Background
Cytosolic PLA
2 specifically hydrolyzes
sn-2 arachidonate from phospholipid
providing the precursors for many different lipid mediators
including prostaglandins and leukotrienes [ 1 2 ] . These
lipid metabolites play a role in acute inflammatory
responses and also regulate normal physiological processes.
Certain prostaglandins are required for female reproduction
and kidney function [ 3 4 5 ] . Because of its important
role in controlling levels of arachidonic acid (AA), much
attention has been focused on the regulation of cPLA
2 activation, with particular emphasis
on the role of its phosphorylation and Ca 2+-mediated
translocation [ 6 7 8 ] .
cPLA
2 is regulated by controlling its
cellular localization and access to membrane-phospholipid
substrate. An amino terminal, calcium-dependent lipid
binding (CaLB or C2) domain regulates Ca 2+-mediated cPLA
2 translocation to intracellular
membranes [ 9 ] . In vitro, membrane docking via the C2
domain is necessary and sufficient for catalysis and
release of AA [ 10 ] . Binding of calcium ions by the cPLA
2 C2 domain is essential for the lipid
association in vitro [ 11 12 ] and translocation in vivo [
13 14 ] . In response to an increase in [Ca 2+]
i , cPLA
2 translocates to the Golgi and ER,
however translocation to Golgi occurs at a lower [Ca 2+]
i [ 15 ] .
Protein kinase pathways play major roles in cPLA
2 activation, and regulation by the
mitogen-activated protein kinase kinase (MEK)
/extracellular-signal regulated kinase (ERK) signaling
pathway has received particular attention. cPLA
2 is phosphorylated by mitogen activated
protein (MAP) kinases, including p42/p44 ERKs and p38, on
Ser 505in vitro [ 16 17 ] and in response to receptor
stimulation [ 16 18 19 20 21 ] . In addition to
phosphorylation by MAP kinase, it has been shown that cPLA
2 is also phosphorylated on Ser 727by
MAPK-interacting kinase I (MNKI) [ 22 ] and on Ser 515by
calcium/calmodulin-dependent protein kinase II [ 23 ] .
Phosphorylation of these sites may also play a role in
regulating cPLA
2 function in certain cell models.
Phosphorylation of Ser 505has been extensively studied
because it is readily detected due to a characteristic
electrophoretic mobility shift when analyzed by SDS-PAGE [
13 16 ] . The importance of Ser 505phosphorylation in
regulating cPLA
2 has been demonstrated in different
cells and in vitro models by using cPLA
2 containing a S505A mutation [ 16 22 ]
. However, the mechanism whereby Ser 505phosphorylation
regulates cPLA
2 function has been elusive. In vitro
studies have demonstrated that dephosphorylated cPLA
2 is catalytically active and that Ser
505phosphorylation increases activity by only ~30 percent [
24 ] . In contrast, cells expressing the cPLA
2 S505A mutation fail to release AA in
response to a low dose of calcium ionophore, but release
similar amounts of AA as cells expressing wild-type cPLA
2 in response to high dose ionophore [
22 ] . From these studies, it has been suggested that cPLA
2 Ser 505phosphorylation may have a role
in regulating translocation [ 22 ] . A previous study
demonstrated translocation of cPLA
2 S505A in response to Ca 2+ionophore,
but did not address the kinetics of translocation,
translocation in response to a physiological agonist, or
differences in targeting [ 25 ] .
To better understand the regulation of cPLA
2 by the MEK1/ERK pathway and Ca 2+, we
investigated the effect of MEK inhibitors on AA release,
cPLA
2 phosphorylation of Ser 505, cPLA
2 translocation kinetics, and [Ca 2+]
i increase in Madin-Darby canine kidney
(MDCK) cells. We found that inhibition of MEK1 by U0126
significantly inhibited AA release and this was correlated
with inhibition of ERK activation. However, MEK inhibition
only partially affected cPLA
2 phosphorylation and had no effect on
the kinetics of Ca 2+-mediated cPLA
2 translocation to membrane. In
addition, using cells expressing wild-type cPLA
2 and cPLA
2 with S505A or S727A mutations, it was
found that translocation kinetics and membrane targeting in
response to ATP or ionomycin was similar to wild-type cPLA
2 . These data suggest that MEK1
inhibition reduces cPLA
2 catalytic activity and AA release
independently of phosphorylation and translocation.
Results
Effect of MEK inhibition on AA release, ERK
activation, and cPLA 2Ser 505phosphorylation
To study the role of the MEK1/ERK pathway in cPLA
2 activation, quiesced MDCK cells were
treated with the MEK1 inhibitor U0126, and the effect on
AA release, ERK activation, and cPLA
2 gel shift determined (Fig. 1). For
equivalence with the imaging studies, cells expressing
EGFP-cPLA
2 were used in all experiments.
EGFP-cPLA
2 was expressed to similar levels as
endogenous enzyme but did not contribute significantly to
AA release in stably transfected cells. However,
EGFP-cPLA
2 is functional since it
dose-dependently catalyzes release of AA when expressed
in cells that lack endogenous cPLA
2 , such as Sf9 cells [ 13 ] and
immortalized mouse lung fibroblasts from cPLA
2 α knock-out mice [ 26 ] . In order
to enhance AA release in cells containing endogenous cPLA
2 , it is necessary to over-express
the enzyme several fold as previously reported [ 16 ] .
Cytosolic PLA
2 has been shown to mediate Ca
2+-induced AA release in MDCK cells treated with ATP and
IONO in experiments using the group IV cPLA
2 α-specific inhibitor pyrrolidine-1 [
27 ] . To measure cPLA
2 mediated AA release, EGFP-cPLA
2 -transfected MDCK cells labeled with
[ 3H]-AA were incubated with 0.3, 1 or 10 μM U0126 for 15
min prior to stimulation with 100 μM ATP, 1 μM IONO, or
10 μM IONO. AA release was measured at 3 min because we
have shown that ATP- and IONO-stimulated AA release peaks
between 3 to 5 min post-stimulation [ 15 ] .
Agonist-induced AA release was inhibited dose-dependently
by U0126 (Fig. 1A) with the highest U0126 concentration
used (10 μM) reducing AA release by 72-80% with all
agonists (Table I). This inhibition was independent of
the total amount of AA released, since AA release
stimulated by 10 μM IONO was 3-fold greater than release
stimulated with 1 μM IONO or 100 μM ATP, but the percent
inhibition by U0126 was similar. Treatment of MDCK cells
with 30 μM PD098059, a less potent inhibitor of MEK [ 28
29 ] , resulted in a ~50% reduction in AA release in
response to 100 μM ATP, 1 μM IONO, and 10 μM IONO (data
not shown). Thus, in MDCK cells, MEK1 inhibition
significantly reduces the ability of cPLA
2 to hydrolyze AA from membrane
phospholipids.
The effect of MEK1 inhibition on activation of p42/p44
ERK measured by immunoblot analysis using
phospho-specific antibodies in cells treated with U0126
and stimulated as above was determined (Fig. 1B). Work in
our laboratory has shown that recognition of ERK by
anti-phospho-ERK antibodies correlates with an increase
in ERK activity [ 21 30 31 ] . Interestingly, the
anti-phospho-ERK immunoblots revealed that ERKs were
constitutively activated in untreated, quiesced MDCK
cells and activation was not enhanced further by ATP or
IONO (Fig. 1B, left panel). ERK activation was diminished
by increasing concentrations of U0126 and was
quantitatively inhibited after 15 min incubation in 10 μM
U0126. U0126 decreased ERK activation following ATP or
IONO stimulation in the same fashion as in unstimulated
cells. Consequently, there was a direct correlation
between the reduction of AA release (Fig. 1A) and
inhibition of ERK activation (Fig. 1B) in MDCK cells
treated with U0126.
Because cPLA
2 is a target of the MEK1/ERK
signaling cascade, we assayed the effect of MEK1
inhibition by U0126 on cPLA
2 phosphorylation by analyzing gel
shift of cPLA
2 . Phosphorylation of Ser 505results
in a retardation of its electrophoretic mobility (gel
shift) [ 13 16 ] . In unstimulated cells, EGFP-tagged and
endogenous cPLA
2 were nearly completely gel shifted,
indicating that most cPLA
2 was phosphorylated on Ser 505(Fig.
1C), which is consistent with the observation that ERKs
are constitutively activated. Incubation with U0126
resulted in a partial reversal of the gel shift although,
at 10 μM U0126, approximately half of cPLA
2 remained phosphorylated on Ser 505.
Thus, unlike the quantitative effect of U0126 on ERK
activation, inhibition of MEK1 with U0126 only partially
reversed the gel shift of cPLA
2 . The reversal of the gel shift was
similar in cells treated with ATP and 1 and 10 μM IONO.
Due to the increased molecular weight of the EGFP-tagged
cPLA
2 , the two forms of cPLA
2 did not separate as well, making the
gel shift more difficult to visualize, but generally
mirrored the gel shift characteristics of the endogenous
cPLA
2 . These results suggest that, in
response to MEK1 inhibition, there is a quantitative,
dose-dependent decrease in AA release that correlates
well with the loss of ERK activation, but not with the
extent of cPLA
2 Ser 505phosphorylation.
To further investigate whether the MEK1/ERK pathway
played a role in regulating AA release independent of Ser
505phosphorylation, we treated cells with anisomycin,
which activates the MAPK homolog p38, but not the
MEK1/ERK pathway [ 32 ] . Activation of p38 in response
to anisomycin treatment was analyzed by immunoblotting
using an anti-phospho-p38 antibody (Fig. 2A). The
immunoblots demonstrate that 30 min treatment in 25 ng/ml
anisomycin resulted in phosphorylation of p38 in
unstimulated MDCK cells and in cells treated with ATP or
IONO. ATP and ionomycin treatment in the absence of
anisomycin only weakly increased p38 phosphorylation. As
expected, the MEK inhibitor U0126 did not significantly
affect anisomycin-stimulated p38 phosphorylation. In
control experiments, anisomycin treatment did not induce
ERK activation nor interfere with inhibition of ERK
activation by 10 μM U0126 treatment (Fig. 2B). We have
previously reported that p38 is also selectively
activated in anisomycin-treated macrophages [ 21 ] .
Importantly, pretreatment of cells for 30 min with
anisomycin resulted in maintenance of the cPLA
2 gel shift in the presence of U0126
in unstimulated cells and in cells stimulated with ATP
and 1 and 10 μM IONO (Fig. 2C). AA release assays show
that, in MDCK cells treated with anisomycin, U0126
resulted in an AA release reduction of ~67-76% (Fig. 2D)
similar to the inhibition observed without anisomycin.
These results demonstrate that activation of the MEK1/ERK
pathway is required for AA release even under conditions
where cPLA
2 Ser 505phosphorylation is
maintained, suggesting an alternative role for the
MEK1/ERK pathway in regulating cPLA
2 .
[Ca 2+] iincrease is independent of MEK1/ERK
pathway
One explanation for the decrease in AA is that U0126
inhibits [Ca 2+]
i mobilization in response to ATP or
IONO, thereby preventing translocation of cPLA
2 . In chick ventricular myocytes,
inhibition of MEK1/ERK by PD98059 inhibits
zinterol-mediated AA release, but also inhibits
zinterol-induced stimulation of [Ca 2+]
i cycling in electrically stimulated
cells [ 33 ] . In MDCK cells, extracellular ATP acts via
P
2Y2 receptors to elicit an IP
3 -mediated [Ca 2+]
i increase [ 34 ] and IONO acts to
increase [Ca 2+]
i by permeabilizing cell membranes to
Ca 2+. To determine the effect of U0126 on intracellular
Ca 2+mobilization by 100 μM ATP or 10 μM IONO, we
utilized single-cell fluorescence microscopy on cells
loaded with the calcium indicator Fura2. Analysis of the
[Ca 2+]
i increase in individual cells reveals
the heterogeneity in the response to ATP, although most
cells exhibited [Ca 2+]
i spikes of similar magnitude and
duration (Fig. 3Aand 3B, thin lines). Analysis of the [Ca
2+]
i increase in several cells (Fig.
3Aand 3B, thick line) revealed that although the duration
of the [Ca 2+]
i increase elicited by ATP in control
cells was the same as in the U0126-treated cells,
approximately 3-4 min, the amplitude of the [Ca 2+]
i increase was slightly higher (~20%)
in the U0126-treated cells. IONO elicited a sustained,
supraphysiological [Ca 2+]
i increase in cells that was also
slightly enhanced by U0126 (Fig. 3Cand 3D). These
experiments demonstrate that U0126 does not decrease [Ca
2+]
i mobilization, and the inhibition of
AA release by U0126 cannot be ascribed to a failure in
[Ca 2+]
i mobilization.
cPLA 2translocation is independent of MEK1/ERK
pathway
The effect of inhibition of the MEK1/ERK pathway by
U0126 on translocation of cPLA
2 was investigated. Although there is
no inhibition of [Ca 2+]
i release by U0126 and little effect
on Ser 505phosphorylation, it is possible that MEK1
inhibition by U0126 prevents cPLA
2 translocation by another mechanism.
To investigate this possibility, cells were transfected
with a wild-type cPLA
2 fused to EYFP (EYFP-cPLA
2 ) and the distribution of EYFP-cPLA
2 was imaged in response to [Ca 2+]
i transients elicited by ATP and
sustained [Ca 2+]
i elevations elicited by IONO, in the
presence and absence of U0126. Following stimulation with
100 μM ATP, there was a rapid translocation of EYFP-cPLA
2 to Golgi that was unaffected by
U0126 (Fig. 4A,4B). In response to physiological agonists
that elicit transient [Ca 2+]
i changes, only a small fraction of
the cPLA
2 translocates. This observation is
consistent with our previous results [ 15 ] and has been
demonstrated by Hirabayashi et al. [ 35 ] . Most studies
of cPLA
2 translocation have utilized
ionophore, which elicits a large, supraphysiological
sustained increase in [Ca 2+]
i [ 14 15 25 35 36 37 38 ] , or
agonists that produce a sustained [Ca 2+]
i increase [ 35 ] . Under these
conditions, a large proportion of cPLA
2 binds to membrane. These studies
show extensive translocation to the endoplasmic reticulum
(ER), nuclear envelope and Golgi [ 14 15 35 36 38 ] . We
found that U0126 also failed to alter extensive EYFP-cPLA
2 translocation to Golgi and ER in
response 10 μM IONO (Fig. 4C,4D). These results
demonstrate that MEK inhibition has no effect on cPLA
2 translocation.
Translocation of phosphorylation site mutants S505A
or S727A is similar as wild-type cPLA 2
Phosphorylation of cPLA
2 on Ser 505has been hypothesized to
play a role in Ca 2+-mediated translocation since Ser
505phosphorylation is required for cPLA
2 -mediated AA release in response to
low-dose, but not high-dose, ionophore [ 22 ] .
Translocation of cPLA
2 S505A in CHO cells has been reported
in response to ionophore stimulation [ 25 ] , but the
effect of Ser 505phosphorylation on the kinetics of
translocation, targeting, and in response to a
physiological agonist was not investigated. MDCK cells
were co-transfected with EYFP-cPLA
2 and a cPLA
2 with a S505A mutation fused to ECFP
(ECFP-cPLA
2 S505A). Using dual EYFP/ECFP
imaging, we were able to directly compare translocation
of both constructs in the same cell. The resting
distribution of EYFP-cPLA
2 was similar to that of ECFP-cPLA
2 S505A and, in response to ATP
followed by IONO, the pattern of translocation of
EYFP-cPLA
2 was similar to ECFP-cPLA
2 S505A (Fig. 5panels A and B, D).
Analysis of the increase in fluorescence at the Golgi
with respect to time demonstrates that the rates of
translocation of cPLA
2 and cPLA
2 S505A elicited by ATP followed by
IONO are very similar (Fig. 5C). As previously reported [
22 ] , the cPLA
2 S727A mutation has a similar
phenotype on AA release as the S505A mutation and Ser
727was found to be phosphorylated in tandem with Ser 505.
Imaging experiments were performed using EYFP-cPLA
2 and ECFP-cPLA
2 S727A and we found that the
distribution of EYFP-cPLA
2 was identical to that of ECFP-cPLA
2 S727A before and after stimulation
with 10 μM IONO (Fig. 7 panels A and B, D). Analysis of
the increase in fluorescence at the Golgi with respect to
time demonstrates that the rates of translocation of cPLA
2 and the cPLA
2 S727A elicited by IONO are very
similar (Fig. 6C).
Discussion
The MEK1/ERK pathway regulates cPLA
2 and ERKs phosphorylate cPLA
2 on Ser 505. The results of this study
demonstrate that this pathway is required for cPLA
2 -mediated AA release independent of
Ser 505phosphorylation and extend our previous work in
macrophages [ 13 ] by demonstrating that this alternative
role of the MEK1/ERK pathway is not involved in regulating
[Ca 2+]
i change or cPLA
2 translocation kinetics or targeting,
but is required for optimal hydrolytic activity and AA
release.
The results shown here demonstrate that inhibition of
MEK with U0126 quantitatively inhibits both ERK
phosphorylation and AA release in MDCK cells in response to
[Ca 2+]
i mobilization. The MEK inhibitor
PD098059, a less potent inhibitor [ 28 29 ] , also
inhibited ATP- and IONO-induced AA release (data not
shown). ERK was found to be constitutively phosphorylated
in our study using MDCK cells from ATCC, in contrast to
what has been reported previously for MDCK-D
1 , a subclone of MDCK selected for
adrenergic receptor expression [ 39 40 ] . However, a
side-by-side comparison of MDCK cells from ATCC and the
MDCK-D
1 subclone (kindly provided by Dr. Paul
Insel, UCSD) demonstrated constitutive activation of ERKs
when both are grown at low density (not shown). However, at
high density ERKs are less active and can be further
activated by phorbol ester (not shown).
In MDCK-D
1 cells, without constitutively active
ERKs, AA release is delayed after [Ca 2+]
i mobilization, and is temporally
correlated with ERK activation [ 40 ] , whereas in MDCK
cells with constitutively active ERKs, AA release is rapid,
with significant AA release measured 30 s after [Ca 2+]
i increase [ 15 ] . This temporal
correlation between AA release and ERK activation has also
been reported in CHO cells in response to PAF stimulation [
35 41 ] . These results support the observations made here
that ERK activity is required for phospholipid hydrolysis
independently of cPLA
2 translocation.
cPLA
2 -mediated AA release must be preceded
by translocation of the enzyme to its membrane substrate
which is a Ca 2+-dependent process and is a function of the
calcium-dependent lipid-binding (C2) domain. cPLA
2 translocates primarily to Golgi in
response to a transient [Ca 2+]
i changes and to Golgi and ER in
response to a sustained [Ca 2+]
i increase [ 15 ] . The reduction in AA
release by MEK inhibition did not involve a failure in [Ca
2+]
i release or translocation. These
results show that translocation is necessary but not
sufficient for optimum hydrolytic activity.
Measuring cPLA
2 translocation is not a trivial matter
when investigating mechanisms of AA release. For example,
cPLA
2 constructs with a S505A mutation have
long been recognized as unable to support AA release in
response to physiological agonists or low-dose ionophore
when transfected in cells. In contrast, cPLA
2 S505A is active in vitro and
phosphorylation only modestly increases the activity of the
enzyme [ 16 17 24 ] . Interestingly, the inhibitory effect
of the S505A mutation on AA release is obviated by a high
[Ca 2+]
i increase. In light of these
observations, it is possible that Ser 505phosphorylation
may alter the [Ca 2+]
i sensitivity of the enzyme, its rate of
translocation, its intracellular targeting or, as has been
previously suggested [ 22 ] , the ability of cPLA
2 to release from a non-membrane
sequestering agent. Although one report has shown that cPLA
2 S505A translocates in CHO cells in
response to ionophore, we were able to directly compare
rates of translocation between cPLA
2 and cPLA
2 S505A to a physiological agonist and
found no difference between translocation rates or
intracellular targeting. We also demonstrated that there
was no difference in translocation rates or intracellular
targeting between wild-type cPLA
2 and cPLA
2 S727A, which has the same phenotype as
S505A with regard to AA release when transfected into
cells. Thus, the role of cPLA
2 phosphorylation in mediating AA
release remains unclear.
Although the alternative mechanism whereby the MEK1/ERK
pathway regulates cPLA
2 is not known, it is possible that it
affects membrane properties and/or cPLA
2 conformation that promotes optimal
hydrolytic activity. It is also possible that the
alternative mechanism is due to phosphorylation of cPLA
2 on a novel site by a kinase that is
downstream of the MEK1/ERK pathway or phosphorylation of a
regulatory protein.
Conclusions
Translocation to membrane is a critical regulatory step
for the action of cPLA
2 because it is necessary for access to
substrate. In this study we demonstrate, however, that
association of cPLA
2 with membrane when phosphorylated on
Ser 505is not sufficient for its full activity in vivo.
This is demonstrated by the results showing that inhibition
of the MEK1/ERK pathway significantly blocks AA release but
has no effect on [Ca 2+]
i mobilization or cPLA
2 translocation and targeting.
Diminution of AA release by MEK1/ERK is also independent of
cPLA
2 phosphorylation on Ser 505.
Consequently, our results demonstrate in living cells that
the translocation process and subsequent catalytic activity
on the membrane are two independently regulated steps.
Materials and Methods
Fluorescent protein-cPLA 2fusion constructs
DNA encoding the full-length human cPLA
2 was cloned into the vector pEGFP-C3
(Clontech) to create pEGFP-cPLA
2 , as previously described [ 15 ] .
The XbaI/PstI fragment from a cPLA
2 α clone containing S505A or S727A
mutations [ 13 ] was inserted into an XbaI/PstI site in
pEGFP-cPLA
2 to generate pEGFP-cPLA
2 S505A and pECFP-cPLA
2 S727A. Different fluorescent-protein
tagged constructs were produced by exchanging the
NheI/BsrGI fragment containing the fluorescent protein
coding sequence between EGFP, EYFP, and ECFP. All
constructs were confirmed by sequencing.
Cell culture
MDCK cells obtained from ATCC were cultured in DMEM
containing 10% FBS, 100 U/ml penicillin, 100 μg/ml
streptomycin, 0.292 mg/ml glutamine (growth medium) in 5%
CO
2 at 37°C. Subconfluent cells (5 × 10
3cells/cm 2) were transfected with 2 μg of the relevant
plasmid using Fugene-6 (Boehringer Mannheim) in DMEM
containing 0.2% BSA, 100 U/ml penicillin, 100 μg/ml
streptomycin, 0.292 mg/ml glutamine (serum-free medium)
following the manufacturer's protocol. Stable lines
expressing EGFP-cPLA
2 were generated by growing
transfected cells in growth medium for 3 d, supplementing
the growth medium with 5 mg/ml Geneticin (antibiotic
G418-sulfate), and culturing for an additional 2 wk in
Geneticin. Cells expressing EGFP fluorescence were
selected using a fluorescence-activated cell sorter. The
EGFP-positive cells were maintained in growth medium
supplemented with 5 mg/ml Geneticin. For imaging studies,
MDCK cells were plated on glass-bottomed 35 mm culture
dishes (MatTek) at 5 × 10 3cells/cm 2in growth medium and
incubated overnight, transfected with the relevant
plasmid(s), changed into serum-free medium to quiesce the
cells, incubated overnight, and used the next day.
Immunoblotting
Stable EGFP-cPLA
2 transfectants were grown on 100 mm
dishes at 5 × 10 3cells/cm 2in growth medium for one day,
then quiesced in serum-free medium overnight. Cells were
scraped into ice-cold lysis buffer: 50 mM HEPES, pH 7.4,
150 mM sodium chloride, 1.5 mM magnesium chloride, 10%
glycerol, 1% Triton X-100, 1 mM EGTA, 200 μM sodium
vanadate, 10 mM tetrasodium pyrophosphate, 100 mM sodium
fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin.
Lysates were centrifuged at 15,000 ×
g for 15 min, and protein
concentration of the supernatant was determined by the
bicinchoninic acid method. Laemmli electrophoresis sample
buffer (5×) was added to the lysates, and
SDS-polyacrylamide gel electrophoresis and immunoblotting
were performed using 35 μg lysate protein,
phospho-specific antibodies for ERK and p38, and rabbit
polyclonal antibody for cPLA
2 [ 21 ] .
Dual imaging microscopy of fluorescent protein
translocation
In order to compare the characteristics of full-length
cPLA
2 and cPLA
2 S505A or cPLA
2 S727A translocation, while
controlling for cell-to-cell heterogeneity, we used a
dual CFP/YFP imaging approach. EYFP-cPLA
2 /ECFP-cPLA
2 S505A- or pECFP-cPLA
2 S727A-transfected MDCK cells grown
on MatTek plates were quiesced overnight in serum-free
medium, washed with and incubated in Hank's balanced salt
solution (HBSS) additionally buffered with 25 mM HEPES pH
7.4 (HHBSS). Cells were imaged using an Olympus inverted
microscope equipped with a 60×, 1.25 NA oil immersion
objective, CFP and YFP emission filters (Chroma) in a
Sutter filter wheel, a dual CFP/YFP dichroic mirror, and
a TILL Imago CCD camera (TILL Photonics). Excitation
light of 430 and 510 nm for CFP and YFP, respectively,
was provided using a Polychrome IV monochromator (TILL
Photonics). TILLvisION software was used for acquisition
and analysis. Bleach values for ECFP and EYFP were
calculated by determining the background-corrected
fluorescence for the entire cell with respect to time and
normalizing each value to the initial value. ECFP/EYFP
fluorescence changes with respect to time for regions of
interest corresponding to an area of Golgi membrane were
determined by calculating the F
t /F
0 , where F
t is the background- and
bleach-corrected ECFP or EYFP fluorescence at time = t
and F
0 is the background-corrected ECFP or
EYFP fluorescence at time = 0 s. Fluorescence was
normalized to the F
0 value, which resulted in F
t /F
0 representing the fraction of total
cell fluorescence at Golgi. Final images were produced
using Adobe Photoshop.
Calcium imaging
MDCK cells grown on MatTek plates were quiesced
overnight in serum-free medium, washed with HHBSS
containing 1 mM probenecid and incubated with 5 μM
Fura2-AM (Calbiochem) in HHBSS, 1 mM probenecid, and 1%
DMSO for 45 min at 37°C. Cells were then washed with
HHBSS containing 1 mM probenecid and imaged after a 30
min incubation for de-esterification of the Fura2-AM.
Single-cell imaging was performed on the Olympus system
described above, but using a 40×, 1.35 NA oil immersion
objective and a Fura2 dichroic mirror and emission filter
(Chroma). Fura2 image pairs illuminated at 340 and 380 nm
were taken at 1 Hz. The [Ca 2+]
i increase is expressed as the ratio
of the background-corrected Fura2 fluorescence at 340 and
380 nm [ 42 ] .
Measurement of AA release
The protocol for determining AA release is essentially
as described [ 15 21 ] . MDCK cells stably expressing
EGFP-cPLA
2 were plated in 12-well plates at 5 ×
10 3cells/cm 2and incubated in growth medium overnight.
Cells were then washed twice with serum-free medium and
incubated with 0.25 μCi [ 3H]-AA/well in serum-free
medium overnight. U0126 or vehicle was added to each well
and the cells were then incubated for 15 min at 37°C in
5% CO
2 . Cells were washed to remove
unincorporated [ 3H]-AA and then incubated in HHBSS
supplemented with 0.05% BSA with either U0126 or vehicle.
Cells were stimulated with the agonist of choice and the
medium was collected at appropriate time points. The
medium was centrifuged at 500 g for 5 min, and the amount
of radioactivity in the supernatant was determined by
scintillation counting. Cells were scraped in 0.5 ml 0.1%
Triton X-100 for determining the total cellular
radioactivity.
Authors' contributions
JHE carried out the Ca and FP imaging studies,
participated in the design and coordination of the study,
and drafted the manuscript. DJF performed the AA release
and Western blot studies and participated in the design and
coordination of the study. CCL conceived of the study,
participated in its design and coordination, and
participated in writing the draft. All authors read and
approved the final manuscript.
