Critical Duration of Intracellular Ca2+ Response
Required for Continuous Translocation and Activation of Cytosolic
Phospholipase A2*
Tetsuya
Hirabayashi
§,
Kazuhiko
Kume
,
Kenzo
Hirose¶,
Takehiko
Yokomizo
,
Masamitsu
Iino¶,
Hiroshi
Itoh§, and
Takao
Shimizu
From the
Department of Biochemistry and Molecular
Biology, ¶ Department of Pharmacology, Faculty of Medicine,
University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 and the
§ Department of Biological Sciences, Faculty of Bioscience
and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho,
Midori-ku, Yokohama 226-8501, Japan
 |
ABSTRACT |
When cells are exposed to certain external
stimuli, arachidonic acid (AA) is released from the membrane and serves
as a precursor of various types of eicosanoids. A
Ca2+-regulated cytosolic phospholipase A2
(cPLA2) plays a dominant role in the release of AA. To
closely examine the relation between Ca2+ response and AA
release by stimulation of G protein-coupled receptors, we established
several lines of Chinese hamster ovary cells expressing platelet-activating factor receptor or leukotriene B4
receptor. Measurement of intracellular Ca2+ concentration
([Ca2+]i) demonstrated that cell lines capable of
releasing AA elicited a sustained [Ca2+]i
increase when stimulated by agonists. The prolonged [Ca2+]i elevation is the result of
Ca2+ entry, because this elevation was blocked by EGTA
treatment or in the presence of Ca2+ channel blockers (SKF
96365 and methoxyverapamil). cPLA2 fused with a green
fluorescent protein (cPLA2-GFP) translocated from the
cytosol to the perinuclear region in response to increases in
[Ca2+]i. When EGTA was added shortly after
[Ca2+]i increase, the cPLA2-GFP
returned to the cytosol, without liberating AA. After a prolonged
[Ca2+]i increase, even by EGTA treatment, the
enzyme was not readily redistributed to the cytosol. Thus, we propose
that a critical time length of [Ca2+]i elevation
is required for continuous membrane localization and full activation of
cPLA2.
 |
INTRODUCTION |
Arachidonic acid (AA)1
is a precursor for biosynthesis of eicosanoids, including
prostaglandins, thromboxanes, leukotrienes, and lipoxins. In the
resting state, the bulk of the AA in mammalian cells is esterified in
glycerophospholipids at the sn-2 position (1). Liberation of
AA occurs mainly by activation of phospholipase A2
(PLA2) in response to varieties of extracellular stimuli
such as cytokines, hormones, neurotransmitters, mitogens, antigens, and
endotoxins (2). Mammalian cells have structurally diverse forms of
PLA2 including secretory PLA2,
Ca2+-independent PLA2, and cytosolic
PLA2 (cPLA2) (3-5). Among these PLA2s, receptor-mediated AA release is primarily attributed
to cPLA2 because the enzyme preferentially hydrolyzes
phospholipids containing AA and is regulated by physiological levels of
intracellular calcium concentrations ([Ca2+]i)
and phosphorylation of Ser-505 by mitogen-activated protein kinase
(MAPK) (6-9). Consistently, stimulated peritoneal macrophages derived
from cPLA2-deficient mice fail to produce eicosanoids (10,
11). Sensitivity of cPLA2 to Ca2+ is mediated
through an amino-terminal CaLB domain which is homologous to the C2
domain of protein kinase C and several other
Ca2+-dependent phospholipid-binding proteins
(6, 12). Immunofluorescent studies have shown that cPLA2
translocates from the cytosol to the nuclear envelope and the
endoplasmic reticulum in response to [Ca2+]i
increase (13, 14). Other proteins involved in eicosanoid production,
such as cyclooxygenase isoforms (15-17), 5-lipoxygenase,
5-lipoxygenase-activating protein, and leukotriene C4
synthase (18-20) are also localized to these sites constitutively or
upon cell stimulation.
The mobilization of Ca2+ can be elicited by ligand binding
to cell-surface receptors that activate phospholipase Cs through heterotrimeric G protein-dependent or protein tyrosine
kinase-dependent pathway. Calcium signaling patterns occur
as single transients, repetitive oscillations, or sustained plateaux.
Although several investigators have noted the importance of
Ca2+ influx in receptor-mediated AA release (21-23),
little is known about how these Ca2+ signaling patterns
regulate cPLA2 translocation and AA release.
To address this question, we monitored Ca2+ signaling
pattern, cPLA2 translocation, and AA release using Chinese
hamster ovary (CHO) cell lines stably expressing platelet-activating
factor (PAF) receptor (24) and/or leukotriene B4
(LTB4) receptor (25). In CHO cells, cPLA2 is
expressed endogenously and coupled to receptor-mediated release of AA
(26, 27). We found that a critical time length of
[Ca2+]i elevation is required for continuous
translocation of cPLA2 to the membrane and for the release
of AA.
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EXPERIMENTAL PROCEDURES |
Materials--
The sources of materials used in this work were
as follows: PAF C-16 from Cayman Chemical (Ann Arbor, MI); cremophore
EL from Sigma; [3H]arachidonic acid,
[3H]WEB 2086, and [3H]leukotriene
B4 from NEN Life Science Products; fura-2 acetoxymethyl from Dojindo (Kumamoto, Japan); SKF 96365, ionomycin, and nifedipine from Calbiochem; methoxyverapamil from RBI (Natick, MA);
anti-cPLA2 monoclonal IgG (4-4B-3C) from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA); and fatty acid-free bovine serum
albumin fraction V from Bayer (Kankakee, IL).
WEB 2086 and leukotriene B4 were generous gifts from
Boehringer Ingelheim (Germany) and Drs. Y. Kobayashi and F. Sato (Tokyo Institute of Technology), respectively.
Plasmid Construction--
For a chimeric protein containing GFP
at the carboxyl terminus of cPLA2, cDNA encoding human
cPLA2 was amplified by polymerase chain reaction with
primers upstream (5'-GGAAGATCTATGTCATTTATAGATCCTT-3') and downstream
(5'-TGCGGTCGACTGCTTTGGGTTTACTTAGA-3') using pSV2-cPLA2 (provided by Drs. I. Kudo and M. Murakami, Showa University, Tokyo) as
a template. The resulting polymerase chain reaction product was cloned
into BglII-SalI sites of pEGFP-N3 vector
(, Palo Alto, CA) to obtain a
cPLA2-GFP fusion construct. The orientation of
cPLA2 and the integrity of the reading frame was verified
by restriction analysis and DNA sequencing.
Cell Culture and Transfection--
CHO-K1 cells were maintained
under 5% CO2 at 37 °C in growth medium (Ham's F12
medium supplemented with 10% fetal calf serum, 50 units/ml penicillin,
and 50 µg/ml streptomycin). CHO cells stably expressing PAF receptor
or LTB4 receptor were generated as described previously
(25, 28). Briefly, cDNAs of guinea pig PAF receptor and human
LTB4 receptor were subcloned into mammalian expression
vectors, pcDNAI/Neo and pcDNA3 (Invitrogen, Carlsbad, CA),
respectively, and transfected into CHO-K1 cells. Clones resistant to
Geneticin (1 mg/ml) were isolated by limiting dilution, tested for
receptor expressions by binding assay, and maintained in the presence
of 0.3 mg/ml Geneticin. PAF-LT cells were generated by co-transfection
of PAF11 cells expressing PAF receptor (see below) with the expression
vector for LTB4 receptor and pPUR encoding the puromycin
resistance gene (), and by selection with puromycin (10 µg/ml, Sigma).
For cPLA2-GFP expression, cells were seeded at a density of
5 × 105 cells/60-mm dishes and transiently
transfected with 2 µg of the expression vector encoding a
cPLA2-GFP fusion protein (pcPLA2-GFP) or a
control vector pEGFP-N3 with LipofectAMINE PLUS (Life Technologies, Inc.), according to the manufacturer's protocol, and used for experiments 48-72 h after transfection. The amount of
cPLA2-GFP was comparable to that of endogenous
cPLA2, as determined by immunoblotting with
anti-cPLA2 antibody (data not shown).
Binding Assay--
Binding assays for the PAF receptor or the
LTB4 receptor on CHO-K1 cells were performed as described
previously (29). Briefly, cells on a 24-well culture plate were
incubated for 1 h at 25 °C with various concentrations of
[3H]WEB 2086 or [3H]LTB4 in the
presence or absence of unlabeled ligand (20 µM WEB 2086 or 4 µM LTB4, respectively). The cells were
washed three times with Hepes/Tyrode's/BSA buffer (140 mM
NaCl, 2.7 mM KCl, 1.8 mM CaCl2,
0.49 mM MgCl2, 5.6 mM
D-glucose, 12 mM NaHCO3, 0.37 mM NaH2PO4, 10 mM Hepes
(pH 7.4), and 0.1% (w/v) fatty acid-free BSA) and then lysed in 1%
Triton X-100. The radioactivity of the cell lysate was determined by
liquid scintillation counting. Kd and
Bmax values were calculated by Scatchard analysis.
Single Cell Calcium Imaging--
4 × 104 cells
were seeded on coverslips (16-mm diameter) in the growth medium and
incubated for 24 h. After another 24 h incubation in
serum-free medium containing 0.1% (w/v) fatty acid-free BSA, the cells
were washed twice with Hepes/Tyrode's/BSA buffer and incubated at
37 °C with 10 µM fura-2 acetoxymethyl ester and 0.01% (w/v) cremophore EL for 1 h in the buffer. After loading, cells were washed with and maintained in the same buffer in the dark at room
temperature. Fura-2 fluorescence intensity was measured by alternating
excitation at 340 and 380 nm and detecting emission at 505 nm with a
40× objective (Nikon UV-fluor), an SIT camera, and an ARGUS-50/CA
image processor (Hamamatsu Photonics, Hamamatsu, Japan). To determine
intracellular free Ca2+ concentrations, the 340/380 ratio
of fluorescence intensities were compared with the ratio for
Ca2+ standard in solution (Molecular Probes, Eugene, OR).
AA Release Assay--
Cells were seeded onto 12-well culture
plates at a density of 8 × 104 cells/well in the
growth medium. After 24 h incubation, the medium was removed, and
the cells were labeled by incubation for 24 h in 0.75 ml of
serum-free medium containing 3.7 kBq of
[3H]arachidonic acid (3.7 TBq/mmol) and 0.1% (w/v)
fatty acid-free BSA. The cells were then washed three times with
Hepes/Tyrode's/BSA buffer and stimulated with ligands or ionomycin in
the same buffer at 37 °C. Radioactivity of supernatants and cell
lysates (in 1% Triton X-100) were measured by liquid scintillation
counting. The amount of the radioactivity released into the supernatant was expressed as a percentage of the total incorporated radioactivity.
Confocal Microscopy--
Cells transfected with
pcPLA2-GFP were seeded on coverslips (14-mm diameter) of
glass-bottomed culture dishes (Matsunami, Osaka, Japan) at a
density of 2.6 × 104 and serum-starved as described
above. The culture medium was replaced with Hepes/Tyrode's/BSA buffer,
and fluorescence images were taken in a FLUOVIEW confocal laser
scanning microscope system (Olympus, Tokyo) with a 100× oil immersion
objective (NA 1.35), 488-nm laser line for excitation and a 520 ± 20-nm band pass filter for emission.
 |
RESULTS |
Agonist-induced Calcium Response and AA Release in CHO Cells
Expressing PAF or LTB4 Receptor--
Guinea pig PAF
receptor and human LTB4 receptor were expressed in CHO-K1
cells that have no detectable endogenous receptors. Six cell lines
PAF11, PAF12, PAF14, LT13, LTF2, and PAF-LT were examined for binding
assay, agonist-induced Ca2+ response (Fig.
1), and AA release. Results obtained
using these cell lines are summarized in Table
I. In cells with low levels of receptor
expression (PAF12 and LT13 cells), stimulation with agonists caused a
rapid and transient [Ca2+]i increase, which
returned to the base line within 2-5 min (Fig. 1, B and
F). These cell lines did not release AA in response to PAF
or LTB4. On the other hand, in cells with high levels of
receptor expression (PAF11, PAF14 and LTF2 cells), stimulation with
agonists induced a sustained [Ca2+]i increase
which remained elevated at least for 5 min (Fig. 1, A, C,
and E) and AA release. In PAF-LT cell line with a high level
of PAF receptor and a low level of LTB4 receptor expression, PAF induced a sustained [Ca2+]i
increase (Fig. 1D) and AA release, whereas LTB4
induced a transient [Ca2+]i increase (Fig.
1G) and little AA release. These results suggest that the
sustained [Ca2+]i increase is necessary for
receptor-mediated AA release from CHO cells.

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Fig. 1.
Ca2+ response in CHO cell lines
with different levels of receptor expression. Fura-2-loaded cells
were stimulated with 100 nM PAF or 1 µM
LTB4 in the presence of 1.8 mM extracellular
Ca2+. [Ca2+]i was calculated from
340/380 ratios obtained from groups of 5-10 cells as described under
"Experimental Procedures."
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Table I
Expression and downstream signals of PAF and LTB4 receptor in
CHO cell lines
Bmax and Kd values for each line
were determined by Scatchard analysis of the binding of
[3H]WEB 2086 or [3H]LTB4 to the cells.
Ca2+ response to 100 nM PAF or 1 µM
LTB4 was measured as described under "Experimental
Procedures." S represents a sustained [Ca2+]i
increase that continued over 5 min; T represents a transient
[Ca2+]i increase that returned to the base line
within 5 min. AA release 10 min after stimulation (100 nM
PAF or 1 µM LTB4) was measured, and the values
were subtracted with background releases without stimulation and
expressed as the means ± S.D. from triplicate determinations.
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Requirement of Calcium Influx for AA Release--
PAF11 cells with
a relatively higher receptor expression were used in the following
studies. 100 nM PAF induced AA release in the time course
shown in Fig. 2A. It had a
2-min lag phase, followed by a linear increase up to 10 min. PAF
induced AA release in a dose-dependent manner, and
ED50 was around 4 × 10-10 M
(Fig. 2B).

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Fig. 2.
AA release from PAF11 cells.
A, time course of PAF-induced AA release from PAF11 cells.
[3H]AA-labeled cells were unstimulated (open
circle) or stimulated (filled circle) with 100 nM PAF at 37 °C. B, dose-response curve for
AA release. Cells were stimulated with PAF for 10 min. The values are
the means ± S.D. of three experiments.
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We next examined contributions of intracellular and extracellular
Ca2+ sources in the agonist-induced Ca2+
response and AA release. PAF stimulation in the presence of either Ca2+ chelator EGTA (10 mM) or Ca2+
channel blockers (50 µM SKF 96365 or 100 µM
methoxyverapamil) (30, 31) evoked a transient
[Ca2+]i increase which returned to the base line
within 2 min (Fig. 3, A-D).
Thus, the initial rise in [Ca2+]i is attributed
to release from the intracellular Ca2+ pool, whereas the
sustained elevation requires Ca2+ influx. Fig.
4A shows that lowering
extracellular Ca2+ concentration or depriving
Ca2+ by EGTA reduced PAF-stimulated AA release, whereas
increasing its concentration enhanced the release. Pretreatment of the
cells with SKF 96365 or methoxyverapamil inhibited PAF-induced AA
release (Fig. 4B), indicating that the Ca2+
influx is required for AA release.

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Fig. 3.
Inhibition of PAF-induced Ca2+
influx by EGTA, SKF 96365, or methoxyverapamil. PAF11 cells were
untreated (A) or pretreated for 5 min with 10 mM
EGTA (B), 50 µM SKF 96365 (C), or
100 µM (±)-methoxyverapamil (D) and
stimulated with 100 nM PAF.
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Fig. 4.
Effects of extracellular Ca2+
concentrations ([Ca2+]o) and Ca2+
channel blockers on PAF-induced AA release. A, PAF11
cells labeled with [3H]AA were incubated for 5 min in the
buffer containing 0.18, 1.8, or 18 mM CaCl2
with or without EGTA and stimulated with 100 nM PAF for 15 min. B, PAF11 cells were pretreated with SKF 96365 or
(±)-methoxyverapamil as described above and stimulated with 100 nM PAF for 10 min. Columns and vertical
bars denote the mean ± S.D. of three experiments.
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Critical Length of [Ca2+]i Elevation for AA
Release--
EGTA was applied at various time points before and after
PAF stimulation in order to terminate [Ca2+]i
elevation. Single cell Ca2+ imaging confirmed that addition
of EGTA after PAF stimulation created a transient
[Ca2+]i increase, which returned to the base line
within 1 min following the addition (Fig.
5A). Fig. 5C shows
that EGTA added 1 min after PAF stimulation substantially inhibited the PAF-induced AA release but did not alter the AA release when added 5 min after the stimulation.

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Fig. 5.
AA release is dependent on duration of
[Ca2+]i rise. A and B,
transient or sustained Ca2+ responses were induced by 100 nM PAF (A) or 2 µM ionomycin
(B) with or without the addition of 10 mM EGTA
at the time point indicated. C and D, EGTA (10 mM) was added to PAF11 cells at various time points before
and after stimulation with 100 nM PAF (C) or 2 µM ionomycin (D). AA release 10 min after
stimulation was determined as in Fig. 2. Data are the means ± S.D. of three experiments. E, as in C, except
that the cells were transiently transfected with GFP expression vector
pEGFP-N3 (open circle) or the expression vector for
cPLA2-GFP (filled circle).
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Since stimulation of the PAF receptor results in various signals
besides [Ca2+]i elevation (32), similar
experiments were carried out using a Ca2+ ionophore
ionomycin to study the role of Ca2+ signaling in a more
direct manner. The prolonged [Ca2+]i increase was
generated by 2 µM ionomycin and had about the same
magnitude as the PAF-stimulated plateau (Fig. 5B). The transient [Ca2+]i increase was created by
ionomycin with the following addition of EGTA, and this increase
returned to the base line within 1 min after the addition (Fig.
5B). The ionomycin-induced sustained
[Ca2+]i rise caused AA release, whereas the brief
[Ca2+]i rise did not (Fig. 5D),
confirming that a prolonged [Ca2+]i increase
(over 2 min) is essential for AA release.
Translocation and Reversal of cPLA2 by the Change in
[Ca2+]i--
Due to its inherent fluorescence
and unique compact structure, green fluorescent protein (GFP) has been
reported in many studies to serve as a valuable reporter in the
localization of various proteins without interfering with their
biological activity (33). We constructed a chimeric protein
cPLA2-GFP by fusing EGFP to the carboxyl terminus of
cPLA2. To ensure that the fusion of GFP did not affect
structure and enzyme activity of cPLA2, the
cPLA2-GFP construct was analyzed for AA release. The
expression of cPLA2-GFP enhanced PAF-induced AA release in
PAF11 cells by about 1.5-fold at 10 min. The correlation of AA release
with the duration of [Ca2+]i increase was
essentially identical to that in untransfected or GFP-transfected cells
(Fig. 5, C and E). In addition, gel shift of
cPLA2-GFP in response to agonist stimulation was comparable with that of endogenous cPLA2 (data not shown). The gel
shift has been established to result from phosphorylation at Ser-505 (7). These results suggest that the fusion of GFP to the carboxyl terminus of cPLA2 does not perturb cPLA2 function.
We then monitored the localization of the cPLA2-GFP in
living cells by confocal laser fluorescence microscopy. In the resting state, cPLA2-GFP was almost homogeneously present in the
cytosol (Fig. 6, A and
B, 0 min). Stimulation of the cells with PAF or ionomycin triggered translocation of cPLA2-GFP to the
perinuclear region within 1 min, and the cPLA2-GFP
fluorescence was retained for over 10 min (Fig. 6, A and
B). When PAF12 cells transiently expressing
cPLA2-GFP were stimulated with PAF, the translocation of
cPLA2-GFP to the perinuclear region was transient and
reversed within 5 min (Fig. 6C). In contrast to
cPLA2-GFP, unconjugated GFP was distributed homogeneously
in the cytosol and nucleus, and its distribution was not affected by
PAF or ionomycin (data not shown).

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Fig. 6.
Translocation of cPLA2-GFP fusion
protein by PAF or ionomycin. PAF11 cells (A and
B) and PAF12 cells (C) transiently transfected
with the expression vector for cPLA2-GFP were serum-starved
overnight and stimulated with 100 nM PAF (A and
C) or 7.5 µM ionomycin (B).
Confocal fluorescence images were recorded at the indicated time. The
scale bars are 20 µm.
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Next, the effect of termination of [Ca2+]i
increase by EGTA on PAF-induced translocation of cPLA2-GFP
was examined. cPLA2-GFP translocation was reversed rapidly
when EGTA was added 1 min after PAF stimulation (Fig.
7A, upper panels). In
contrast, the reversal was much slower when EGTA was added 5 min after
stimulation; a large portion of cPLA2-GFP was retained at
the perinuclear region for another 5 min after the addition of EGTA
(Fig. 7A, lower panels). Similar redistributions of
cPLA2-GFP were obtained by application of ionomycin instead
of PAF (Fig. 7B). These results agree with the finding that
AA release was inhibited when EGTA was added 1 min after PAF
application, while it was not inhibited after 5 min (Fig.
5C), which means that a prolonged
[Ca2+]i increase for about 5 min induces stable
localization of cPLA2 at the perinuclear region and
continuous hydrolysis of AA from membrane phospholipids.

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Fig. 7.
Redistribution of cPLA2-GFP by
EGTA. PAF11 cells expressing cPLA2-GFP were treated as
described in Fig. 6. 10 mM EGTA was added 1 (upper
panels) or 5 min (lower panels) after stimulation with
100 nM PAF (A) or 10 µM ionomycin
(B). The scale bars are 20 µm.
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DISCUSSION |
cPLA2 plays a major role in releasing AA upon cell
stimuli and in production of eicosanoids (10, 11, 26). This enzyme is
activated by [Ca2+]i increases in physiological
ranges. Although previous reports have shown by immunohistochemical and
biochemical methods that cPLA2 is translocated to membranes
in response to [Ca2+]i increases, the dynamic
change of the enzyme localization in the living cell has not been
reported. The question we addressed here was how different
Ca2+ signaling patterns control cPLA2
translocation and AA release. We found that a critical time length of
[Ca2+]i elevation is required for continuous
membrane localization and full activation of cPLA2.
Optimal concentrations of PAF and LTB4 triggered comparable
maximal increases in [Ca2+]i in CHO cell lines
expressing PAF and/or LTB4 receptor. However, duration of
the [Ca2+]i increase varied with the cell line
(Fig. 1). Correlations between Ca2+ signaling pattern
(transient or sustained) and response in AA release (Table I) raised
the possibility that the agonist-induced Ca2+ signaling
pattern is responsible for the release of AA. Calcium mobilization from
internal stores and subsequent Ca2+ entry from the
extracellular space are the two major components of Ca2+
signaling following activation of cell-surface receptors. The inability
of PAF to induce AA release in Ca2+-free medium (Fig.
4A) or in the presence of Ca2+ channel blockers
(Fig. 4B) indicates the critical role of Ca2+
influx in AA release. This is consistent with the previous
observations, in which AA release has been linked to Ca2+
influx (21). Ca2+ entry across the plasma membrane occurs
via voltage-, store-, or receptor-operated channels. Although the
primary route of Ca2+ influx in CHO cells remains to be
elucidated, 100 µM nifedipine, an L-type
voltage-dependent Ca2+ channel blocker, had no
apparent effects on the Ca2+ influx induced by PAF or
LTB4 (data not shown).
Stimulation of PAF receptor exerts various responses, including
inhibition of adenylyl cyclase, activations of MAPK,
phosphatidylinositol 3-kinase, phospholipases C and D, and tyrosine
kinases, as well as increase in [Ca2+]i (24, 32,
34, 35). Although MAPK was activated by PAF and LTB4 to
about the same extent in PAF-LT cells (data not shown),
LTB4 did not induce AA release (Table I), suggesting that
MAPK activation with a transient [Ca2+]i increase
is insufficient to induce AA release. The experiments using ionomycin
and EGTA confirmed that a sustained [Ca2+]i
increase is essential for AA release in CHO cells (Fig. 5D).
The difference in the extent of AA release between PAF and ionomycin
(Fig. 5, C and D) is probably due to
phosphorylation of cPLA2 by MAPK. Whereas PAF induced MAPK
activation (34) and mobility shift of cPLA2 in
SDS-polyacrylamide gel electrophoresis (36), ionomycin did not activate
MAPK or promote the gel shift of cPLA2 (data not shown).
These results are consistent with the previous reports that
phosphorylation of cPLA2 increases its intrinsic enzymatic
activity (7, 37, 38) and augments Ca2+-induced AA release
(26, 39). It was recently reported that, in mouse peritoneal
macrophages, the effects of phosphorylation of cPLA2 on AA
release differ depending on whether calcium response is transient or
sustained (40). Further studies are necessary to identify fully the
individual and combined roles of calcium and phosphorylation for the
activation of cPLA2 in various types of cells.
Calcium-mediated translocation of heterologously expressed
cPLA2 to the nuclear envelope and endoplasmic reticulum was
shown previously in CHO cells using an anti-cPLA2 antibody
(14). Because immunohistochemical studies provide only a static picture
and have a possible loss in signal linearity due to fixation,
permeabilization, and staining of the cells, we took advantage of the
GFP fusion protein and digital imaging technique to monitor
cPLA2 translocation in real time in living cells. Although
the endoplasmic reticulum forms a fine reticular network throughout the
cytoplasm and around the nuclear envelope in CHO cells (41),
cPLA2-GFP was translocated upon
[Ca2+]i increase to a restricted space at the
perinuclear region including the nuclear envelope (Figs. 6 and 7). This
observation may be consistent with the study in rat alveolar epithelial
cells, in which stimulation with a calcium ionophore A23187 induced
translocation of cPLA2 to the nuclear membrane fraction and
preferential loss of AA from this membrane fraction (42). Therefore,
functional activation of cPLA2 takes place after
translocation to the perinuclear region. Mechanisms involved in the
preferential targeting of the enzyme to this site are under investigation.
Another important finding of the present study is that a brief
[Ca2+]i rise caused a transient translocation of
cPLA2 without AA release, whereas an increase in
[Ca2+]i of longer duration (~5 min) induced a
prolonged translocation of cPLA2 and AA release even after
[Ca2+]i returned to the resting level (Figs. 5
and 7). To explain these observations, we propose a two-step mechanism
for cPLA2 activation. In the initial step,
cPLA2 is translocated to the perinuclear region upon
increase in [Ca2+]i and weakly binds to the
membranes. The enzyme is not yet activated, and the translocation can
be readily reversed upon decrease in [Ca2+]i. In
the second step, the binding of cPLA2 to the membranes becomes stronger with a marked reduction in the dissociation rate. Concomitantly, the enzyme is activated. The transfer from the first
step to the second requires a critical length of continuous [Ca2+]i increase. The critical duration of
[Ca2+]i increase was ~2 min in PAF11 cells but
may differ depending on the cell type and the level of
cPLA2 expression. Possible mechanisms by which
cPLA2 is transferred from the weak-binding state to the
strong-binding state include conformational change of cPLA2
by phosphorylation (7), dissociation of inhibitory proteins such as p11
(43), and interaction with anchoring or activating factors (44). The
requirement of critical duration of [Ca2+]i
increase for the activation of cPLA2 may serve as a safety
mechanism to discriminate appropriate signals from transient fluctuations of [Ca2+]i. Once the
[Ca2+]i transient exceeds the critical duration,
the strong-binding state of cPLA2 allows the cells to
produce AA for prolonged periods even after
[Ca2+]i returns to the resting value.
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ACKNOWLEDGEMENTS |
We thank Drs. H. Hanaka and T. Izumi for
plasmids; Dr. Y. Kaziro and other laboratory members (University of
Tokyo and Tokyo Institute of Technology) for valuable discussions; and
M. Ohara for comments.
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FOOTNOTES |
*
This work was supported in part by grants-in-aid from the
Ministry of Education, Science, Sports and Culture, CREST of Japan Science and Technology, and the Human Science Foundation.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: Dept. of
Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.:
81-3-5802-2925; Fax: 81-3-3813-8732; E-mail:
tshimizu{at}m.u-tokyo.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
AA, arachidonic
acid;
CHO, Chinese hamster ovary;
[Ca2+]i, intracellular Ca2+ concentration;
PLA2, phospholipase A2;
cPLA2, cytosolic
phospholipase A2;
MAPK, mitogen-activated protein kinase;
PAF, platelet-activating factor;
LTB4, leukotriene
B4;
GFP, green fluorescent protein;
BSA, bovine serum
albumin.
 |
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