Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee, Memphis, Tennessee 38163
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ABSTRACT |
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Nigericin decreases intracellular pH (pHi) and stimulates prostanoid (PG) synthesis in endothelial cells from cerebral microvessels of newborn pigs. Nigericin-induced PG production was abolished by protein tyrosine kinase (PTK) inhibitors and amplified by phorbol 12-myristate 13-acetate (PMA) or protein tyrosine phosphatase (PTP) inhibitors. Nigericin-induced PG production in PMA-primed cells was potentiated by PTP inhibitors and abrogated by PTK inhibitors. Phospholipase A2 (PLA2) activity was stimulated by nigericin in a phosphorylation-dependent manner. Nigericin's effects on PG production and PLA2 activity were reproduced by ionomycin, which activates cytosolic PLA2 (cPLA2). cPLA2 was immunodetected in endothelial cell lysates. We found no evidence that nigericin's effects are mediated via mitogen-activated protein (MAP) kinase [extracellularly regulated kinase 1 (ERK1) and ERK2] activation: although nigericin stimulated detergent-soluble MAP kinase, its effects were not amplified by PMA or PTP inhibitors. Phosphorylation-dependent stimulation of PG synthesis was also observed when pHi was decreased by sodium propionate or a high level of CO2. Altogether, our data indicate that nigericin and decreased pHi stimulate PG synthesis by a protein phosphorylation-dependent mechanism involving cross talk between pathways mediated by PTK and PTP and by protein kinase C; cPLA2 appears to be a key enzyme affected by nigericin and decreased pHi.
prostaglandins; phospholipase A2; cyclooxygenase; vascular endothelium; protein phosphorylation
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INTRODUCTION |
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HYPERCAPNIA IS AN IMPORTANT physiological stimulus that regulates cerebral blood flow. In newborn pigs, the vasodilation response of pial arterioles to hypercapnia is accompanied by an increased production of major endothelium-derived vasodilator prostanoids (PGs) prostacyclin and prostaglandin E2 (PGE2); cerebral vascular responses to hypercapnia are completely prevented by indomethacin (17). Primary cultures of endothelial cells from cerebral microvessels of newborn pigs respond to high CO2 levels by increasing the production of prostacyclin and PGE2 (15). We have demonstrated that the effects of hypercapnia on endothelial PG production can be reproduced by compounds that rapidly decrease intracellular pH (pHi; nigericin and sodium propionate), whereas decreasing extracellular pH does not affect PG production (14). Therefore, the rapid decrease in pHi caused by hypercapnia is essential for stimulation of endothelial PG production (14). Nigericin, a K+ and H+ ionophore, rapidly decreases pHi in cerebral microvascular endothelial cells [pH 7.2 and 6.9 in absence and presence, respectively, of nigericin (5 µM)] and stimulates PG production 1.5- to 2-fold (14). Therefore, nigericin appears to be a useful tool for investigating mechanisms by which endothelial PG production is stimulated on a rapid decrease in pHi.
Phospholipase A2 (PLA2) and cyclooxygenase (COX) are rate-limiting enzymes in PG synthesis. Rapid changes in PG production might be due to a rapid alteration in the activity of either enzyme. PLA2, which releases arachidonic acid from the sn-2 position of membrane phospholipids, is represented by several distinct types (10, 19). Ca2+-dependent types of PLA2 [the 14-kDa secreted enzyme (sPLA2) and the 85-kDa cytosolic enzyme (cPLA2)] are regulated by changes in the intracellular Ca2+ level ([Ca2+]i) in response to different stimuli (16, 19, 22). In addition, the activity of cPLA2 is rapidly regulated by protein phosphorylation (16, 19). A Ca2+-independent PLA2 (CaIPLA2; also called iPLA2) that has been recently identified in many cell types also contributes to PG production in a variety of cell types (1, 3, 13, 18). We have recently demonstrated that COX-2, a major functional constitutive isoform in cerebral microvascular endothelial cells from newborn pigs, is posttranslationally activated by tyrosine phosphorylation (24). Therefore, endothelial PG production could be rapidly regulated at the level of either PLA2 (Ca2+; phosphorylation) or COX-2 (tyrosine phosphorylation) or both.
In this paper, we investigated whether changes in [Ca2+]i and/or protein phosphorylation contribute to the stimulation of PG production in response to the nigericin-induced decrease in pHi in cultured endothelial cells from cerebral microvessels of newborn pigs.
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MATERIALS AND METHODS |
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Protocols involving animals were approved by the Animal Care and Use Committee at the University of Tennessee-Memphis. All procedures were done by aseptic techniques.
Materials. Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD) and Sigma (St. Louis, MO). Matrigel (growth factor reduced) was purchased from Becton Dickinson (Bedford, MA). Nigericin, ionomycin (Ca2+ salt), A-23187, genistein, sodium orthovanadate, phorbol 12-myristate 13-acetate (PMA), and myelin basic protein were obtained from Sigma; bromoenol lactone (HELSS) was from Biomol. Fura 2-AM and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR). Phenylarsine oxide (PAO) and tyrphostin 47 were from Biomol. Protein G-Sepharose was from Pharmacia Biotech (Piscataway, NJ). Arachidonic acid was from Cayman Chemical (Ann Arbor, MI). [5,6,8,9,11, 12,14,15-3H]arachidonic acid (200 Ci/mmol) was from Amersham.
Cultured cells. Primary cultures of cerebral microvascular endothelial cells from newborn pigs were established as previously described (15). Endothelial cells were obtained from isolated cerebral microvessels (60-300 µm) by collagenase-dispase treatment (1 mg/ml for 2 h at 37°C). Digested cells were separated by Percoll density gradient and plated onto 12-well Costar plates coated with Matrigel or onto Matrigel-coated glass coverslips (9 × 35 mm) within a Leighton tissue culture tube (16 × 93 mm). Endothelial cells were cultured in DMEM with 20% fetal bovine serum (FBS), 30 µg/ml endothelial cell growth supplement (ECGS), 1 U/ml heparin, and an antibiotic-antimycotic mixture in a 5% CO2-air incubator at 37°C for 5-6 days to reach confluence. Primary cultures consisted of >95% endothelial cells, identified by cellular morphology and fluorescence staining with antibodies to von Willebrand factor.
Stock cultures of Swiss 3T3 fibroblasts from the American Type Culture Collection were maintained in DMEM supplemented with 10% FBS (24). For experimental purposes, cells were plated onto 12-well cell culture plates (105 cells/well) and grown in DMEM with 20% FBS in a 5% CO2-air incubator at 37°C for 4-5 days to reach confluence. All experiments were performed on confluent quiescent cells. To achieve quiescence, cells were exposed to a serum-depleted medium (0% FBS; 0% ECGS) for 15-20 h before the experiment.Measurement of
[Ca2+]i.
For the measurement of
[Ca2+]i,
endothelial cells grown on Matrigel-coated coverslips were loaded with
the fluorescent Ca2+-sensitive dye
fura 2-AM (5 µg/ml) in the presence of Pluronic F-127 (0.01%) in
Ca2+-Krebs buffer (in mM: 5.0 KCl,
0.6 MgSO4, 1.8 CaCl2, 120 NaCl, 6 glucose, 10 HEPES; pH 7.4) for 15 min at 37°C (14). The coverslips were washed
twice with the buffer, placed into an LS-50 spectrofluorometer (Perkin-Elmer; 2-ml cuvette), and superfused with
Ca2+-Krebs buffer at 37°C
(perfusion rate, 0.8 ml/min). The fluorescence intensity of fura 2 was
measured with the excitation wavelength pair of 340/380 nm and an
emission wavelength of 510 nm (14). To determine the effects of
nigericin on
[Ca2+]i,
cells were superfused with nigericin
(105 M) in
Ca2+-Krebs buffer for 10 min. For
calibration purposes, ionomycin (5 × 10
6 M) was applied at the
end of each experiment; this was followed by the
Ca2+-free Krebs containing 4 mM EGTA.
PG production.
Cells were rinsed with PBS and incubated for 10-30 min in 1 ml of
artificial cerebrospinal fluid (aCSF), an incubation medium similar to
cortical CSF (in mM: 3.0 KCl, 1.5 MgCl2, 1.5 CaCl2, 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO3
equilibrated with 5% CO2 and 21%
O2 to pH 7.4-7.5;
PCO2, 32-36 mmHg; PO2, 100-120 mmHg). To achieve
the hypercapnic condition, aCSF was preequilibrated with 14%
CO2 and 21%
O2 (pH,
PCO2, and
PO2 were in the range of
7.00-7.10, 70-80 mmHg, and 100-120 mmHg, respectively)
(25). Nigericin or ionomycin was added directly to the
incubation medium. To determine whether CaIPLA2 contributes to the basal
or nigericin-induced PG synthesis, cells were pretreated for 15 min
with 5-10 µM HELSS (13). To evaluate the effects of protein
phosphorylation on PG production, we preincubated cells for 30 min with
protein tyrosine kinase (PTK) inhibitors (300 µM genistein and
tyrphostin) (24), protein tyrosine phosphatase (PTP) inhibitors (1 mM
sodium orthovanadate and 10 µM PAO) (24), or the protein kinase C
(PKC) activator PMA (1 µM); all inhibitors were also included in the
incubation medium. To determine the effects of protein tyrosine
phosphorylation on PMA-induced PG synthesis, cells were preincubated
first with PMA for 15 min and then with inhibitors of protein kinase
and protein phosphatase for an additional 20 min; all inhibitors were also included in the incubation medium. The active doses
of the inhibitors as well as the treatment conditions for endothelial cells were determined based on our previously published data (24). After a 15-min incubation at 37°C, the medium was aspirated and stored at 20°C for PG determination. For protein
determination, cells were extracted with 0.1 N HCl; protein was
detected with the Micro BCA assay (Pierce Chemical, Rockford, IL). The
viability of the cells (indicated by the protein mass of the cells
attached to the wells at the end of the experiment) was not altered by any of the treatments.
COX activity.
To evaluate COX activity in intact cells, we determined PG production
(prostacyclin and PGE2) from the
exogenous substrate, arachidonic acid (24). Cells were washed twice
with PBS and incubated with 10 µM arachidonic acid in 1 ml of aCSF
for 10 min at 37°C without or with nigericin
(106-10
4
M). The incubation medium was aspirated and stored at
20°C
for PG determination.
PG assays.
Concentrations of 6-ketoprostaglandin
F1
(6-keto-PGF1
; the stable
hydrolysis product of prostacyclin) and
PGE2 in the cell incubation medium
were determined by RIA as described previously (13). The PG
concentration was normalized to cell protein.
PLA2 activity.
PLA2 activity was detected as a
release of arachidonic acid from intact cells (20). Cells grown on
12-well culture plates were loaded overnight with
[3H]arachidonic acid
(0.5 µCi/ml) in serum-free DMEM. To prevent further conversion of
arachidonic acid, cells were treated with 104 M indomethacin for 30 min immediately before the experiment. When indicated, cells were
pretreated with 1 mM sodium orthovanadate for 30 min or with 0.5-5
µM PMA for 10 min; these compounds were also included in the
incubation medium. Control and pretreated cells were washed three times
to remove unincorporated tracer and then incubated in serum-free DMEM
without or with nigericin or ionomycin (concentrations as indicated in
legends for Figs. 4 and 5) for 30 min. The medium was
aspirated, cleared by centrifugation, and used for detection of
released
[3H]arachidonic acid.
The cell monolayer was lysed with 1% Triton X-100 for 30 min and used
to estimate total incorporation of
[3H]arachidonic acid
into cells. Incorporated and released
[3H]arachidonic acid
was detected in samples by liquid scintillation. In control
unstimulated cells, 3-4% of the total incorporated arachidonic
acid was released under basal conditions.
Detection of cPLA2 and mitogen-activated protein kinase proteins. Quiescent cells were extracted by agitation on ice for 30 min with the extraction buffer [1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS in PBS] containing protease and phosphatase inhibitors (200 µM leupeptin, 40 µg/ml aprotinin, 200 µM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, 1 mM sodium-EDTA, 1 mM sodium fluoride, 100 µg/ml trypsin inhibitor). Detergent-soluble proteins were collected by aspiration, mixed with two volumes of 3× concentrated Laemmli sample buffer (0.12 M Tris · HCl, pH 6.8, 8% SDS, 4.5% dithiothreitol, 30% glycerol, 0.02% bromphenol blue), and boiled for 10 min. Detergent-insoluble proteins were scraped and solubilized with the Laemmli sample buffer (10 min, 100°C). The amount of protein in the samples was quantified by dot-blot staining with amido black as described previously (24). Detergent-soluble and detergent-insoluble proteins (20-50 µg protein/lane) were separated by SDS-7.5% PAGE and transferred to nitrocellulose membranes. Membranes were probed with 1) a monoclonal antibody to the amino-terminal domain of cPLA2 of human origin (Santa Cruz Biotechnology; dilution 1:2,000) and then a peroxidase-conjugated donkey anti-mouse IgG (dilution 1:10,000; Jackson Immunoresearch, West Grove, PA) and 2) a polyclonal antibody to the carboxy-terminal domain of ERK1 (p44) or ERK2 (p42) of rat origin (Santa Cruz Biotechnology; dilution 1:5,000) and then a peroxidase-conjugated donkey anti-rabbit IgG (dilution 1:10,000; Jackson Immunoresearch).
Detection of MAP kinase activity.
Detergent-soluble and detergent-insoluble fractions of endothelial
cells were collected with the extraction buffer with the protease and
phosphatase inhibitors described above. Detergent-soluble proteins were
collected by aspiration. Detergent-insoluble proteins were partially
solubilized by sonication of extracted cell debris in the extraction
buffer and clarified by centrifugation. The amount of protein in the
samples was quantified by dot-blot staining with amido black as
described previously (24). Mitogen-activated protein (MAP) kinase
activity was detected as described previously (30) with modifications.
Briefly, MAP kinase was immunoprecipitated from detergent-soluble and
detergent-insoluble fractions (200-300 µg protein; 500 µl) by
ERK1 and ERK2 antibodies (dilution 1:100; Santa Cruz Biotechnology) and
25 µl of protein G-Sepharose for 2 h on ice. Immunoprecipitated ERK1
and ERK2 were pelleted by centrifugation. To determine MAP kinase
activity, Sepharose-conjugated proteins were incubated with 10 µg of
myelin basic protein, 10 µM cold ATP, and 2.5 µCi of
[-32P]ATP (10 Ci/mmol; Amersham) in 50 µl of the kinase buffer (in mM: 30 Tris · HCl, 20 MgCl2, 2 MnCl2; pH 8.0). The reaction was terminated by Laemmli sample buffer (10 min, 100°C). Samples were resolved by electrophoresis in 14% gels. MAP kinase activity, expressed as 32P incorporation
into myelin basic protein, was quantitated by phosphorimager analysis.
Statistical analysis. Data are presented as means ± SE of absolute values or percentages of control. Statistical significance was assessed with Student's t-test. A level of P < 0.05 was considered significant.
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RESULTS |
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Effect of nigericin on PG production and COX activity.
Nigericin
(106-10
5
M), added directly into the incubation medium, rapidly (in 10-15
min) increased PG production (both prostacyclin and
PGE2) by endothelial cells two-
to threefold (Fig.
1A).
However, nigericin
(10
6-10
5
M) did not alter the COX activity measured as PG production from exogenous arachidonic acid (Fig.
1B). These data indicate that COX is
not affected by nigericin.
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Effect of extracellular
Ca2+ removal on
PG production.
Stimulation of PG production by nigericin was observed in the presence
of the Ca2+ (1.5 mM) normally
included in the incubation medium (aCSF). We investigated whether
removal of Ca2+ from the
incubation medium affects basal or nigericin-stimulated PG production.
In the absence of extracellular
Ca2+ (nominally
Ca2+-free media), the production
of 6-keto-PGF1 and
PGE2 was decreased by 40-50%
(Fig. 2). However, nigericin (5 and 10 µM) stimulated the production of prostacyclin in a
Ca2+-free medium (1.5 ± 0.3- and
2.2 ± 0.4-fold, respectively) to the same extent as in a
Ca2+-containing medium (1.7 ± 0.2- and 2.1 ± 0.2-fold, respectively) (Fig. 2).
Similarly, the production of PGE2
was stimulated by nigericin in a
Ca2+-free medium (2.4 ± 0.4- and 2.9 ± 0.3-fold in the presence of 5 and 10 µM
nigericin, respectively) to the same extent as in a
Ca2+-containing medium (2.4 ± 0.4- and 2.5 ± 0.3-fold, respectively) (Fig. 2). Therefore,
stimulation of PG production by nigericin does not require
extracellular Ca2+.
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Effect of nigericin on
[Ca2+]i
in endothelial cells.
When endothelial cells were loaded with fura 2-AM in a
Ca2+-Krebs solution, nigericin
(105 M) caused a rapid
decrease in the original
[Ca2+]i
tracing that was maintained over a 10-min period of nigericin application (Fig.
3A).
After removal of nigericin from the incubation medium, the cells
responded to ionomycin (10
6
M) by increasing
[Ca2+]i
(Fig. 3A). We have demonstrated
earlier that intracellular acidification significantly increases the
dissociation constant (Kd) of
Ca2+ binding to fura 2 (under our
experimental conditions,
Kd = 713
89 × pH) (12), which may account for the
apparent decrease in the original
[Ca2+]i
tracing on nigericin application. Using
pHi values for control and
nigericin-treated cerebral microvascular endothelial cells (12) as well
as pH-corrected
Kd values for
fura 2 (12), we determined
[Ca2+]i
values based on the ratio of fluorescence at 340 nm to that at 380 nm
(340/380 ratio) in three separate experiments as 50 ± 3 and 48 ± 2 nM in the absence and presence of nigericin, respectively. Therefore, our data demonstrate that nigericin does not increase [Ca2+]i
in endothelial cells from cerebral microvessels of newborn pigs. To
investigate whether nigericin affects
Ca2+ entry, endothelial cells were
loaded with fura 2 in a Ca2+-free
Krebs solution and then transferred to a
Ca2+-Krebs solution with or
without nigericin (10
5 M).
Control cells responded to extracellular
Ca2+ by rapidly increasing
[Ca2+]i,
whereas the subsequent removal of
Ca2+ from the incubation medium
resulted in a rapid decrease in
[Ca2+]i;
this cycle could be repeatedly reproduced without desensitization (Fig.
3B). When nigericin
(10
5 M) was added to the
incubation medium, Ca2+ entry was
apparently inhibited; the removal of nigericin did not restore the
ability of endothelial cells to respond to extracellular Ca2+ within at least 15-20
min (Fig. 3B).
[Ca2+]i
values (calculated as described above) were determined as 52 ± 3 and 54 ± 2 nM in the absence and presence of nigericin,
respectively. The ability of endothelial cells to respond to ionomycin
(5 × 10
6 M) by
increasing
[Ca2+]i
remained intact (Fig. 3B). These
data indicate that nigericin does not increase
Ca2+ entry or the
[Ca2+]i
level in cerebral microvascular endothelial cells.
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Role of CaIPLA2 in the production of
endothelial PG.
Because the endothelial effects of nigericin are not associated with
the increase in
[Ca2+]i,
we investigated whether CaIPLA2
contributes to nigericin-stimulated PG production. We used HELSS, an
inhibitor of CaIPLA2 that has been
shown to selectively inhibit the enzyme at concentrations of 1-5
µM (2, 10, 18). Cells were preincubated with 5 µM HELSS for 15 min.
HELSS did not alter basal or nigericin-stimulated PG production by
endothelial cells (Table 1). Similarly, 10 µM HELSS did not affect endothelial PG production (data not shown). As a negative control for inhibitor selectivity, we determined the
effects of HELSS on PG production stimulated by
Ca2+ ionophore A-23187. A-23187
(107 M) added directly into
the incubation medium increased PG synthesis 2.5- to 3-fold in both
control and HELSS-pretreated cells in 15 min (Table 1), thus confirming
that Ca2+-dependent PG production
is not affected by HELSS. These results may indicate that
CaIPLA2 is not involved in basal
and nigericin-induced PG production by cerebral microvascular
endothelial cells.
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Effect of increased protein phosphorylation on nigericin- and
ionomycin-induced PG synthesis.
We investigated the effects of protein tyrosine phosphorylation on the
responses to nigericin. Nigericin-stimulated PG production was
inhibited by genistein (300 µM), a PTK inhibitor (Fig.
4). Similarly, genistein inhibited PG
production in response to Ca2+
ionophores ionomycin and A-23187 (Fig. 4). We further investigated the
effects of PTK-PTP- and PKC-dependent phosphorylation on
PG production in cerebral microvascular endothelial cells and in Swiss
3T3 fibroblasts in response to nigericin and ionomycin. As we
demonstrated previously, PTP inhibitors rapidly stimulate COX activity
in cerebral microvascular endothelial cells from newborn piglets, but
not in Swiss 3T3 fibroblasts (26). As a result, the basal level of PG
production by endothelial cells (but not 3T3 cells) pretreated for 30 min with sodium orthovanadate (1 mM) or PAO (10 µM) is elevated (Fig.
5, A and
B). In endothelial cells pretreated
with PTP inhibitors, nigericin
(105 M) caused a 7- to 12-fold increase in PG production compared with a 1.5- to 2-fold
increase in control untreated cells (Fig. 5A). The greatly amplified response
of PG synthesis to nigericin was also observed in Swiss 3T3 fibroblasts
treated with PTP inhibitors (Fig.
5B). The PKC activator PMA
(0.5-5 µM) increased basal PG production in endothelial cells
(3- to 4-fold) (Fig. 5A), but not in
Swiss 3T3 fibroblasts (Fig. 5B).
However, both cell types pretreated with PMA (1 µM) demonstrated an
amplified response to nigericin (13- to 15-fold increase above the
control basal level) (Fig. 5, A and
B).
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Effect of nigericin on COX activity.
We investigated the effects of nigericin on COX activity in cerebral
microvascular endothelial cells primed with protein phosphorylation (Fig. 7). In cells pretreated with PTP
inhibitors, COX activity was increased (Fig. 7), confirming our
previous data (26). In cells treated with PMA (5 µM), the COX
activity was unaltered (Fig. 7). Nigericin
(105 M) did not alter COX
activity in cells treated with either modulator of protein
phosphorylation (Fig. 7). Increasing the nigericin concentration up to
10
4 M also had no effect on
the COX activity (data not shown). Therefore, nigericin does not alter
the COX activity in cerebral microvascular endothelial cells primed
with protein phosphorylation.
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Effect of nigericin and ionomycin on PLA2
activity.
The effects of nigericin on PLA2
activity were determined in endothelial cells (control or primed with
protein phosphorylation) (Fig.
8B).
Short pretreatment (20-30 min) of endothelial cells with sodium
orthovanadate (1 mM) and PAO (10 µM), but not with PMA (1 µM),
increased basal PLA2 activity
(Fig. 8B). In control cells,
nigericin (105 M) slightly
increased PLA2 activity (Fig.
8B). In cells pretreated with sodium
orthovanadate or PMA (but not PAO), the effects of nigericin on
PLA2 activity were amplified (Fig.
8B). It is known that protein
phosphorylation greatly potentiates the activation of
cPLA2 by ionomycin (19). In our
experiments, ionomycin (10
6
M) stimulated PLA2 activity.
Responses to ionomycin were greatly potentiated in cells pretreated
with sodium orthovanadate or PMA, but not with PAO (Fig.
8B). The close similarity between
the phosphorylation-dependent effects of ionomycin and nigericin on
PLA2 activity may indicate that
cPLA2 is affected by nigericin.
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Effects of propionate and hypercapnia on PG synthesis in cells
primed with protein phosphorylation.
Propionate (80 mM) and hypercapnia (14%
CO2) rapidly decrease
pHi in endothelial cells to the
same extent as 5 µM nigericin (pH 6.9-7.0) (12). We compared the
effects of nigericin (5 µM), propionate (80 µM), and hypercapnia
(14% CO2, extracellular pH = 7.1)
on the production of prostacyclin and
PGE2 (Fig.
9) in endothelial cells with an increased
level of protein phosphorylation. In control untreated cells, all
agents stimulated PG production 1.5- to 2-fold. Similar to the effects
of nigericin, the effects of propionate and hypercapnia on the
production of prostacyclin and
PGE2 were greatly amplified in
endothelial cells pretreated with sodium orthovanadate, PAO, and PMA
(Fig. 9). These data indicate that the decrease in
pHi is a major factor that
potentiates the effects of nigericin in cells primed with protein
phosphorylation.
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MAP kinase in cerebral endothelial cells.
MAP kinase, as ERK1 (44 kDa) and ERK2 (42 kDa) proteins (4), and MAP
kinase activity were detected in cell lysates (Fig. 10, A
and B). About 80% of the total MAP
kinase protein (Fig. 10B) and MAP
kinase activity (data not shown) is soluble in the detergent-containing buffer for immunoprecipitation (radioimmunoprecipitation assay buffer), and 20% of the MAP kinase remains insoluble.
Endothelial MAP kinase is rapidly (in 30 min) activated in cells with
upregulated protein phosphorylation (Fig.
10A). Our data demonstrate a
distinct functional diversity between detergent-soluble and
detergent-insoluble MAP kinases with respect to protein
phosphorylation. Sodium orthovanadate greatly increases MAP kinase
activity in the detergent-soluble fraction (3- to 4-fold), with much
less effect (1.5- to 2-fold stimulation) on detergent-insoluble MAP
kinase. In contrast, PAO stimulates exclusively a detergent-insoluble
MAP kinase (2- to 2.5-fold increase), with no effect on
detergent-soluble MAP kinase. PAO also caused translocation of MAP
kinase from the detergent-soluble to the detergent-insoluble
endothelial fraction: soluble MAP kinase protein decreased to 40%,
whereas detergent-insoluble MAP kinase protein increased to 60% (Fig.
10B). No protein redistribution in
the cells treated with sodium orthovanadate and PMA was observed (Fig.
10B). PMA stimulated MAP kinase
activity in both fractions evenly (Fig.
10A). Tyrphostin did not alter the
PMA-induced stimulation of MAP kinase (data not shown), indicating no
interaction between protein tyrosine- and PKC-mediated phosphorylation
in MAP kinase activation.
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DISCUSSION |
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In newborn pigs, as well as in newborn human babies, endothelially derived PGs are major factors that control cerebral vascular tone (17). Therefore, mechanisms that rapidly regulate PG synthesis are of special physiological importance in the newborn cerebral circulation. Using primary cultures of endothelial cells from cerebral microvessels of newborn pigs, we investigated mechanisms of increased endothelial PG production in response to hypercapnia (high level of CO2). Our major finding is that the decreased pHi caused by nigericin, propionate, and hypercapnia stimulates PG production in endothelial cells by targeting PLA2 via a protein phosphorylation-dependent mechanism. Protein tyrosine phosphorylation and PKC-mediated phosphorylation greatly potentiate the ability of intracellular acidification to stimulate PG production and PLA2 activity, whereas inhibition of PTK abrogates the stimulation. The effects of PTK-,PTP-, and PKC-mediated phosphorylation are additive, and tyrosine phosphorylation may be essential for maximum effect. cPLA2 appears to be a key enzyme affected by nigericin and decreased pHi.
Primary cultures of endothelial cells from the cerebral cortices of newborn pigs respond to high CO2 by an immediate increase in PG synthesis (14, 25). Although hypercapnia decreases both extracellular pH and pHi, the decrease in pHi (from 7.2 under normocapnic conditions to 7.0-6.9 on exposure to 14% CO2) is a leading factor in increasing endothelial PG synthesis (14). Similar to high CO2, pharmacological compounds that rapidly decrease pHi (nigericin and sodium propionate) also rapidly stimulate endothelial PG synthesis (14). In contrast, agents that selectively alter extracellular pH do not affect PG production (14). Nigericin, a K+ and H+ ionophore, is a powerful tool for selectively decreasing pHi in a variety of cultured cells. In cerebral microvascular endothelial cells from newborn pigs, nigericin (5 µM) decreases pHi to 7.0-6.9 and rapidly stimulates PG production (14). We used nigericin as a tool to investigate the mechanisms by which the rapid decrease in pHi stimulates PG production.
COX and PLA2 are key rate-limiting enzymes in PG production. Because reactivity to hypercapnia includes a variety of PGs (15), we assume that the key enzyme(s) in PG synthesis rather than individual prostaglandin synthases is involved in the response to intracellular acidification.
In cerebral microvascular endothelial cells from newborn piglets,
COX-2, a major constitutively functioning COX isoform, is posttranslationally activated by tyrosine phosphorylation (24). Therefore, rapid responses to nigericin could be associated with COX
activation. However, nigericin in a wide range of concentrations (106-10
4
M) did not alter COX activity in endothelial cells. When cells were
pretreated with PTP inhibitors to upregulate protein tyrosine phosphorylation, basal COX activity was stimulated (consistent with our
previous findings), but nigericin did not cause any additional changes.
Swiss 3T3 fibroblasts do not functionally express COX-2 and do not
respond to tyrosine phosphorylation by COX activation (24). However,
nigericin stimulated PG production in Swiss 3T3 cells even more
effectively than in cerebral endothelial cells. Altogether, our data
demonstrate that COX is not affected by nigericin.
PLA2 cleaves arachidonic acid by hydrolyzing membrane phospholipids at the sn-2 position, thus largely contributing to PG synthesis (6, 7, 10, 19, 20). Several types of PLA2 have been described, including CaIPLA2 and Ca2+-dependent PLA2 (sPLA2 and cPLA2) (3, 7, 10, 18-20). First, we investigated whether the effects of nigericin on PG production are Ca2+ dependent. The effects of nigericin do not require extracellular Ca2+: removal of Ca2+ from the incubation medium did not prevent activation of PG production. However, similarity between the effects of nigericin and ionomycin on endothelial PG production indicated a possibility that nigericin may elevate [Ca2+]i. Indeed, in some cell types (pancreatic acinar cells, gastric parietal cells, platelets, and neutrophils) intracellular acidification by nigericin triggered Ca2+ mobilization from intracellular stores independently of extracellular Ca2+ (12, 22, 30). In contrast, in endothelial cells (8), fibroblasts (9), and vascular smooth muscle cells (2, 28), intracellular alkalinization elevates cytosolic Ca2+ via release from intracellular stores. As we have previously reported, in endothelial cells from cerebral microvessels of newborn pigs, intracellular acidification by high CO2 decreased an original [Ca2+]i tracing; when the tracing was corrected for the pHi effect on Kd for fura 2, no changes in [Ca2+]i were found (14). Our present data demonstrate that the application of nigericin to cerebral endothelial cells also decreased the 340/380 ratio independently of extracellular [Ca2+]i; however, we have found no changes in absolute [Ca2+]i (calculated using pHi-corrected Kd values for fura 2) in nigericin-stimulated cells. Altogether, these data indicate that the effects of intracellular acidification on endothelial PG production are not mediated by Ca2+.
We addressed the possibility that nigericin stimulated PG production by targeting CaIPLA2. It has been shown that CaIPLA2 contributes to PG production in a cell-specific and a signal-specific manner (1, 3, 13, 18). HELSS, a CaIPLA2 selective inhibitor, at concentrations of 5 µM that maximally inhibit the enzyme in a variety of cell types (1, 3, 13), did not alter basal or nigericin-stimulated PG production in cerebral endothelial cells. These data do not confirm that CaIPLA2 is involved in the mechanism by which nigericin stimulated endothelial PG production.
The effects of nigericin on PG production are modulated by protein phosphorylation. Genistein abrogated the stimulation of PG production, indicating that the basal level of protein tyrosine phosphorylation is essential for the effects of nigericin. In concert with these data, the effects of nigericin on PG production were greatly amplified in cells with increased protein phosphorylation. Priming cells with either a PTP inhibitor (sodium orthovanadate or PAO) or a PKC activator (PMA) greatly increased the responses to nigericin. Amplification of responses to nigericin in cells primed with protein phosphorylation is not limited to cerebral microvascular endothelial cells but is also observed in other cell types, such as Swiss 3T3 fibroblasts. The detection of PLA2 activity unveiled a similar pattern: nigericin rapidly stimulated enzyme activity in cells with upregulated protein phosphorylation.
We investigated the relationship between PKC and protein tyrosine phosphorylation in priming endothelial cells for a nigericin response. Nigericin's stimulation of PG production in PMA-primed cells was greatly potentiated by PTP inhibitors (vanadate and PAO) and completely abolished by PTK inhibitors (tyrphostin and genistein). These data indicate a close interaction between a PTK(s) or PTP(s) and a PKC-dependent pathway(s) involved in the mechanism of nigericin's stimulation of PG production. PTK is possibly downstream of PKC in the mechanism of nigericin-stimulated PLA2 activity.
cPLA2 is the only
well-characterized PLA2 that is
posttranslationally activated by protein phosphorylation via diverse
cell-specific signaling pathways that may involve PKC, MAP kinase, and
PTK (4, 6, 10, 11, 16, 19, 21, 23, 26, 32). A characteristic feature of
the response is that phosphorylation of
cPLA2 is not sufficient for full
activation of the enzyme; the enzyme also requires an additional
stimulus that is necessary for a translocation of
cPLA2 to the cell membrane to have
full access to substrate phospholipids (6, 19, 23, 27). It has been
shown that ionomycin serves as such an additional signal, facilitating
the Ca2+-dependent translocation
of cPLA2 to the membrane via an as
yet unknown mechanism (19). To elucidate whether nigericin could also
promote full activation of cPLA2,
we compared the effects of nigericin and ionomycin on PG production in
cells primed with protein phosphorylation. Our data demonstrate that in
cerebral microvascular endothelial cells stimulation of PG production
by ionomycin was blocked by PTK inhibition and amplified by PTK- and
PKC-mediated phosphorylation. We also found a striking similarity between the effects of ionomycin and nigericin in Swiss 3T3 fibroblasts primed with PTP inhibitors or PMA. Therefore, nigericin, as well as
ionomycin, may serve as an additional signal that promotes full
activation of cPLA2. These data
demonstrate that the effects of ionomycin on PG production and
cPLA2 activity in endothelial cells and Swiss 3T3 fibroblasts primed with protein phosphorylation could be fully reproduced by nigericin. Because nigericin does not
increase
[Ca2+]i,
our finding may suggest that the increase in
Ca2+ influx caused by ionomycin
could be irrelevant to the ability of ionomycin to activate
cPLA2. Indeed, an increase in
[Ca2+]i
was not required for cPLA2
activation in PMA-primed macrophages and neutrophils (26). The
contribution of a novel,
Ca2+-independent
cPLA2 homologue
(cPLA2-) (31) to
nigericin-induced phospholipase activation should be considered.
Our data indicate that the significant part of cPLA2 (~30% of the total amount) is associated with the detergent-insoluble cytoskeleton. The association of cPLA2 with the endothelial cytoskeleton is regulated by selected protein tyrosine phosphorylation pathways. The amount of detergent-insoluble cPLA2 is increased up to 60% when protein phosphorylation is upregulated with PAO, but not with sodium orthovanadate or PMA. The association of cPLA2 with the cytoskeleton is subject to a dynamic regulation. It appears that nigericin caused rapid dissociation of cPLA2 from the cytoskeleton and translocation of the enzyme to other cellular compartments.
What is the mechanism by which protein phosphorylation is involved in nigericin's stimulation of phospholipase activity? In some, but not all, cell types, cPLA2 is phosphorylated and activated by MAP kinase (4, 5, 11, 21, 23). MAP kinase activity itself is regulated by both serine/threonine and tyrosine phosphorylation and, therefore, could serve as an integrating effector in cell signaling (5). We investigated the possibility that MAP kinase is involved in the mechanism of the endothelial response to nigericin. We detected two structurally and functionally distinct pools of ERK1 and ERK2 kinase (detergent soluble and detergent insoluble) in cerebral microvascular endothelial cells. Under basal conditions, ~80% of the total ERK1 and ERK2 kinase protein and activity can be solubilized by detergents (1% NP-40, 0.5% sodium deoxycholate, and 1% SDS in PBS), whereas ~20% of MAP kinase remains detergent insoluble. According to our preliminary data, the detergent-insoluble fraction also contains a variety of cytoskeletal proteins (actin, tubulin, and paxillin). We found that both soluble and cytoskeletal fractions of ERK1 and ERK2 kinase are activated by protein phosphorylation. PMA stimulates ERK1 and ERK2 kinase activity in both fractions two- to threefold. However, PTP inhibitors exhibit strong selectivity to subcellular fractions of MAP kinase. Sodium orthovanadate stimulates soluble MAP kinase to a greater extent than detergent-insoluble cytoskeletal MAP kinase (3.5- and 1.5-fold, respectively), whereas PAO activates exclusively cytoskeletal ERK1 and ERK2 kinase. PAO also induces translocation of MAP kinase to the cytoskeleton, thus increasing the amount of the detergent-insoluble fraction to up to 60% of the total MAP kinase protein. Although endothelial MAP kinase activity is regulated by both serine and threonine phosphorylation, we did not observe cross talk between tyrosine kinase- and PKC-mediated pathways. MAP kinase activation by PMA was not altered by inhibitors of PTK or PTP (data not shown). Therefore, it is unlikely that MAP kinase provides an interaction between tyrosine kinase- and PKC-mediated pathways in the stimulation of endothelial PG synthesis.
Nigericin increased ERK1 and ERK2 activity in the detergent-soluble fraction and decreased their activity in the detergent-insoluble cytoskeleton, thus indicating the translocation of MAP kinase from the cytoskeleton to other compartments. A dissociation of MAP kinase from the cytoskeleton was also observed in nigericin-stimulated cells pretreated with sodium orthovanadate and PAO, but not with PMA. Therefore, it appears that nigericin may cause cytoskeletal rearrangements that result in the dissociation of MAP kinase and, probably, other proteins from the cytoskeleton. However, no potentiation of the effects of nigericin on MAP kinase activity has been observed in cells primed with protein phosphorylation. Therefore, MAP kinases ERK1 and ERK2 are not involved in the mechanism of nigericin-induced activation of PLA2 and PG synthesis.
Other agents that rapidly decrease pHi in cerebral microvascular endothelial cells in a manner similar to that of nigericin, such as sodium propionate and increased CO2, also stimulate PG production in a protein phosphorylation-dependent manner. The effects of sodium propionate and 14% CO2 on PG production in cerebral endothelial cells were greatly amplified in cells primed with PTP inhibitors or PMA. These data indicate that decreased pHi is a leading factor in the ability of nigericin to stimulate PG production via a protein phosphorylation-dependent mechanism.
Taken together, our data demonstrate that the intracellular acidification pH caused by nigericin and high CO2 levels stimulates PG production in cerebral microvascular endothelial cells via a protein phosphorylation-dependent mechanism that may involve cross talk between PTK(s) or PTP(s) and PKC. cPLA2, a potential target enzyme in the mechanism of stimulation of PG production by decreased pHi, is immunodetected in cell lysates. The significant part of immunodetectable cPLA2 is tightly associated with the endothelial cytoskeleton. Intracellular acidification may cause a rearrangement of the endothelial cytoskeleton that results in the dissociation of cPLA2 from the cytoskeleton and translocation of the enzyme to other compartments. Phosphorylation-mediated activation of cPLA2 by nigericin does not appear to involve MAP kinase (ERK1 and ERK2).
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ACKNOWLEDGEMENTS |
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We thank A. Fedinec and M. Jackson for their technical assistance, D. Morse for the illustrations, and J. Emerson-Cobb for editorial assistance.
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FOOTNOTES |
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This research was supported, in part, by National Heart, Lung, and Blood Institute Grants HL-42851 and HL-34059. H. Parfenova was supported by a grant-in-aid from the Southeast Affiliate of the American Heart Association.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Parfenova, Dept. of Physiology, Univ. of Tennessee-Memphis, 894 Union Ave., Memphis, TN 38163.
Received 24 February 1999; accepted in final form 10 June 1999.
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