Toxicology Letters 196 (2010) 168–174
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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Activation of phospholipase D involved in both injury and survival in A549
alveolar epithelial cells exposed to H
2
O
2
Ming Wu
a
, Qi Wang
a
, Jiang-Yun Luo
d
, Bo Jiang
b
, Xu-Yun Li
c
, Ru-Kun Chen
a
, Yun-Bi Lu
d,∗
a
Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, China
b
Department of Clinical Pharmacy, Second Affiliated Hospital, School of Medicine, Zhejiang University, China
c
Department of Physiology, School of Medicine, Zhejiang University, China
d
Department of Pharmacology, School of Medicine, Zhejiang University, Yu Hang Tang Road 388, Hangzhou, Zhejiang 310058, China
article info
Article history:
Received 8 March 2010
Received in revised form 15 April 2010
Accepted 16 April 2010
Available online 24 April 2010
Keywords:
Hydrogen peroxide
Phospholipase D
Phosphatidic acid
A549 cell
abstract
To determine the role of the phospholipase D (PLD) pathway in injury and survival of alveolar epithelial
cells, A549 cells were exposed to H
2
O
2
(500 M) which resulted in time-dependent injury and bi-phasic
increase of PLD activity at 5 min and at 3 h, respectively. n-Butanol (0.5%) inhibited PLD activation,
attenuated cell injury at 5 min of H
2
O
2
exposure, but enhanced injury at 3 h of exposure. This activa-
tion was inhibited by treatment with catalase (500 units/ml). Exogenous phosphatidic acid mimicked
the effects of PLD activation, and diphenyliodonium (NADPH oxidase inhibitor) reversed the decline in
cell viability induced by H
2
O
2
exposure. Propranolol (phosphatidic acid phospholydrolase inhibitor) and
quinacrine (phospholipase A2 inhibitor) had weak effects on H
2
O
2
-induced PLD activation but reversed
H
2
O
2
-induced injury. We speculate that PLD activation at the initiation of H
2
O
2
exposure predominantly
results in NAPDH oxidase activation, which mediates A549 cell injury, but turns to mediating cell survival
as the H
2
O
2
attack continues, which might be mainly due to the accumulation of intracellular phosphatidic
acid.
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Acute lung injury and acute respiratory distress syndrome are
characterized by alveolar epithelial and alveolar-capillary dam-
age which result in nonhydrostatic pulmonary edema and severe
hypoxemia (Lucas et al., 2009; Manicone, 2009). Oxidative dam-
age plays an important role in the loss of integrity of the epithelial
barrier which leads to the influx of protein-rich edema fluid and
accumulation of neutrophils in the alveolar space (Lucas et al.,
2009; Manicone, 2009). In particular, a mass of reactive oxygen
species (ROS) released by neutrophils in the alveolar space con-
tribute greatly to alveolar tissue injury (Mendez and Hubmayr,
2005; Tsushima et al., 2009). However, a new paradigm of redox
signaling has emerged recently, whereby some oxidants are con-
sidered to function as intracellular signaling molecules which can
contribute to either cell death or survival (Giorgio et al., 2007;
Trachootham et al., 2008).
Phospholipase D (E.C.3.1.4.4; PLD) is ubiquitous in mammalian
cells and is activated by various extracellular stimuli, including
H
2
O
2
, in different cell types. H
2
O
2
stimulates PLD activity by a
∗
Corresponding author. Tel.: +86 571 88208223; fax: +86 571 88208022.
E-mail address: yunbi@zju.edu.cn (Y.-B. Lu).
poorly understood, probably direct or indirect signaling pathway
(Min et al., 2001; Natarajan et al., 1993; Oh et al., 2000; Xiao et
al., 2005). Two mammalian PLD isozymes, PLD1 and PLD2, have
been identified, characterized and cloned (Cockcroft, 2001; Jenkins
and Frohman, 2005). PLD catalyzes the hydrolysis of phosphatidyl-
choline and other membrane phospholipids to phosphatidic acid
(PA) and choline. PA can be subsequently converted to lyso-PA by
phospholipase A2 (PLA2) or to diacylglycerol (DAG) by PA phos-
pholydrolase (PAP), where PA is considered to be the main effector
of the functions of PLD in cells (Cockcroft, 2001; Cockcroft and
Frohman, 2009; Exton, 2002; Jenkins and Frohman, 2005). In the
presence of primary alcohols, PLD catalyzes a transphosphatidyla-
tion reaction producing phosphtidylalcohols at the expense of PA;
this feature provides a tool to implicate PLD in cellular responses
(Exton, 2002).
PLD and its metabolites are involved in various cellular func-
tions, such as activation of NADPH oxidase (oxidative burst),
membrane trafficking, exocytosis (Cockcroft et al., 2002), phago-
cytosis (Corrotte et al., 2006), cell adhesion and chemotaxis
(Gomez-Cambronero et al., 2007), cytoskeletal reorganization, cell
proliferation, apoptosis, and survival (Cockcroft and Frohman,
2009). In inflammatory cells or phagocytic cells, PLD plays a role
in stimulation of NADPH during the respiratory oxidative burst.
PLD functions both directly, by generating PA, which binds to and
0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2010.04.014