activation in BPAECs (44, 82, and

69% of inhibition at concentrations of 1, 3, and 5 lM,

respectively) (Fig. 3b). These results revealed that the

PLA2-specific inhibitors effectively attenuated the MeHg-

induced PLD activation in BPAECs, suggesting the

involvement of upstream cPLA2 activation in the MeHg-

induced activation of PLD in BPAECs.

COX-Specific Inhibitors Attenuate MeHg-Induced PLD

Activation in BPAECs

As the earlier experiment of this study showed that MeHg-

induced PLA2 activation and caused the release of AA, we

further investigated whether the COX-generated eicosa-

noids would be involved in the MeHg-induced PLD

activation in BPAECs. In order to establish the role of

COX-generated eicosanoids in the MeHg-induced PLD

activation in BPAECs, here, the well-established general-

COX inhibitors (ibuprofen and CDC) were utilized. Cells

were pre-treated for 1 h with MEM alone or MEM con-

taining the chosen COX inhibitors (100 and 300 lM for

ibuprofen and 10 and 25 lM for CDC) and then treated for

30 min with MeHg (5 lM). Ibuprofen caused effective and

significant attenuation of the MeHg-induced PLD activa-

tion in BPAECs (9 and 30% at inhibition for concentrations

Fig. 2 MeHg activates PLA2 in dose- and time-dependent manner in

BPAECs. BPAEC monolayers (5 9 105 cells/35-mm dish) were

labeled with [3H]AA (5 lCi) in complete EC medium for 12 h.

Following [3H]AA labeling, the cells were treated with MEM alone or

MEM containing different concentrations (1, 5, and 10 lM) of MeHg

for 30 and 60 min (a) or for different time periods (0–60 min; b). At

the end of incubation, [3H]AA released into the medium was

determined as described under ‘‘Materials and Methods’’ section.

Data represent mean ± SD calculated from three independent

experiments. *Significantly different at P \ 0.05 as compared to

cells treated with MEM alone

Fig. 3 PLA2-specific inhibitors attenuate MeHg-induced PLD acti-

vation in BPAECs. BPAEC monolayers (5 9 105 cells/35-mm dish)

were labeled with [32P]orthophosphate (5 lCi/ml) in phosphate-free

MEM for 6–14 h. Following [32P]orthophosphate labeling, the cells

were pretreated for 1 h with MEM alone or MEM containing

AACOCF3 (1, 5, and 10 lM; a) or BEL (1, 5, and 10 lM; b) and then

subjected to treatment with MEM alone or MEM containing MeHg

(5 lM) for 30 min in presence of 0.05% (v/v) 1-butanol. At the end of

incubation, [32P]PBt formed was determined as described under

‘‘Materials and Methods’’ section. Data represent mean ± SD of three

independent experiments. *Significantly different at P \ 0.05 as

compared to cells treated with MEM alone. **Significantly different

at P \ 0.05 as compared to cells treated with MEM-containing MeHg

alone


123

of 100 and 300 lM, respectively) (Fig. 4a). CDC also

offered effective and significant attenuation of the MeHg-

induced PLD activation in BPAECs (28 and 51% of inhi-

bition at concentrations of 10 and 25 lM, respectively)

(Fig. 4b). These results revealed that the general-COX

inhibitors effectively attenuated the MeHg-induced PLD

activation in BPAECs, suggesting the involvement of

COX-generated eicosanoids in the MeHg-induced

upstream activation of PLD in BPAECs.

General-LOX Inhibitors Attenuate MeHg-Induced PLD

Activation in BPAECs

Earlier experiments of this study revealed that the COX-

generated eicosanoids were involved in the MeHg-induced

PLD activation, and therefore we further investigated to

determine whether the related LOX-generated eicosanoids

would be involved in the MeHg-induced PLD activation in

BPAECs. In order to establish the role of LOX-generated

eicosanoids in the MeHg-induced PLD activation in

BPAECs, the well-established general-LOX inhibitors (ETI

and ETYA) were used. Cells were pre-treated for 1 h with

MEM alone or MEM containing the chosen LOX inhibitors

(10, 50, and 100 lM) and then treated for 30 min with

MeHg (5 lM). ETI caused effective and significant atten-

uation of the MeHg-induced PLD activation in BPAECs

(54, 79, and 77% of inhibition at concentrations of 10, 50,

and 100 lM, respectively) (Fig. 5a). ETYA also offered

effective and significant attenuation of the MeHg-induced

PLD activation in BPAECs (1, 27, and 52% of inhibition at

concentrations of 10, 50, and 100 lM, respectively)

(Fig. 5b). These results revealed that the general-LOX

inhibitors effectively attenuated the MeHg-induced PLD

activation in BPAECs, suggesting the involvement of the

LOX-generated eicosanoids in the MeHg-induced

upstream activation of PLD in BPAECs.

12-LOX-Specific Inhibitors Attenuate MeHg-Induced

PLD Activation in BPAECs

Earlier experiments of this study revealed that the LOX-

generated eicosanoids were involved in the MeHg-induced

PLD activation, and therefore we further investigated to

demonstrate whether the 12-LOX-generated eicosanoids

would be responsible for the MeHg-induced PLD

activation in BPAECs. In order to establish the role of

Fig. 4 COX-specific inhibitors attenuate MeHg-induced PLD acti-




were pretreated for 1 h with MEM alone or MEM containing

ibuprofen (100 and 300 lM; a) or CDC (5, 10, and 25 lM; b) and

then subjected to treatment with MEM alone or MEM containing

MeHg (5 lM) for 30 min in presence of 0.05% (v/v) 1-butanol. At the

end of incubation, [32P]PBt formed was determined as described

under Materials and Methods. Data represent mean ± SD of three



at P \ 0.05 as compared to cells treated with MEM containing MeHg

alone

Fig. 5 LOX-specific inhibitors attenuate MeHg-induced PLD acti-




were pretreated for 1 h with MEM alone or MEM containing ETI (10,

50, and 100 lM; a) or ETYA (10, 50, and 100 lM; b) and then

subjected to treatment with MEM alone or MEM-containing MeHg



‘‘Materials and Methods’’ sectiont at P \ 0.05 as compared to cells

treated with MEM alone. **Significantly different at P \ 0.05 as

compared to cells treated with MEM-containing MeHg alone


123

12-LOX-generated eicosanoids in the MeHg-induced PLD

activation in BPAECs, here, the well-established 12-LOX

inhibitors (baicalein and caffeic acid) were used. Cells

were pre-treated for 1 h with MEM alone or MEM con-

taining the chosen 12-LOX-specific inhibitors (5 lM for

baicalein and 10, 50, and 100 lM for caffeic acid) and then

treated for 30 min with MeHg (5 lM). Baicalein caused

effective and significant attenuation of the MeHg-induced

PLD activation in BPAECs (87% of inhibition for 5 lM)

(Fig. 6a). Caffeic acid also offered effective and significant

attenuation of the MeHg-induced PLD activation in

BPAECs (66, 75, and 79% of inhibition at concentrations

of 20, 50, and 100 lM, respectively) (Fig. 6b). These

results revealed that the 12-LOX-specific inhibitors sig-

nificantly attenuated the MeHg-induced PLD activation in

BPAECs, suggesting major involvement of 12-LOX-gen-

erated eicosanoids in the MeHg-induced activation of PLD

in BPAECs.

MeHg Induces COX- and LOX-Catalyzed Formation

of Eicosanoids in BPAECs

Earlier in this study, the involvement of PLA2 activity and

the COX- and LOX-generated eicosanoids were shown to

be involved in the MeHg-induced PLD activation in

BPAECs. Therefore, we further determined the extent of

COX- and LOX-generated eicosanoids in BPAECs fol-

lowing MeHg exposure. Treatment of ECs with MeHg

(5 lM) for 15, 30, and 60 min induced significant increase

(27-, 31-, and 48-fold) in PGE2 release as compared with

Fig. 6 12-LOX-specific inhibitors attenuate MeHg-induced PLD

activation in BPAECs. BPAEC monolayers (5 9 105 cells/35-mm

dish) were labeled with [32P]orthophosphate (5 lCi/ml) in phosphate-

free MEM for 6–14 h. Following [32P]orthophosphate labeling, the

cells were pretreated for 1 h with MEM alone or MEM containing

baicalein (5 lM; a) or caffeic acid (20, 50, and 100 lM; b) and then




‘‘Materials and Methods’’ section. Data represent mean ± SD of three



at P \ 0.05 as compared to cells treated with MEM-containing MeHg

alone

Fig. 7 MeHg induces COX- and LOX-catalyzed formation of

eicosanoids in BPAECs. BPAEC monolayers (5 9 105 cells/35-mm

dish) were treated with MEM alone or MEM containing different

concentrations (1, 5, and 10 lM) of MeHg for desired amount of time

and the formation of PGE2 (a), 5-HETE (b), 12-HETE (c), and

15-HETE (d) was measured as described under ‘‘Materials and

Methods’’ section. Data represent mean ± SD calculated from three


compared to cells treated with MEM alone


123

the same in the control untreated cells (Fig. 7a). MeHg (1,

5, 10 lM), at 30 min of incubation, induced significant

5-HETE formation (2-, 1.5-, and 1.1-fold) as compared

with the same in the control untreated cells (Fig. 2a).

MeHg (5 lM) at 15, 30, and 60 min of treatment induced

significant increase in the 12-HETE formation (1.4-, 1.3-,

and 1.4-fold) as compared with the same in the control

untreated cells (Fig. 7c). MeHg (5 lM) at 15, 30, and

60 min of treatment induced significant increase in the

15-HETE formation (13-, 13-, and 12-fold) as compared

with the same in the control untreated cells (Fig. 7d). These

results revealed that MeHg enhanced the extent of forma-

tion of the COX- and LOX-generated eicosanoids in

BPAECs.

COX- and LOX-Specific Inhibitors Attenuate MeHg-

Induced LDH Release in BPAECs

Earlier in this study, MeHg was shown to activate PLA2

and generate eicosanoids in BPAECs. Our earlier studies

have also shown that cPLA2 inhibitor (AACOCF3) and

iPLA2 inhibitor (BEL) attenuate MeHg-induced cytotox-

icity in BPAECs [26, 27]. Therefore, here, we investigated

to demonstrate whether the formation of PA and the sub-

sequent COX- and LOX-generated eicosanoids would

be responsible for the MeHg-induced cytotoxicity in

BPAECs. In order to establish the role of COX- and LOX-

generated eicosanoids in the MeHg-induced cytotoxicity in

BPAECs, the well-established general-COX and LOX

inhibitors (indomethacin and ETYA) were used. Cells were

pre-treated for 1 h with MEM alone or MEM containing

the chosen inhibitors (100 lM) and then treated for 30 min

with MeHg (5 lM). Indomethacin offered effective and

significant attenuation of the MeHg-induced LDH leak in

BPAECs (45% of inhibition at a concentration of 100 lM)

(Fig. 8a). ETYA also offered effective and significant

attenuation of the MeHg-induced LDH leak in BPAECs

(73% of inhibition at a concentration of 100 lM) (Fig. 8b).

These results revealed that the COX- and LOX-specific

inhibitors effectively attenuated the MeHg-induced LDH

leak in BPAECs, suggesting the involvement of COX- and

LOX-generated eicosanoids in the MeHg-induced cyto-

toxicity in BPAECs.

PLA2-Specific Inhibitor and 1-Butanol, the Quencher

of PLD-Generated PA, Attenuate MeHg-Induced LDH

Release in BPAECs

Earlier in the study, MeHg was shown to induce cytotox-

icity which was reversed by the COX- and LOX-specific

inhibitors in BPAECs. In order to further understand the

role of the PLA2 and PLD in the MeHg-induced cytotox-

icity, the protective effects of well-established general

PLA2 inhibitor (quinacrine, 2 lM) and 1-butanol (2%), the

PLD-generated PA quencher were used. Cells were pre-

treated for 1 h with MEM alone or MEM containing the

chosen inhibitors and then treated for 30 min with MeHg

(10 lM). Quinacrine offered effective and significant

attenuation of the MeHg-induced LDH leak in BPAECs

(33% of inhibition at a concentration of 2 lM) (Fig. 9a).

1-Butanol also offered effective and significant attenuation

of the MeHg-induced LDH leak in BPAECs (92% of

inhibition at a concentration of 2%) (Fig. 9b). These results

revealed that PLA2 inhibition and quenching of the PLD-

generated PA effectively attenuated the MeHg-induced

LDH leak in BPAECs, suggesting the involvement of PLA2

and PLD activation in the MeHg-induced cytotoxicity in

BPAECs.

MeHg Induces Serine Phosphorylation of cPLA2

in BPAECs

As the earlier experiment of this study demonstrated the

MeHg-induced activation of cPLA2 in BPAECs, we

investigated whether the enzyme activation would be

associated with the serine phosphorylation of cPLA2. SDS-

PAGE and Western blot analysis of proteins revealed that

MeHg (5–25 lM) induced serine phosphorylation in a

Fig. 8 COX- and LOX-specific inhibitors attenuate MeHg-induced

LDH release in BPAECs. BPAEC monolayers (2.5 9 105 cells/17.5-

mm dish) were pretreated for 1 h with MEM alone or MEM

containing indomethacin (100 lM; a) or ETYA (100 lM; b) and then


(5 lM) for 60 min. At the end of incubation period, release of LDH

into the medium was determined spectrophotometrically as described

under ‘‘Materials and Methods’’ section. Data represent mean ± SD

calculated from three independent experiments. *Significantly differ-

ent at P \ 0.05 as compared to cells treated with MEM alone.

**Significantly different at P \ 0.05 as compared to cells treated with

MEM containing MeHg alone


123

dose- and time-dependent manner (0–30 min) in BPAECs.

MeHg-induced serine phosphorylation of cPLA2 was

maximum at 10 lM dose (Fig. 10a). In addition, the extent

of serine phosphorylation of cPLA2 was the highest at

5 min of incubation and then declined from 5 to 30 min

(Fig. 10b) in BPAECs as compared to the same in the

control untreated cells. From these results, it was evident

that MeHg induced serine phosphorylation of cPLA2 in a

dose- and time-dependent fashion in BPAECs (Fig. 10).

Discussion

The results of this study revealed that the MeHg-induced

activation of PLD in BPAECs was regulated upstream by

the activation of PLA2 and eicosanoids generated by COXs

and LOXs. Furthermore, the results showed that the MeHg-

induced cytotoxicity in BPAECs was mediated by the

bioactive lipids generated by PLA2, COXs, LOXs, and

PLD. Overall, this study demonstrated that the bioactive

lipid signaling cascades, operated by the phospholipases

and lipid oxygenases, regulated the MeHg-induced vascu-

lar EC cytotoxicity.

Endothelium of the blood vessels plays a fundamental

role in the structure and function of the vasculature and

maintains the homeostasis of the circulation. Cellular

membrane phospholipids are crucial for the structure and

function of the living cells. Phospholipases including PLA2

and PLD are essential enzymes in the housekeeping of cells

as well as in the generation of bioactive lipid messengers

which play a vital role in cellular signaling [22, 30]. Hence,

it is conceivable to surmise that MeHg exerts its toxic

effects on the vascular endothelium leading to the modu-

lation or alteration of the functions of the blood vessel.

Phospholipase A2 is an important membrane phospho-

lipid-hydrolyzing enzyme which releases the unsaturated

fatty acid (typically AA) esterified at the sn-2 position of

the phospholipid [22]. Lipid oxygenases, including the

COXs and LOXs, utilize the PLA2-released free AA as the

substrate and convert it into potent bioactive AA metabo-

lites (eicosanoids) including the prostaglandins and leu-

kotrienes [20]. COX-1 and COX-2, the two isoforms of

mammalian COX convert AA into prostaglandin H2

(PGH2), which acts as a precursor for further metabolic

conversion into thromboxane A2 (TXA2), prostacyclin

(PGI2), and PGE2 in the vascular ECs [19]. LOXs are of

three main types viz., 5-LOX, 12-LOX, and 15-LOX which

convert the free AA into the hydroperoxyeicosatetraenoic

acids (5-HPETE, 12-HPETE, and 15-HPETE), which are

further reduced to the hydroxyeicosatetraenoic acids

(5-HETE, 12-HETE, and 15-HETE) by the cellular gluta-

thione peroxidase [19]. The HPETEs act as precursors for

the LOX-generated leukotrienes. The eicosanoids, gener-

ated by COXs and LOXs, have been established to play

crucial roles in the inflammatory cascades and their for-

mation is tightly regulated by the activity of PLA2 [22].

The LOX-derived eicosanoids are potent bioactive lipid

signaling molecules in mammalian cells including the

vascular ECs [19]. Both PLA2 and the eicosanoids are

emerging as critical players in cardiovascular diseases [30].

Hence, the unregulated activation of PLA2, mediated by

certain agonists (e.g., environmental toxicants), can

endanger the vascular endothelial structure and function,

and ultimately the blood vessel function.

Mammalian cells contain three major classes of PLA2:

(i) cytosolic calcium-dependent PLA2 (cPLA2), (ii) intra-

cellular calcium-independent PLA2 (iPLA2), and (iii)

secretory calcium-dependent PLA2 (sPLA2) [30]. Several

agonists have been identified that activate PLA2 in differ-

ent systems in vitro and in vivo [20]. Regulation of PLA2 is

apparently complex. The regulation of cPLA2 activation

has been extensively investigated, wherein the mitogen-

activated protein kinases (MAPKs), protein kinase A

(PKA), and protein kinase C (PKC) have been shown to

play important roles [20]. Results of this study revealed

that MeHg induced the serine phosphorylation of cPLA2 in

BPAECs, possibly through the upstream regulation by

MAPKs. On the other hand, the regulation of iPLA2 and

Fig. 9 PLA2-specific inhibitor and 1-butanol, the quencher of PLD-

generated PA, attenuate MeHg-induced LDH release in BPAECs.

BPAEC monolayers (2.5 9 105 cells/17.5-mm dish) were pretreated

for 1 h with MEM alone or MEM containing quinacrine (2 lM; a) or

1-butanol (2%; b) and then subjected to treatment with MEM alone or

MEM-containing MeHg (10 lM) for 60 min. At the end of incubation

period, LDH in the medium was determined spectrophotometrically

as described under ‘‘Materials and Methods’’ section. Data represent

mean ± SD calculated from three independent experiments. *Signif-

icantly different at P \ 0.05 as compared to cells treated with MEM

alone. **Significantly different at P \ 0.05 as compared to cells

treated with MEM containing MeHg alone


123

sPLA2 is not known in detail. Studies reveal that lipid

peroxidation activates sPLA2 [31]. ROS have been shown

to cause the activation of iPLA2 leading to the release of

AA in macrophages [32]. Hydrogen peroxide has been

reported to cause the release of AA in astrocytes that is

mediated by the activation of cPLA2 and iPLA2 [33].

Overall, these reports corroborate that PLA2 activity in

mammalian cells is regulated by signaling cascades, ROS,

and oxidative stress. Earlier, we have shown that MeHg

causes the formation of ROS, induces oxidative stress, and

activates PLA2 activity through ROS production and thiol

depletion in BPAECs [15, 26, 27]. As observed in this

study, cPLA2 activation by MeHg in BPAECs could be

mediated by the ROS generation, oxidative stress, and

thiol-redox alteration. Needless to mention, the involve-

ment of other signaling kinases such as the MAPKs, PKA,

and PKC in the regulation of MeHg-induced cPLA2 acti-

vation in the vascular ECs is not ruled out.

MeHg has been reported to cause toxicity in astrocytes

and neurons that is mediated by ROS, oxidative stress, and

loss of GSH [34, 35]. Mepacrine, a well-known PLA2

inhibitor, has been shown to protect against the MeHg-

induced cytotoxicity in the cerebellar granule cells, sug-

gesting the role of PLA2 activation in the MeHg-induced

neurotoxicity [36]. Earlier, we have also shown that inor-

ganic mercury- and MeHg-induced cytotoxicity in

BPAECs is protected by the cPLA2 and iPLA2 inhibitors

and inorganic mercury induces the formation of eicosa-

noids in BPAECs [26, 27], suggesting the role of PLA2 in

the mercury-induced cytotoxicity in the vascular ECs.

Along these lines, the results of the current study also

demonstrated that quinacrine attenuated the MeHg-induced

cytotoxicity in BPAECs. Thus, the findings of this

study reinforced the role of PLA2 in the mediation of

MeHg-induced cytotoxicity in the vascular ECs. Activation

of cPLA2, and probably iPLA2, might have contributed to

the cytotoxicity of MeHg through the formation and action

of eicosanoids generated by COXs and LOXs as the eico-

sanoids (prostaglandins, HPETEs, and leukotrienes) are

known as potent bioactive lipid mediators. This was further

supported by the results of this study that MeHg induced the

formation of PGE2 and 5-, 12-, and 15-HPETEs and the

COX- and LOX-specific inhibitors offered protection

against the MeHg cytotoxicity in BPAECs. Eicosanoids,

including the COX-generated prostanoids (prostaglandins,

thromboxane, and prostacyclin), are emerging as the

important mediators of inflammation in vascular endothelial

dysfunction and atherosclerosis [37, 38].

In mammalian cells, two predominant isoforms of PLD,

namely PLD1 and PLD2, have been identified, cloned, and

characterized [16, 19]. Vascular ECs have also been

reported to contain both PLD1 and PLD2 [16]. Distinct

cofactors including Arf, Rho, Cdc42, phosphatidylinositol

4,5-bisphosphate, and detergents have been reported to

cause PLD activation in vitro specific to the isoform [19].

ROS and oxidants have been shown to activate PLD in the

cultured cellular systems including the vascular ECs [16].

Signaling kinases such as the p38 MAPK, extracellular

signal-regulated kinases (ERKs), and Src kinases have

been identified to play a role in the regulation of the oxi-

dant-mediated activation of PLD in BPAECs [16]. Also,

the activation of PLD by several agonists has been

observed to be regulated by cellular calcium, PKC, het-

erotrimeric G proteins, small molecular weight G proteins,

protein tyrosine kinases, and protein tyrosine phosphatases

[16]. Earlier, we have shown that the MeHg-induced PLD

Fig. 10 MeHg induces serine phosphorylation of cPLA2 in BPAECs.

BPAECs monolayers (5 9 105 cells/35-mm dish) were subjected to

treatment with MEM alone or MEM containing MeHg (5, 10, and

25 lM) for 30 min (a) or for different time periods (0–30 min; b). At

the end of incubation period, cPLA2 and phosphoserine-cPLA2 were

probed using Western blotting as described under Materials and

Methods. The intensities of the blots were quantified. Data represent

mean ± SD calculated from three independent experiments. *Signif-

icantly different at P \ 0.05 as compared to cells treated with MEM

alone


123

activation in BPAECs is regulated by calcium and the

thiol-redox perturbation being associated with the ROS

generation [15, 28]. Therefore, calcium, ROS, and the

thiol-redox perturbation could have contributed to the

MeHg-induced PLD activation that was observed in this

study.

PGF2a-stimulated activation of PLD in MC3T3-E1 cells

has been observed to be associated with DAG formation

[39]. G proteins have been reported to be involved in the

PGF2a-induced PLD activation in the osteoblast-like cells

[40]. Another COX-generated eicosanoid, PGD2, has been

shown to activate PLD in the osteoblast-like cells through

the calcium/calmodulin signaling [41]. Earlier, we have

shown that the calcium/calmodulin signaling regulates the

MeHg-induced activation of PLD in BPAECs [28]. Thus, it

could be surmised, from the results of the current study,

that eicosanoids might be the possible bioactive lipids in

mediating the MeHg-induced activation of PLD in

BPAECs through calcium/calmodulin signaling. Linoleic

acid hydroperoxide, generated by the soybean LOX,

has been shown to activate PLD in BPAECs [42]. PGF2a-

induced PLD activation in rat luteal cells has been shown to

be attenuated by the LOX-specific inhibitors such as

nordihydroguaeretic acid and ETYA, suggesting the role of

LOX therein [43]. Earlier, we have also reported that the

vitamin C-induced activation of PLD in the lung micro-

vascular ECs is attenuated by the COX- and LOX-specific

inhibitors, suggesting the regulation of prooxidant-induced

PLD activation by COXs and LOXs [19]. The results of this

study were in agreement with these reports, and revealed

that both COX- and LOX-generated eicosanoids, derived

from the PLA2-released AA from the membrane phospho-

lipids, appeared as key regulators in the MeHg-induced

PLD activation in BPAECs (Scheme 1). In addition, the

results of the current study demonstrated that PLD-gener-

ated PA also played a crucial role in the MeHg-induced

cytotoxicity in BPAECs as revealed from the experiments

where the primary alcohol, 1-butanol, protected the MeHg-

induced cytotoxicity by quenching the PLD-generated PA

and rendering it physiologically inactive.

The roles of phospholipases and COXs in the vascular

diseases and ischemic injury of the tissue are becoming

increasingly evident [44, 45]. The three important isoforms

of LOX (5-LOX, 12-LOX, and 15-LOX) have been shown

to be associated with several human diseases including the

myocardial diseases [46–49]. The role of PLD in vascular

disorders is becoming increasingly evident [16]. MeHg is

an established heavy metal toxicant that depletes the cel-

lular thiols and causes the generation of ROS [15]. As

demonstrated in this study, the activation and cross-talk

among PLA2, COXs, LOXs, and PLD to generate the

potent bioactive lipid messengers that caused the cytotox-

icity in the cultured vascular ECs under MeHg exposure,

offered a possible lipid signaling mechanism of the mer-

cury-induced vascular diseases.

Acknowledgments This work was supported by the funding from

the International Academy of Oral Medicine and Toxicology (IA-

OMT), Davis Heart and Lung Research Institute, the Division of

Pulmonary, Allergy, Critical Care, and Sleep Medicine, and the

National Institutes of Health (HL 093463).

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