Bradykinin-stimulated cPLA2
phosphorylation is protein kinase C dependent in rabbit CCD cells
Mark A.
Lal1,
Chris R. J.
Kennedy2,
Pierre R.
Proulx2, and
Richard L.
Hébert1,3
Departments of 1 Physiology,
2 Biochemistry, and
3 Medicine, Faculty of
Medicine, University of Ottawa, Ontario, Canada K1H 8M5
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ABSTRACT |
We have used an
established cell line of rabbit cortical collecting duct (RCCD)
epithelial cells representing a mixed population of principal and
intercalated cell types to determine which phospholipase A2
(PLA2) enzyme therein is
responsible for bradykinin (BK)-stimulated arachidonic acid (AA)
release and how its activation is regulated. BK-stimulated AA release
was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of
cytosolic PLA2
(cPLA2). Examination of PLA2 activity in vitro
demonstrated that BK stimulation resulted in a greater than twofold
increase in PLA2 activity and that
this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2.
To determine a possible role for protein kinase C (PKC) in the
BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects
on AA release, cPLA2 activity, and
phosphorylation. Ro31-8220 reduced BK-stimulated AA release and
cPLA2 activity by 51 and 58%,
respectively. cPLA2 activity
stimulated by phorbol ester [phorbol 12-myristate
13-acetate (PMA)] displayed a similar degree of activation and
was associated with an increase in serine phosphorylation identical to
that caused by BK. The phosphorylation-induced activation of this
enzyme was confirmed by the phosphatase-mediated reversal of both BK-
and PMA-stimulated cPLA2 activity.
In addition, we have also found that PMA stimulation did not cause a
synergistic potentiation of BK-stimulated AA release as did calcium
ionophore. This occurred despite membrane PKC activity increasing 93%
in response to PMA vs. 42% in response to BK. These data, taken
together, indicate that cPLA2 is
the enzyme responsible for BK-mediated AA release, and, moreover, they
indicate that PKC is involved in the onset responses of
cPLA2 to BK.
cytosolic phospholipase A2; rabbit cortical collecting duct cells
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INTRODUCTION |
THE MAMMALIAN KIDNEY, in particular the collecting duct
segment of the nephron, represents a major site of renal sodium and water reabsorption (6). Of the many agonists known to regulate collecting duct function, the nonapeptide bradykinin (BK) assumes a
particular importance (6). This peptide is a potent vasodilator, natriuretic, and diuretic that has been shown to specifically inhibit
vasopressin-stimulated water permeability in the rabbit cortical
collecting duct (RCCD) while increasing intracellular Ca2+ mobilization, inositol
trisphosphate generation, and protein kinase C (PKC) activity (1, 12,
37). Ultimately, it appears that it is the production of prostaglandins
(largely prostaglandin E2)
mediated by BK signaling that is responsible for the inhibition of
water permeability (36).
Availability of the precursor molecule, arachidonic acid (AA), is
thought to represent the rate-limiting step for the production of the
prostaglandins. This fatty acid is found mainly at the sn-2 position of membrane
phospholipids and is readily cleaved off by phospholipase
A2
(PLA2) activity known to be
exhibited in at least three groups of enzymes: the secretory
PLA2 enzymes with a molecular mass
of ~14 kDa, dependent on a high concentration of
Ca2+; the
Ca2+-independent
PLA2 types; and the 85-kDa
cytosolic PLA2
(cPLA2), dependent on a low
concentration of Ca2+ (25, 26).
Over the past years, cPLA2 has
received a great deal of attention because it is AA specific and
appears to represent the enzyme that is distinctively regulated by cell
signaling mechanisms downstream of receptor occupation. Since its
purification by Clark et al. (10),
cPLA2 has been demonstrated to
display both Ca2+- and
phosphorylation-dependent activation (23). This enzyme possesses a
Ca2+ lipid binding (CaLB) domain,
which, in the presence of micromolar Ca2+ concentrations, allows
translocation to membranes (9), particularly those of the nuclear
envelope and endoplasmic reticulum (18, 35). Furthermore, it has been
demonstrated in transfected Chinese hamster ovary cells overexpressing
cPLA2 that phosphorylation on
serine-505 by mitogen-activated protein kinase, secondary to PKC
stimulation, is a necessary component for enzyme activation (24). In
contrast to these and other studies (3, 23, 27, 31, 32, 43, 45) that
support a role for PKC in mediating cPLA2 phosphorylation in response
to various agonists, a number of reports describe a lack of any role
for this enzyme (5, 11, 16, 18, 39, 41). It appears that regulation of
cPLA2 phosphorylation by PKC may
depend on the specific agonist-receptor coupling and the cell type
involved. With regard to BK-mediated AA release and
cPLA2 involvement in the
collecting duct, to date there have been no studies other than
extensive ones on Madin-Darby canine kidney (MDCK) cells (19, 20, 38,
42). A role for PKC in mediating BK-stimulated AA release
in MDCK cells has been implicated to varying degrees, depending on the
method of study (14, 42). Recent evidence obtained in our laboratory
has denied a role for PKC in the onset responses of BK
stimulation leading to AA release in MDCK cells (unpublished
results).
In the present study, we have used a recently characterized RCCD cell
line to study the regulation of AA release by this nephron segment.
These transfected RCCD cells represent a mixed population of principal
-intercalated and
-intercalated epithelial cell types as assessed
by specific antibody recognition. They have retained both their
morphological and hormonal response properties and possess the
signaling machinery present downstream of angiotensin signaling (8).
When RCCD cells were treated with BK, we found that this peptide was
able to specifically activate the
cPLA2 isoform and that serine
phosphorylation was at least partially involved in this response. Our
results reveal that cPLA2 is the major enzyme responsible for AA release in RCCD cells and that its
activity is dependent on PKC recruitment as an onset response to BK
stimulation.
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EXPERIMENTAL PROCEDURES |
Materials.
BK, the Ca2+ ionophore A-23187,
potato acid phosphatase, and
1-stearoyl-2-arachidonyl-sn-glycerol
were purchased from Sigma Chemical (Mississauga, ON, Canada). The agent
RHC-80267, phorbol 12-myristate 13-acetate (PMA), and arachidonyl
trifluoromethyl ketone (AACOCF3)
were obtained from Biomol Research Laboratories (Plymouth Meeting, PA),
whereas Ro31-8220 was supplied by Calbiochem-Novabiochem (La Jolla,
CA). Phosphatidylcholine
(1-stearoyl-2-L-[
-arachidonyl-5,6,8,9,11,12,14,15-3H]phosphatidylcholine)
was obtained from DuPont NEN (Mississauga, ON).
[5,6,8,9,11,12,14,15-3H]AA,
biodegradable counting scintillant, horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin (Ig), enhanced chemiluminescence (ECL) Hyperfilm, Hybond nitrocellulose, ECL reagents, and PKC assay kit
were from Amersham (Oakville, ON). Protein A-agarose for
immunoprecipitation was purchased from Boehringer Mannheim (Laval, PQ,
Canada). The rabbit polyclonal antibody to phosphoserine, a product of
Dimension Laboratories (Mississauga, ON), was purified from rabbit
antiserum by phosphoserine-specific affinity chromatography and shows
no significant cross-reactivity to either phosphothreonine or
phosphotyrosine. The Genetics Institute (Boston, MA) kindly provided
the 85-kDa cPLA2 standard protein
and rabbit polyclonal antibody to
cPLA2, which is directed against
amino acids 42-58, located within the CaLB domain.
Cell culture.
RCCD cells, used between passages
3 and
25, were cultured in Dulbecco's
modified Eagle's medium (DMEM)-F-12 medium supplemented with 10%
fetal calf serum, 0.4% Pen/Strep solution (GIBCO), 15 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, 44 mM sodium bicarbonate, insulin-transferrin-sodium selenite
medium supplement (Sigma), 50 nM hydrocortisone, and 2.5 nM
3,5,3'-triiodothyronine. Cells were maintained in an atmosphere
of 5% CO2 at 37°C and were passaged after 2-3 days. Experiments were performed with RCCD cells cultured in 12-well dishes (for measurement of AA release) or 100 × 20-mm dishes (for PLA2
activity assays, immunoprecipitations, and PKC activity assays) before
formation of a complete monolayer of cells.
Measurement of AA release.
Cells grown in 12-well dishes were serum starved overnight in DMEM-F-12
medium containing 0.05% (wt/vol) bovine serum albumin (BSA) and 0.3 µCi [3H]AA. After
~24 h of incubation, the labeled medium was aspirated. This was
followed by two washes with Hanks' balanced salt solution (HBSS)
containing 0.05% BSA and a 30-min preincubation. Cells were
subsequently stimulated for 15 min at 37°C with various agents. When examining the effect of a specific inhibitor, we included the
agent during both the preincubation and stimulatory period. After
stimulation, medium was immediately removed and centrifuged at 5,000 g for 5 min to pellet any cellular
debris. An aliquot of the supernatant was measured for
[3H]AA release by
scintillation counting. Results were normalized for total label
incorporated into the cells by dividing the disintegrations per minute
(dpm) [3H]AA released
by the total dpm
[3H]AA incorporated
into the cells [determined by solubilizing the cells in 5%
sodium dodecyl sulfate (SDS)].
Determination of PLA2 activity.
Cells grown in 100 × 20-mm dishes were serum starved overnight in
DMEM-F12 medium containing 0.05% BSA. The next day they were washed
twice with HBSS + 0.05% BSA and preincubated for 30 min at 37°C
before stimulation. Cells that were subject to the action of inhibitors
were exposed to these agents during this preincubation and also during
stimulation. After a 2-min stimulation, medium was rapidly aspirated
and cells were washed twice with ice-cold wash buffer containing 50 mM
tris(hydroxymethyl)aminomethane (Tris) hydrochloride (pH 7.5), 250 mM
sucrose, and 1 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA). They were then scraped off the plates into wash buffer,
centrifuged at 1,000 g for 5 min, and
resuspended in lysis buffer composed of 50 mM
Tris · HCl (pH 7.5), 250 mM sucrose, 1 mM EGTA,
protease inhibitors [in µg/ml: 100 benzamidine, 20 leupeptin, 2 phenylmethylsulfonyl fluoride (PMSF), 30 bacitracin, 100 aprotinin], phosphatase inhibitors (in mM: 10 sodium vanadate, 10 sodium pyrophosphate, 1 levamisole), and 5 mM dithiothreitol (DTT). The
resuspended cells were then sonicated on ice by two pulses of 10 s
each, with the use of the small probe of an Ultrasonics cell disrupter
set at 5, and protein concentrations were determined by the Bio-Rad
protein assay method with BSA as a standard. For those experiments
examining the phosphorylation-dependent activation of
cPLA2, cells were resuspended in
the same lysis buffer (without phosphatase inhibitors) as described
above, supplemented with 1 U/ml potato acid phosphatase, and adjusted
to a final pH of 6.1. After sonication, lysates were incubated at
30°C for 30 min, after which the pH was returned to 7.5.
Total cell lysates were subsequently assayed for
PLA2 activity, according to the
protocol described by Leslie (22). Briefly, lysates were incubated in
assay buffer [50 mM Tris · HCl (pH 7.5), 250 mM
sucrose, 0.05% BSA, 1 mM
Ca2+] containing 30 µM
1-stearoyl-2-arachidonyl phosphatidylcholine and 55,000 dpm
1-stearoyl-2-[arachidonyl-3H]phosphatidylcholine
tracer. Incubations were carried out at 37°C and terminated after 1 h by addition of 2.5 ml Dole reagent (2-propanol-heptane-0.5 M
H2SO4;
20:5:1, vol/vol/vol) (13). This was followed by the addition of 1.5 ml
heptane containing 20 µg cold AA. To obtain visibly separate phases,
1 ml of H2O was added, and an
aliquot of the top layer was purified by silicic acid column
chromatography. Columns were then washed with diethyl ether, and the
final collected eluent was dried under nitrogen and analyzed by liquid
scintillation spectrometry.
Immunoprecipitation.
Cells were stimulated under the same conditions as used for the
PLA2 activity assay described
above. After stimulation, they were washed and scraped off the plates
with ice-cold buffer solution containing 50 mM
Tris · HCl (pH 7.5) and 1 mM EGTA. Cells were centrifuged at 1,000 g, resuspended,
and then lysed in immunoprecipitation buffer [50 mM
Tris · HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 20 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 30 µg/ml
bacitracin, 1 mM sodium vanadate, 10 mM sodium pyrophosphate, and 1 mM
levamisole]. Protein concentrations were determined and diluted
to 50 µg/500 µl of immunoprecipitation buffer in a fresh centrifuge
tube. To this aliquot, 25 µl protein A-agarose suspension were added,
and the solution was subsequently rocked on a platform for 3 h at
4°C to reduce background that might have been caused by nonspecific adsorption of cellular debris. After a low-speed centrifugation, the
resultant supernatant was transferred to a new centrifuge tube,
incubated with 4 µl of cPLA2
antibody for 1 h, and left overnight after the addition of 25 µl
protein A-agarose. The beads were then pelleted and washed twice with
500 µl immunoprecipitation buffer, twice with 500 µl
wash buffer 1 [50 mM
Tris · HCl (pH 7.5), 500 mM NaCl, 0.1% NP-40, 0.05%
sodium deoxycholate], and once with 500 µl
wash buffer 2 [50 mM
Tris · HCl (pH 7.5), 0.1% NP-40, 0.05% sodium
deoxycholate]. After the last wash, the immunoprecipitates were
dried with strips of filter paper, extracted in Laemmli sample buffer,
boiled for 5 min, and then subjected to 7.5% SDS-polyacrylamide gel
electrophoresis (PAGE). Gels were then transferred to nitrocellulose membranes and blocked overnight with 5% nonfat skim milk in
Tris-buffered saline (TBS).
Immunoblotting.
Nitrocellulose membranes were incubated with a rabbit antibody to
phosphoserine (1:8,000) for 1 h. Blots were washed repeatedly with 0.1% Tween 20-TBS (TTBS) and then incubated with horseradish peroxidase-conjugated donkey anti-rabbit Ig (1:2,000) for 1 h. After
being extensively washed with TTBS, blots were developed with ECL
reagents according to the manufacturer's specifications.
PKC assay.
Cells were stimulated as described in Determination of
PLA2
activity and then subsequently washed and
scraped off the plates into ice-cold phosphate-buffered saline (pH
7.5). After a low-speed centrifugation, cells were resuspended in
homogenization buffer [50 mM Tris · HCl (pH
7.5), 20 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 1 mM EGTA,
1 mM DTT] and sonicated on ice. To separate cytosol and
particulate fractions, the cell lysate was subjected to a
centrifugation at 100,000 g for 60 min. The high-speed pellet was resuspended in homogenization buffer,
and protein was determined. PKC activity was measured according to the
protocol described in the Amersham kit, which is based on the
phosphorylation of a PKC-specific peptide. The activity is expressed as
the amount of phosphate transferred to a PKC-specific substrate (pmol
phosphate transferred · mg
protein
1 · min
1)
and represents the activities of the conventional and
novel isozymes.
Statistics.
Data are expressed as averages of duplicate determinations from
individual experiments and are presented as means ± SE where n
4 or means ± SD where
n = 3 experiments.
Statistical significance was accepted at
P < 0.05 as determined by Student's
t-test.
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RESULTS |
BK stimulates AA release through cPLA2
activation.
On stimulation with BK for 15 min, RCCD cells released AA in a
dose-dependent manner (Fig. 1). Previous
results from our laboratory with MDCK-D1 cells derived from canine
kidney distal tubule/collecting duct have revealed that BK-mediated AA
release occurs through the action of
cPLA2 (20). Because such
information was not yet available for RCCD cells, we set out to
characterize the enzyme responsible for AA release in these cells.

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Fig. 1.
Dose-dependent arachidonic acid (AA) release in response to bradykinin
(BK). Rabbit cortical collecting duct (RCCD) cells were labeled
overnight with 0.3 µCi
[3H]AA in Dulbecco's
modified Eagle's (DMEM)-F-12 defined medium containing 0.05% (wt/vol)
bovine serum albumin (BSA). Cells were subsequently washed twice with
Hanks' balanced salt solution (HBSS) + 0.05% BSA and then
preincubated for 30 min before a 15-min stimulation with indicated
concentrations of BK.
[3H]AA released into
medium and total cell label incorporated into cells were counted.
Amount of label released was divided by total incorporation and was
expressed as multifold increases in
[3H]AA release
compared with control (n = 3 experiments).
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By Western blotting with a polyclonal antibody to
cPLA2, we could demonstrate that
RCCD cells express cPLA2 protein
(Fig. 2). To examine whether this enzyme is
responsible for mediating BK-induced AA release, we employed several
strategies. The agent AACOCF3, an
analogue of AA that inhibits the 85-kDa
cPLA2, was first tested (33). As
shown in Fig. 3, BK-stimulated AA release was 92% inhibited by 50 µM
AACOCF3, whereas an inhibitor of
diacylglycerol lipase (RHC-80267) was without significant effect. The
latter result precluded the combined action of phospholipase C (PLC) and diacylglycerol lipase as a major pathway for releasing AA. It has
been shown recently that AACOCF3
also inhibits Ca2+-independent
PLA2 (2). However, when cells were
treated with haloenol lactone suicide substrate, a specific inhibitor
of Ca2+-independent
PLA2 (15), there was no
significant attenuation of BK-stimulated AA release. To demonstrate
more definitively that cPLA2 is
directly responsible for BK-stimulated AA release, we first measured in
vitro PLA2 activity. Results (not
shown) from such assays revealed that the
PLA2 activity of RCCD cells could
be activated by submillimolar Ca2+
concentration and was insensitive to the reducing agent DTT, thus
eliminating the likelihood of the involvement of 14-kDa
PLA2 as the isoform responsible
for mediating the response to BK. Second, we observed (cf. Table
1) that maximal BK (100 nM) stimulation resulted in a 2.0-fold increase in
PLA2 activity from 13.9 ± 1.5 to 27.9 ± 2.5 pmol · min
1 · mg
1
as assayed in vitro. This increase in enzyme activity was completely eliminated with the addition of an antibody to
cPLA2. The evidence as a whole
demonstrates that cPLA2 is the
major enzyme mediating BK-stimulated AA release.

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Fig. 2.
RCCD cells express cytosolic phospholipase
A2
(cPLA2). RCCD cells were lysed
in buffer [50 mM Tris · HCl (pH 7.5), 150 mM
NaCl, 1 mM EGTA, 20 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride], boiled for 5 min in Laemmli
sample buffer, and subsequently separated by 7.5% SDS-polyacrylamide
gel electrophoresis (PAGE). Gels were then transferred to
nitrocellulose membranes, blocked overnight with 5% nonfat skim milk
in Tris-buffered saline, and probed with a
cPLA2 antibody (1:2,000) for 1 h.
Blots were then treated as described under
EXPERIMENTAL PROCEDURES.
Lane 1, 10 ng
cPLA2 standard;
lanes 2-4,
2 µg, 5 µg, and 10 µg of cell lysate protein, respectively.
Values to left of blot are in kDa.
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Fig. 3.
Effect of arachidonyl trifluoromethyl ketone
(AACOCF3; an inhibitor of
cPLA2) and RHC-80267 (an
inhibitor of diacylglycerol lipase) on BK-stimulated AA release. RCCD
cells were labeled overnight with 0.3 µCi
[3H]AA in DMEM-F-12
defined medium containing 0.05% (wt/vol) BSA, then washed twice with
HBSS, followed by a 30-min pretreatment with 50 µM
AACOCF3 or 10 µM RHC-80267.
Cells were subsequently stimulated with 1 nM BK in presence of
inhibitors. Medium was removed and counted for
[3H]AA release by
cells. Amount of label released was normalized for total label
incorporated into cells and is presented as a percent inhibition of
BK-stimulated AA release. Data are expressed as means ± SE of 4 independent experiments performed in duplicate.
* P < 0.005 vs. BK alone.
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BK-mediated AA release is PKC dependent.
Taking into consideration the role of
cPLA2 in BK-stimulated AA release,
we next tested whether downstream BK-receptor signaling events could be
mimicked by PMA activation of PKC and the
Ca2+ ionophore A-23187. Results
summarized in Fig.
4A
illustrate that 100 nM PMA alone did not result in an increased release
of AA compared with control; however, when PMA was presented to cells together with 100 nM A-23187, there was a clear synergy of release above that caused by A-23187 alone (compare 4.15 ± 0.21-fold for A-23187 with 9.08 ± 0.72-fold for PMA + A-23187). Furthermore, to
test whether BK-stimulated AA release was dependent on PKC, we used the
bisindolylmaleimide Ro31-8220 as a PKC-selective inhibitor (44). As
seen in Fig. 4A, BK-stimulated AA
release was inhibited 51% by 5 µM Ro31-8220. Higher concentrations
of Ro31-8220 did not result in greater inhibition. Confirming the role
for PKC in regulating AA release, the synergy seen with PMA + A-23187 could also be totally blocked by Ro31-8220, returning AA release to
levels seen for A-23187 alone (compare 2.59 ± 0.26-fold for A-23187/Ro31-8220 with 2.91 ± 0.05-fold for PMA + A-23187/Ro31-8220). Interestingly, Ro31-8220 was also able to inhibit
release by A-23187 alone (Fig. 4A),
which suggests that PKC is involved to some extent in mediating this
response to ionophore. This result is similar to that reported by Lin
et al. (23) and Qiu and Leslie (32).

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Fig. 4.
BK-stimulated AA release is protein kinase C (PKC) dependent.
A: RCCD cells were labeled overnight
with 0.3 µCi [3H]AA
in DMEM-F-12 defined medium containing 0.05% (wt/vol) BSA, then washed
twice with HBSS + 0.05% BSA, followed by a 30-min pretreatment with 5 µM Ro31-8220. Cells were subsequently stimulated for 15 min in
continued presence of inhibitor by 1 nM BK, 100 nM phorbol 12-myristate
13-acetate (PMA), 100 nM A-23187, or 100 nM PMA + 100 nM A-23187.
B: cells were labeled and washed as
for A, followed by a 30-min
preincubation before a 15-min stimulation with 100 nM BK, 100 nM BK + 100 nM PMA, 100 nM A-23187, or 100 nM BK + 100 nM A-23187. Amount of
label released was normalized by dividing by total label incorporated
into cells and was expressed as multifold increases in
[3H]AA release
compared with control. Results are expressed as means ± SE of 4 (A) or 5 (B) independent experiments
performed in duplicate. A:
* P < 0.05 compared with its
respective control. B: * P < 0.05 vs. BK.
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Slivka and Insel (38) previously reported that BK-stimulated AA release
is increased by treatment of MDCK cells together with phorbol ester.
Recent studies from our laboratory on these same cells
confirm these earlier findings (unpublished observations). However, the
present results shown in Fig. 4B
reveal that PMA treatment of RCCD cells is unable to potentiate the
release of AA caused by BK (compare 4.24 ± 0.57-fold for BK with
3.51 ± 0.51-fold for BK + PMA). It is possible that with our
present cells BK engenders a limited
Ca2+ signal and responses cannot
be enhanced by PMA unless ionophore is added (compare 4.24 ± 0.57-fold for BK with 9.48 ± 1.14-fold for BK + A-23187).
Interestingly, the inability of PMA to increase BK-stimulated AA
release prevails, despite our finding that, with respect to control,
PMA caused a 93% increase in membrane PKC activity compared with only
42% for BK (Fig. 5).

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Fig. 5.
Effect of BK and PMA on PKC activity. RCCD cells were serum starved
overnight in DMEM-F-12 defined medium supplemented with 0.05% (wt/vol)
BSA. Cells were preincubated with HBSS + 0.05% BSA, followed
by a 2-min stimulation with 100 nM BK or 100 nM PMA. Membrane PKC
activity was measured as described in EXPERIMENTAL
PROCEDURES and is expressed as pmol peptide
phosphorylated · min 1 · mg
protein assayed 1. Values
are presented as means ± SE of 5 independent experiments performed
in duplicate. * P < 0.001 vs.
basal; ** P < 0.005 vs. BK.
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We next examined the effects of BK and PMA on
cPLA2 activity to see whether they
corresponded to the observed changes in PKC activity. Although the
increase in PKC activity for PMA-treated cells was more than double the
increase found for BK-treated cells, our results, shown in Fig.
6A,
demonstrate that there was no significant difference between the
ability of either agonist to stimulate cPLA2 activity (compare 30.9 ± 2.97 for BK with 36.3 ± 8.08 pmol · min
1 · mg
1
for PMA). The specific role of PKC was confirmed by the 58% inhibition in BK-stimulated cPLA2 activity
observed on treatment with Ro31-8220. This was similar to the 51%
inhibition seen for AA release (Fig. 4A).

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Fig. 6.
PKC is responsible for increasing
PLA2 activity and phosphorylation
by BK. RCCD cells were incubated overnight in DMEM-F-12 defined medium
containing 0.05% (wt/vol) BSA. Cells were preincubated with
(A) or without
(B) 5 µM Ro31-8220 for 30 min
before stimulation with 100 nM BK or 100 nM PMA for 2 min. Total cell
lysate PLA2 activity was
determined as described under EXPERIMENTAL
PROCEDURES. Results are expressed as means ± SE of
4 (A) or means ± SD of 3 (B) separate experiments assayed in
duplicate. * P < 0.05 vs.
basal control; ** P < 0.05 compared with its respective agonist-treated control.
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BK-induced cPLA2 activation occurs
through PKC-mediated phosphorylation.
The activation of cPLA2 by BK or
PMA was thought to be the result of enhanced enzyme phosphorylation. To
determine whether this modification was responsible for the observed
increase in cPLA2 activity, we
treated lysates from both stimulated and unstimulated cells with potato
acid phosphatase. Our results, depicted in Fig. 6B, illustrate that both BK- and
PMA-induced cPLA2 activities were
indeed a result of a phosphorylation event, since phosphatase exposure completely abrogated these responses.
Lin et al. (24) demonstrated that phosphorylation of
cPLA2 on serine-505 is responsible
for increased cPLA2 catalytic
activity. In some cases, PKC appears to be involved either in direct
phosphorylation of cPLA2 (27) or
secondarily by activation of a mitogen-activated protein kinase (24,
31). To specifically determine whether BK treatment causes changes in
the level of enzyme serine phosphorylation, we immunoprecipitated
cPLA2, performed SDS-PAGE, and
subsequently immunoblotted membranes with a rabbit phosphoserine
antibody. Results summarized in Fig.
7A show a
representative blot, and those in Fig.
7B show the averaged optical density
of scans from three such experiments. Both BK and PMA caused an
approximately twofold increase in
cPLA2 phosphorylation, and this
phosphorylation was completely reduced to control levels by Ro31-8220
treatment. The almost identical responses of both
cPLA2 activation and
phosphorylation to BK and PMA, considered together with the
differential activation of PKC by either agonist, suggest that PKC need
not be fully activated to allow the same result or that specific PKC
isozymes are mediating the activation of
cPLA2.

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Fig. 7.
Serine phosphorylation of cPLA2
with BK or PMA stimulation is reversed by Ro31-8220. RCCD cells were
incubated overnight in DMEM-F-12 defined medium containing 0.05%
(wt/vol) BSA. The next day, cells were preincubated with or without 5 µM Ro31-8220 for 30 min before stimulation with 100 nM BK or 100 nM
PMA for 2 min. cPLA2 was
immunoprecipitated, using a polyclonal
cPLA2 antibody, and was resolved
on SDS-PAGE (7.5%). C, control. A:
serine phosphorylation was detected, using a rabbit polyclonal antibody
to phosphoserine residues
(top). Total
cPLA2 present in each lane was
similar, as detected by stripping membrane and reblotting membrane with
cPLA2 antibody
(bottom). Position of molecular mass
markers is indicated to the left of each blot.
B: densitometry analysis of 3 such
experiments.
|
|
 |
DISCUSSION |
AA release and subsequent production of eicosanoids represent key
signaling events for salt and water balance in the mammalian kidney.
Accordingly, the nonapeptide BK is able to modulate kidney water
balance largely through its ability to increase prostaglandin production in the collecting duct segment of the nephron (6). The aim
of the present study was to arrive at a better understanding of how
BK-mediated signaling events regulate AA release in the collecting
duct, and this was achieved with the aid of an immortalized RCCD cell
line. Increasing evidence indicates that agonist-stimulated release of
AA occurs via activation of cPLA2.
Several mechanisms have been proposed for the activation of
cPLA2 and consequent AA release,
namely, direct receptor-G protein coupling to
cPLA2 (4, 21), modulation by
products of PLC and/or phospholipase D (20), and dependence on
phosphorylation (25, 35, 37) and a rise in cytosolic
Ca2+ (3, 7, 23, 28).
Results shown in Figs. 1-3 and Table 1 provide substantial
evidence in support of the conclusion that
cPLA2 is the enzyme largely responsible for BK-stimulated AA release in RCCD cells. In addition to
cPLA2, the rabbit kidney has been
shown to possess secretory PLA2
enzymes as well as a novel 28-kDa
Ca2+-independent
PLA2 recently purified by Portilla
and Dai (30). A role for the disulfide bond-containing
secretory isoforms was eliminated, because BK-stimulated
PLA2 activity was insensitive to
the reducing agent DTT and because
PLA2 could be activated by
Ca2+ in the submillimolar range.
The novel 28-kDa Ca2+-independent
PLA2 was not responsible for
BK-stimulated PLA2 activity, since
we found that omitting Ca2+ from
the assay buffer completely abolished the stimulated activity. These
other PLA2 isoforms may indeed be
present in RCCD cells, but they clearly are not responsible for
BK-stimulated AA release.
To determine whether PKC was involved in the BK-stimulated increase in
AA release and PLA2 activity, we
used PMA to activate and Ro31-8220 to inhibit PKC activity. The agent,
Ro31-8220, a member of the bisindolylmaleimide class of PKC inhibitors,
blunts the activity of a partially purified rat brain preparation
containing PKC-
, -
1,
-
2, -
, -
, -
, and -
,
with a 50% inhibitory concentration of 23 nM (44). In RCCD cells,
BK-stimulated AA release was partly dependent on PKC, since treatment
with Ro31-8220 resulted in >50% inhibition of this response.
Activation of PKC by PMA alone did not mimic BK in its ability to
release AA. However, when cells were treated with PMA + A-23187
together, there was a synergistic increase in AA release. The inability
of PMA to induce AA release on its own is not surprising, since it does
not generate a Ca2+ signal, a
result that has been reported for other systems (5, 23, 38, 40, 45).
Indeed, when cPLA2 activity was
determined in the presence of an assay buffer containing
Ca2+, PMA could be seen to
increase the activity of this enzyme. The mechanism of the PKC- and
Ca2+-dependent synergy of AA
release is probably a result of PKC-mediated phosphorylation of
cPLA2 and its
Ca2+-dependent translocation to
membrane substrates (23). For the purpose of determining which of these
two signals limits BK-stimulated AA release, we incubated BK in the
presence of either PMA or A-23187. In contrast to results on MDCK cells
(38), simultaneous addition of BK + PMA did not result in a
potentiation of AA release. It appears that PKC activation does not
limit the ability of BK to cause AA release but rather that
Ca2+ may be limiting, since BK + A-23187 treatment resulted in a potentiation of BK-stimulated AA
release that was similar to that resulting from PMA + A-23187 treatment
(cf. Fig. 4, A and
B). Although
Ca2+ is critically important to AA
hydrolysis by cPLA2, it should also be appreciated that A-23187 treatment does not likely mimic the
physiological release of Ca2+ by
agonists such as BK, since, in the prior case, both extracellular and
intracellular Ca2+ stores would
indiscriminately raise intracellular
Ca2+ concentration. The ionophore,
in addition to its ability to recruit PKC activity (Fig.
4A) and mediate
Ca2+ influx, may also modify the
integrity of the phospholipid bilayer, thereby altering
cPLA2 binding to its substrate. It
is therefore difficult to firmly establish whether ionophore alters
cPLA2 activity in ways other than
via its effect on Ca2+ influx.
In view of previous reports that
cPLA2 phosphorylation on serine
residues is critical for its catalytic activity and AA release (23, 24,
31) and our determination that phosphatase was able to blunt this
enzyme's activation, we sought to verify by Western blot whether
serine phosphorylation occurred in BK- and PMA-stimulated RCCD cells.
Our results show that BK, like PMA, caused an almost twofold increase
in cPLA2 serine phosphorylation compared with control and that these responses could be completely blocked by Ro31-8220. Consideration of this and our other results on AA
release and cPLA2 activity brings
to mind interesting possibilities regarding the regulation of this
enzyme. Although Ro31-8220 completely blocked BK-induced
cPLA2 phosphorylation, it was
unable to completely block cPLA2
activity. Therefore, in addition to serine phosphorylation, BK may
activate cPLA2 by other
mechanisms, possibly via nonserine phosphorylations of
cPLA2 or by effects on a
PLA2-activating protein or
PLA2-inhibitory protein (26).
Another explanation perhaps may be more likely, given the results of
the serine phosphorylation. Previous studies based on gel-shift
mobility and phosphatase treatment of
cPLA2 have demonstrated that basal
cPLA2 can occur with varying degrees of phosphorylation (3, 5, 23, 31). Under basal conditions
(i.e., in the absence of BK stimulation), as seen in Fig. 7, RCCD
cPLA2 appears to display a
significant amount of phosphorylation. Thus some of the RCCD
cPLA2 could actually be primed for
activation, just needing a Ca2+
signal for translocation to the membrane substrate. In fact, cPLA2 appears to be at least
partially active under basal conditions, given the modest yet
statistically insignificant decrease in activity on phosphatase
treatment. Bradykinin stimulation, however, is able to enhance
cPLA2 activity via its activation
of PKC, thereby causing recruitment of basally phosphorylated enzyme as
well as amounts of freshly phosphorylated enzyme to the membrane.
On the basis of the similarities of
cPLA2 activation and
phosphorylation and the AA release by BK and PMA, it would seem
reasonable that these agonists would increase PKC activity by the same
amount. However, we found that PMA caused significantly greater
membrane-associated PKC activity than BK. The question then arises as
to why further PKC activation by PMA does not result in greater
activation and phosphorylation of
cPLA2. It is possible that
cPLA2, which already exists in a
partially phosphorylated state, becomes further maximally phosphorylated when only a portion of the PKC pool is activated. Another attractive possibility is that specific PKC isozymes may be
responsible for regulating cPLA2
activation. At least 13 PKC isozymes have been identified and
characterized with respect to their structure and cofactor regulation
(29), while evidence exists for isozyme-selective activation, substrate
preference, and localization (17). In our scenario, BK may specifically activate only a subset of PKC isozymes, whereas PMA activates a more
complete selection as reflected by its activation of PKC. However,
these additional PKC isozymes would not contribute to cPLA2 activation. Support for the
involvement of specific PKC isozymes in regulating
cPLA2 function is provided by
Godson et al. (14) and Wang et al. (40), who have demonstrated that PKC-
is the main PKC isozyme involved in phorbol ester-mediated arachidonate release in MDCK cells and
1-adrenergic activation of
cPLA2 in FRTL-5 thyroid cells,
respectively. Which PKC isozymes are present and involved after BK
stimulation of RCCD cells is unknown but is the subject of current
investigation.
In summary, the results of this research demonstrate that BK-stimulated
AA release in an RCCD cell line is mediated through the activation of
cPLA2 and that this process is
largely dependent on cPLA2
phosphorylation via a PKC-dependent route. Interestingly, despite
evidence from our laboratory that
cPLA2 is also the enzyme responsible for BK-mediated AA release in MDCK cells, PKC in this case
is not involved in onset responses to this hormone (unpublished observations). However, a role for PKC in regulating
cPLA2 can be seen with PKC
downregulation in MDCK cells, which results in reduced AA release,
cPLA2 activity, and
cPLA2 serine phosphorylation (19). Thus it appears that BK
signaling in MDCK and RCCD cells, although activating
cPLA2, has a different dependence
on PKC with respect to the onset response. The reason for such
differences in BK-dependent signaling may be a result of variable PKC
isoform involvement, a possibility that also is being currently
examined.
 |
ACKNOWLEDGEMENTS |
This study was supported by the Kidney Foundation of Canada.
 |
FOOTNOTES |
Address for reprint requests: R. L. Hébert, Dept. of Physiology,
Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, ON,
Canada K1H 8M5.
Received 15 April 1997; accepted in final form 7 August 1997.
 |
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