(Hypertension. 2000;36:201.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Effects of Bradykinin on Prostaglandin I2 Synthesis in Human Vascular Endothelial Cells
From the Second Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Correspondence to Sumio Komatsu, MD, Second Department of Medicine, Kyoto Prefectural University of Medicine, 465, Kajii-cho, Kawaramachi-Hirokoji agaru, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail sawada{at}koto.kpu-m.ac.jp
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Abstract—The effects of bradykinin on the regulatory mechanisms of prostacyclin synthesis in endothelial cells were investigated in association with intracellular Ca2+ kinetics, cytosolic phospholipase A2 (cPLA2) activity, and mRNA expression of cPLA2 and prostaglandin H synthase (PGHS) isoforms. Bradykinin enhanced prostacyclin release from endothelial cells time-dependently, but pretreatment with EGTA H-7 or HOE 140 inhibited bradykinin-induced prostacyclin release. Bradykinin increased both the influx of extracellular Ca2+ and Ca2+ release from the intracellular Ca2+ storage sites. These reactions occurred within 5 minutes after bradykinin stimulation. Within 15 minutes, bradykinin activated cPLA2 to 1.3-fold the control level. The constitutive expressions of mRNA of cPLA2, PGHS-1, and PGHS-2 was 87, 562, and 47 amol/µg RNA, respectively. With the stimulation of bradykinin, cPLA2 mRNA increased to 746 amol/µg RNA in 15 minutes, PGHS-1 mRNA increased to 10 608 amol/µg RNA, and PGHS-2 mRNA increased to 22 400 amol/µg RNA in 180 minutes. Pretreatment with cycloheximide superinduced cPLA2 and PGHS-2 mRNA expression but almost completely inhibited PGHS-1. Pretreatment with EGTA had effects similar to pretreatment with cycloheximide in the case of cPLA2 and PGHS-1 but did not affect PGHS-2. These findings suggest that the elevation of cPLA2 activity caused by the increase of intracellular Ca2+ concentration is important in the early phase of bradykinin-induced prostacyclin synthesis and that the mechanisms regulating cPLA2 are different from those regulating PGHS isoforms in endothelial cells.
Key Words: bradykinin • prostacyclin • kinetics, calcium • phospholipases A • prostaglandin H synthase • polymerase chain reaction
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Introduction |
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Bradykinin (BK) has blood pressure–lowering effects that can be induced by either the direct action of BK on target organs or the secretion of secondary agents. BK in plasma mainly acts on endothelial cells and stimulates them to release various agents associated with vascular tonus and blood coagulation, such as prostaglandins, endothelium-derived relaxation factor, catecholamines, and cytokines, resulting in the maintenance of circulation homeostasis. BK is resolved into inactive peptides by 2 kininases, carboxypeptidase N (kininase I) and dipeptide hydrase (kininase II). Kininase II is the same enzyme as angiotensin I–converting enzyme (ACE) and is secreted by endothelial cells. The clinical observation that ACE inhibitors lower blood pressure, even under low renin conditions,1 suggests a renin-angiotensin system–independent mechanism for evoking their actions. From reports that the blood pressure–lowering effect of ACE inhibitors correlates with the increase of intravenous concentration of kinin and prostacyclin (prostaglandin I2, PGI2), it appears that this mechanism is due to the local accumulation of BK and BK-induced vasodilators, especially PGI2, rather than to the reduction of vasoconstrictor angiotensin II;2 however, the effects of ACE inhibition on BK and prostaglandins are unclear. PGI2 is synthesized in response to release of arachidonic acid (AA) from phospholipids followed by transformation of AA to PGH2 by prostaglandin H synthase (PGHS=cyclooxygenase; COX) and subsequent transformation of PGH2 by PGI2 synthase. Recent studies have revealed that PLA2 and PGHS have several isoforms and that many agents affect their expression at the level of genome transcription or protein translation. However, there have been few reports about the relation between BK-induced PGI2 synthesis and signal transduction mechanisms in endothelial cells. In the present study, we investigated the effects of BK on the regulatory mechanisms of PGI2 generation in human umbilical vein endothelial cells (HUVEC) with reference to intracellular Ca2+ kinetics, cytosolic PLA2 activity, and mRNA expression of cPLA2 and PGHS isozymes.
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Methods |
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Culture of HUVEC
Endothelial cells were obtained from human umbilical cord vein and cultured according to our previously described method.3 We used primary cultured HUVEC that formed confluent monolayers in the following experiments. Cells were identified as vascular endothelial cells by electron microscopic findings of Weibel-Palade bodies in their cytoplasm and detection of ACE activity.
Assay of PGI2 and Inositol 1,4,5-Trisphosphate
Concentration
The concentration of PGI2 and inositol
1,4,5-trisphosphate (IP3) were measured with the
use of previously described methods.3 Briefly, the amount
of PGI2 released from HUVEC was measured as the
stable metabolite 6-keto prostaglandin
F1
with the use of a
[3H] 6-keto prostaglandin
F1 radioimmunoassay kit (New England Nuclear),
and the IP3 in HUVEC was measured with the use of
the D-myo-inositol 1,4,5-trisphosphate [3H]
Biotrack radioimmunoassay system (Amersham Pharmacia Biotech).
Measurement of cPLA2 Activity
After incubation in tissue culture medium (TCM) for 12
hours, the cells were scraped from the dish and collected into glass
test tubes. The contents were centrifuged at 3000g
for 5 minutes at 4°C. The cells were resuspended in TCM and
centrifuged under the same conditions. The cells were finally
suspended in TCM at a final concentration of 105
cells/mL and sonicated at 4°C for 3x10 seconds. The sonicated cells
were centrifuged at 3000g for 10 minutes to remove
unlysed cells. The resulting lysate was then centrifuged at
10 000g for 60 minutes. The supernatant (soluble fraction)
was transferred to another container and used as a source for the assay
of cPLA2. The activity of
cPLA2 was measured by the method of
Kramer,4 with some modification. Each substrate was
dried under nitrogen gas and suspended in distilled water by sonication
at room temperature. The standard incubation system for the assay of
cPLA2 contained, in 0.25 mL, 50 mmol/L HEPES
(pH 7.4), 150 mmol/L NaCl, 1 mmol/L
CaCl2, 2 mmol/L 2-mercaptoethanol,
2x10-6 mol/L
1-palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine,
10-6 mol/L dioleoyl
glycerol and enzyme preparation. The incubation was for 15 to 180
minutes at 37°C in the presence of BK (final concentration
10-5 mol/L). The reaction
was stopped by adding 1.25 mL of Dole’s reagent. After 0.5 mL of
H2O and 0.75 mL of n-heptane were added, the
contents were spun in a vortex mixer for 5 minutes and
centrifuged at 3000g for 5 minutes. An aliquot (0.8
mL) of the upper phase was pipetted into another tube containing 1 mL
of heptane and 100 mg of silicic acid powder. After the tubes were
shaken in a vortex mixer for 5 minutes and centrifuged as
above, the heptane phase was transferred into scintillation vials. The
radioactivity was measured by liquid scintillation spectrometry
(TRI-CARB 460C Automatic Liquid Scintillation System, Packard
Instrument Co Inc) with 10 mL of Atomlight (New England Nuclear) as a
scintillator agent.
Measurement of Intracellular Ca2+
Concentration
Intracellular Ca2+ concentration
([Ca2+]i) was measured by
the method of Grynkiewicz et al,5 with some modification.
The cells were scraped from the dish and collected in
centrifugation tubes. After being suspended in 3 mL of
buffer A containing 1.0% BSA, the cells were incubated in the presence
of 3.2x10-6 mol/L
fura-2/AM at 37°C for 45 minutes. After resuspension in 1.0%
BSA-containing solution (cell count 106/mL), they
were centrifuged at 250g for 10 minutes. The
fluorescence intensity from the endothelial
cell suspensions then was recorded in UV-compatible cuvettes with a
Ca2+ analyzer (CAF-100: Japan
Spectroscopic Co, Ltd) at excitation wave lengths of 340 nm and 380 nm
and at an emission wavelength of 510 nm.
Ca2+ Uptake
After the cells were washed 3 times with buffer A, they were
incubated with buffer A containing 1.85x105
Bq/mL of 45CaCl2
(7.1484x108 Bq/mg CaCl2,
New England Nuclear) for 10 minutes. After incubation, the cells were
washed 3 times with buffer A, and 1.0 mL of 6% trichloroacetic acid
then was added to the dish. The cells were then rubbed off the dish
with a rubber spatula, and the radioactivity of intracellular
45Ca was measured by means of liquid
scintillation spectrometry.
Measurement of Ca2+ Release From Ca2+
Storage Sites
First, the cells were scraped from the dish and
permeabilized with saponin according to our previously
described method.3 The saponized HUVEC were subsequently
incubated for 15 minutes after the addition of
45Ca2+, phosphocreatine
(PC), CPK, succinic acid disodium salt (Suc), and adenosine
5'-triphosphate magnesium salt (ATP). After the supernatant was removed
by centrifugation, the cells were resuspended in
3x10-8 mol/L
Ca2+-EGTA buffer solution containing PC, CPK,
Suc, and ATP and then were apportioned into several tubes
(2x105 cells/200 µL). After stimulation of the
cells with the test agent for a certain period, 200 µL of cell
suspension was filtered on a filter by means of a diaphragm vacuum pump
and 1225 Sampling Manifold (Millipore Co) with 30 mL of a rinsing
buffer solution. The radioactivity of HUVEC on the vacuum pump filter
was measured as residual 45Ca in the cells by
means of liquid scintillation spectrometry. The
Ca2+ release from Ca2+
storage sites induced by agents was calculated at 10 seconds and
compared with the Ca2+ release induced by
Ca2+ ionophore A23187.
Quantitative Measurement of mRNA
Synthesis of Primers for Genomic DNA Preparation, Reverse
Transcription Reaction, and Competitive Polymerase Chain
Reaction
Primers used for genomic DNA (gDNA) amplification, reverse
transcription, and for competitive polymerase chain reaction (PCR) were
synthesized on the Gene Assembler Plus (Pharmacia LKB Biochrom Ltd).
The sequences of 5'- and 3'- flanking oligonucleotide
primers used for PCR amplification of gene and mRNA transcription were
5'-GGAAGAAGCAGTTGCCAGATGCCCAGCTCC-3' (PGHS-1-Fw),
5'-TTGTCTCCATAAATGTGGCCGAGGTCTACC-3' (PGHS-1-Rv),
5'-GTGTTCTATTATGAGATTCTGAACTCCCCA-3' (PLA2-Fw),
5'-TAGAATGAGGCAGACAAAAGTTGGAAGGCC-3'
(PLA2-Rv), 5'-ATGTTCCACCCGCAGTACAG-3'
(PGHS-2-Fw), and 5'-GCCCTTCACGTTATTGCAGAT-3' (PGHS-2-Rv).
RNA Isolation and Reverse Transcription
Total cellular RNA was isolated from HUVEC and was reverse
transcribed to complementary DNA (cDNA) by Moloney murine leukemia
virus reverse transcriptase according to our previously published
method.6 The concentrations of total RNA were determined
by spectrophotometer at a 260-nm wave length.
Amplification of Genomic PGHS-1 DNA and Its Purification for
Internal Standard of Competitive PCR
Genomic DNA samples were extracted from heparinized blood and
amplified by PCR following the method of Saiki et
al7 in the presence of 25 pmol of each primer
(PGHS-1-Fw and -Rv), 100 µmol of each dNTP, 20 µg genomic DNA,
1xbuffer (5 mmol/L Tris-HCl [pH 8.3], 20 mmol/L
MgCl2, 50 mmol/L KCl, 0.01% gelatin), and 5
U of AmpliTaq (Takara Co), for a total volume of 200 µL. With the use
of the Gene ATAQ controller (Pharmacia LKB Biochrom Ltd), 40 PCR cycles
were performed as follows: denaturation at 94°C for 1 minute,
annealing at 62°C for 2 minutes, and extension at 72°C for 3
minutes. Five microliters of the reaction mixture containing the PCR
product was subjected to electrophoresis on 2% agarose gel for
confirmation of the product size. The remaining 195 µL was loaded
onto a 10% polyacrylamide gel
(acrylamide/bis-acrylamide: vol/vol,
29:1) and electrophoresed for 40 minutes. The segment of the gel
containing the DNA band was cut out by visualizing the
ethidium-bromide–stained gels in long-wavelength ultraviolet light.
The gel slice then was transferred to a microfuge tube containing
elution buffer (10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L EDTA,
0.2% SDS, 0.3 mol/L NaCl). After the tube was incubated at 37°C for
12 hours, the supernatant was transferred to another microfuge tube.
Two volumes of 100% ethanol were added to the tube at 4°C, and the
tube was stored at -20°C for 30 minutes. The DNA was recovered as a
pellet by centrifugation at 12 000g for 10
minutes at 4°C in a microfuge. Then the pellet was rinsed twice with
70% ethanol and redissolved in 50 µL of
dH2O.
Synthesis of Mutant-cDNA of cPLA2 and PGHS-2 by
Site-Directed Mutagenesis With PCR Used for Internal Standard of
Competitive PCR
Complementary DNA of a mutant-cDNA of
cPLA2 and PGHS-2 were prepared by PCR-based,
site-directed mutagenesis following the method of Higuchi et
al.8 The mutagenic primer of sequence
5'-GTGTTCTATTATGAGATTCTGAACTCCCCAGA-GAAAGCTTGCTCTC-3'
(PLA2-mFw),
5'-ATGTTCCACCCGCAGTACAGAAAG-TATCACAAGCTT-3'
(PGHS-2-mFw) was synthesized on the Gene Assembler Plus. Amplification
of PLA2- or PGHS-2–cDNA with each mutagenic
primer and Rv-primer by PCR showed a single change, C to T (italicized
in PLA2-mFw) or G to A (italicized in
PGHS-2-mFw), which created a unique Hind III site. The
amplification and purification then were performed.
Competitive PCR for Quantification of PGHS-1 or -2 and
cPLA2 mRNA With Amplified Genomic DNA or Mutant cDNA
Fragments Used as Internal Standard
In a series of tubes, PCR amplification was performed on
reaction mixtures (10 µL) containing 1 µL of cDNA of PGHS-1,
PGHS-2, or cPLA2 and an increasing concentration
of amplified genomic DNA of PGHS-1, mutant-cDNA of PGHS-2 (0.01, 0.1,
1.0, 10, 100, 1000 amol), or mutant-cDNA of cPLA2
(0.25, 1.0, 16, 256, 4096 amol),
250x10-6 mol/L of each
dNTP plus 3.7x105 Bq of
[-32P] dCTP
(1.11x1014 Bq/mmol: New England Nuclear),
0.5x10-6 mol/L of each paired primer (Fw and
Rv), 0.25 U of Taq DNA polymerase (AmpliTaq), in PCR buffer (100
mmol/L Tris-HCl [pH 8.3], 500 mmol/L KCl, 15 mmol/L
MgCl2, 0.01% [wt/vol] gelatin). The PCR cycle
was repeated 40 times following the protocol described above. Before
loading the PCR products on gels for electrophoresis,
cPLA2 or PGHS-2 PCR products were digested by
10 U of Hind III for 60 minutes at 37°C. The PCR
products of mutant-cDNA of cPLA2 and PGHS-2
were divided into 2 fragments by Hind III digestion, but the
PCR products of cDNA of native cPLA2 and
PGHS-2 did not have the Hind III–digested site. In this
manner, competitive PCR products of native and mutant-cDNA of
cPLA2 and PGHS-2 were differentiated by digesting
them with Hind III before electrophoresis. The samples then
were loaded onto an 8% polyacrylamide gel
(acrylamide/bis-acrylamide: vol/vol,
29:1) and analyzed by electrophoresis. The gel was stained with
ethidium bromide (0.4 µg/mL) and photographed. The bands
corresponding to the amplified products of the internal standard
(gDNA or digested mutant c-DNA) and mRNA were cut out, and their
radioactivity was counted. The data were analyzed by plotting
the logarithm of the gDNA cpm/cDNA cpm ratio as a function of the
logarithm of the gRNA concentration.
Statistical Analysis
Data are expressed as mean±SEM. ANOVA was used to make
comparisons. For F ratios significant at the level of 
5%,
Duncan’s multiple range test was applied to determine differences
between any 2 groups. Differences of 5% (P<0.05) were
considered statistically significant. Percent changes, for which a
normal distribution cannot be assumed, were compared by means of the
Kruskal-Wallis nonparametric method for ANOVA. Whenever the
2 test results were significant, the
Mann-Whitney test was used to determine the significance of the
differences between pairs of means.
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Results |
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The addition of BK increased PGI2 release dose-dependently at concentrations from 10-10 mol/L through 10-4 mol/L, and the release reached a plateau at the concentration of 10-5 mol/L, so we used this concentration (10-5 mol/L) of BK in the following experiments. The stimulation of BK remarkably increased PGI2 generation within 15 minutes, and the effect was sustained through 180 minutes. This increase was not inhibited by pretreatment with the B1 receptor antagonist des-Arg9-BK(10-4 mol/L) or the PGHS-2 inhibitor NS-398 (10-6 mol/L) but was inhibited by pretreatment with the B2 receptor antagonist HOE 140 (10-4 mol/L) and was reduced to the constitutive level by pretreatment with the extracellular Ca2+ chelator EGTA (10-3 mol/L) or protein kinase C (PKC) inhibitor H-7 (10-5 mol/L). Pretreatment with a phospholipase C (PLC) inhibitor, U-73122 (10-5 mol/L), or a diacylglycerol (DAG) lipase inhibitor, RHC80267 (5x10-5 mol/L), reduced PGI2 release to the level under the constitutive generation and pretreatment with a PLA2 inhibitor, mepacrine (10-4 mol/L), or a PGHS inhibitor, aspirin (10-3 mol/L), reduced PGI2 release to almost zero in control as well as in BK stimulation experiments (Table). The concentration of BK in incubation fluid measured by radioimmunoassay-dextran coated charcoal method at 15, 60, 180 minutes after BK addition was 1.12±0.06 (10-5 mol/L), 1.16±0.02 (10-5 mol/L), and 1.10±0.11 (10-5 mol/L), respectively. These data were not statistically different from each other.
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Within 15 minutes of BK addition, the cytosolic PLA2 activity was increased to 132±10.3% of control. A further increase to 199.4±6.5% of control was found at 60 minutes, and this high level was maintained for 180 minutes.
The [Ca2+]i in HUVEC was 86.37±8.34 nmol/L in the steady state. BK caused a transitory increase in [Ca2+]i, with the peak at 30 seconds after addition and the return to basal level within 5 minutes (Figure 1A). BK also increased 45Ca uptake to 145±5.3% that of control. Pretreatment with EGTA (10-3 mol/L) remarkably decreased the increase of both [Ca2+]i and 45Ca uptake induced by BK (Figure 1B).
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The generation of IP3 in HUVEC was increased by BK from 0.213±0.038 (pmol/2x105 cells) to 0.476±0.052 (pmol/2x105 cells). Pretreatment with EGTA (10-3 mol/L) inhibited the BK-induced IP3 generation (Figure 2A). The addition of IP3 (10-5 mol/L) increased the 45Ca release from the intracellular calcium storage sites in saponized HUVEC. The increase of 45Ca release induced by IP3 was 35.7% less than that caused by the calcium ionophore A23187 (10-5 mol/L) (Figure 2B).
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The constitutive expression of cPLA2 mRNA was 86.8±6.8 amol/µg RNA in HUVEC. BK increased it to 746.5±43.3 amol/µg RNA at 15 minutes after stimulation, and the same level of mRNA expression was maintained for 180 minutes. The constitutive expression of PGHS-1 mRNA was 562.2±21.8 amol/µg RNA in HUVEC. Whereas BK had increased the expression only to 773.2±15.4 amol/µg RNA after 15 minutes of stimulation, it reached to 10 608±467.4 amol/µg RNA in 180 minutes. The constitutive expression of PGHS-2 mRNA was only 46.8±3.0 amol/µg RNA in HUVEC. BK did not affect its expression until 60 minutes after stimulation; however, the increase reached 22 400±2650 amol/µg RNA at 180 minutes (Figure 3). Pretreatment with cycloheximide (5 µg/mL) for 60 minutes superinduced the mRNA expression of cPLA2 and PGHS-2 to 2183±193 and 6891±547 amol/µg RNA, respectively, but it suppressed the mRNA expression of PGHS-1 to 16.6±2.5 amol/µg RNA. Pretreatment with EGTA (10-3 mol/L) for 60 minutes superinduced the mRNA expression of cPLA2 to 2718±309 amol/µg RNA, but it suppressed that of PGHS-1 to 1.1±0.8. The mRNA expression of PGHS-2 was increased by EGTA pretreatment from 46.8±3.0 amol/µg RNA to 89.5±23.7 amol/µg RNA; however, the difference of PGHS-2 mRNA expression between control and EGTA pretreatment was not statistically significant (Figure 4).
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Discussion |
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ACE inhibitors are the most widely used drugs in the treatment of hypertensive patients. Their pharmacological effects depend not only on the reduction of the vasoconstrictor angiotensin II but also the local accumulation of kinins and kinin-related PGI2 release, and the importance of the latter mechanisms has become apparent recently. Several kinds of BK-receptor subtypes have been identified and characterized according to their pharmacological and biochemical properties. Above all, the B2 receptor is widely distributed over human tissues, especially smooth muscle cells and neurons, and mediates most of the cellular actions of BK.9 In the present study, pretreatment with the B1-receptor antagonist des-Arg9-bradykinin did not inhibit the increase of BK-induced PGI2 generation in HUVEC, but pretreatment with B2-receptor antagonist HOE 140 inhibited the BK-induced increase in HUVEC. The increased effect of BK on PGI2 release was sustained for 3 hours after stimulation, but the increase rate of PGI2 release declined after 15 minutes of BK stimulation. Because the concentration of BK in incubation fluid did not change from the beginning of incubation to 180 minutes after BK addition, this suggests that the cause of the rate of decline of PGI2 release was not due to the reduction of BK concentration in incubation fluid but to other factors, for instance, downregulation of BK receptors induced by long-term exposure of BK. Pretreatment with the calcium chelator EGTA, PKC inhibitor H-7, PLC inhibitor U-73122, or DAG lipase inhibitor RHC80267 remarkably reduced BK-induced PGI2 release, but the effects of the latter 2 inhibitors were more suppressive. These findings suggest that PLC and DAG affect BK-induced PGI2 synthesis in HUVEC not only by PKC but also the PKC-independent pathway. From these results, we speculate that BK acts on HUVEC through B2 receptors and that extracellular Ca2+ and protein phosphorylation related to PKC, PLC, and DAG play important roles in the process of BK-induced PGI2 synthesis.
The release of AA from phospholipids in the cell membrane is the first
step of PGI2 generation, and
PLA2 acts mainly in this step.
PLA2 has several isoforms. The genomic structures
of type I and II of secretory PLA2
(sPLA2) and cytosolic PLA2
(cPLA2) have been determined. The release of AA
from phospholipids of cell membranes is chiefly dependent on
cPLA2 and partially on
sPLA2.10 In the present study,
BK elevated cPLA2 activity to 132% of control
within 15 minutes after stimulation, and this level of activity was
maintained for 180 minutes. It is well known that
cPLA2 activity is affected by intracellular
Ca2+ kinetics. BK increased
[Ca2+]i transitorily to
240% of control. Pretreatment with the extracellular
Ca2+ chelator EGTA reduced BK-induced
Ca2+ influx and
[Ca2+]i increase, but
this BK-induced [Ca2+]i
increase was not completely inhibited, and we could observe a small
amount of increase in
[Ca2+]i despite EGTA
pretreatment. This increase in
[Ca2+]i was thought to be
mobilized from Ca2+ storage sites in cytosol. The
intracellular Ca2+ storage sites consist of
mitochondrial and nonmitochondrial sites, and the latter, especially
for IP3-induced Ca2+
release, is a main source of
[Ca2+]i. In our
present study, BK significantly increased the concentration of
cytosolic IP3 in HUVEC, and the addition of
IP3
(10-5 mol/L) promoted the
45Ca release from the Ca2+
storage sites in saponized HUVEC. These results suggest that the
BK-induced [Ca2+]i
increase consists of not only Ca2+ influx but
also IP3-induced Ca2+
release from the [Ca2+]i
storage sites in HUVEC. This elevated
[Ca2+]i in turn
activates cPLA2. It was recently reported
that cPLA2 contains the CaLB domain, a
Ca2+-dependent translocation domain in its
N-terminal,11 and that cPLA2
translocates from cytoplasm to cell membrane in the gradient of
[Ca2+]i
concentration.12 It was also reported that the
phosphorylation by MAP kinase raised
PLA2 activity to severalfold that of
control.13 These mechanisms raised
cPLA2 activity, thereby promoting the release of
AA from membrane phospholipids and its subsequent conversion into
PGI2 through the PGHS pathway.
The synthesis of PGI2 can also be controlled by altering the activity or expression of the enzyme. Because the increase effect of BK on PGI2 generation is sustained for 3 hours after stimulation, we believe that not only the raise of enzymatic activity but also the quantitative increase of cPLA2 or PGHS protein through mRNA transcription is involved in BK-induced PGI2 synthesis in HUVEC. PGHS is another rate-limiting enzyme in the biosynthesis of prostanoids. Recently, 2 isoforms of PGHS, PGHS-1 (COX-1) and PGHS-2 (COX-2), were detected, and their gene structures and proteins were examined. PGHS-1 is expressed constitutively in most tissues, but PGHS-2 is highly and rapidly induced in response to cell activation.14 In our present study, pretreatment with the PGHS-2 inhibitor NS-398 did not affect BK-induced PGI2 release until 3 hours after BK stimulation, and the mRNA of PGHS-1, quantified by competitive PCR, was more markedly expressed than that of cPLA2 or PGHS-2. This finding indicates that PGI2 generation of the constitutive phase and early phase of BK stimulation in HUVEC depends on PGHS-1, not PGHS-2. The importance of PGHS-1 in the constitutive generation of prostanoids has been reported in most tissues including endothelial cells,15 and our results support these findings.
The stimulation of BK provided interesting results. The mRNA expression
of cPLA2 increased to 8-fold that of control
at 15 minutes after BK stimulation and maintained this level for 180
minutes. In contrast, that of PGHS-1 was unchanged until 60 minutes
after BK stimulation but then had increased remarkably by 180 minutes.
Although many investigators have evaluated the effects of substances on
PGHS-1 mRNA expression, an increase of PGHS-1 mRNA was reported in a
few studies, and their ratios of increase in expression of PGHS-1 mRNA
were only 2-fold to 4-fold that of control level.14 It
was, therefore, assumed that the role of this enzyme was to produce
prostaglandins that regulate normal cellular processes.
Contrary to these former reports, in our study the level of PGHS-1 mRNA
expression increased to 18-fold that of control at 180 minutes.
Thus, it is possible that PGHS-1 acts as not only a constitutive enzyme
but also an inducible enzyme in the regulation of
PGI2 synthesis in BK-stimulated HUVEC. The
expression of PGHS-2 mRNA was only 44 amol/µg RNA in an unstimulated
HUVEC but had increased to 500-fold of control level at 180 minutes.
The changes in PGHS-2 mRNA induced by BK were typical of an inducible
enzyme. From our present findings, we speculate that there are 3
steps in the process of BK-induced PGI2 synthesis
in HUVEC. The first step is an immediate activation of
cPLA2 mediated by increase of intracellular
Ca2+ and protein phosphorylation
of the already-existing enzyme. The second is a new synthesis of
cPLA2 protein by gene transcription. In these 2
steps, the main reaction is the supply of AA by
cPLA2 and the synthesis of
PGI2 by constitutively existing PGHS-1. The third
step is the gene transcription and synthesis of new proteins of the
PGHS isoforms. We observed no increase in PGHS-1 and PGHS-2 mRNA until
180 minutes. This time lag of BK-induced mRNA expression of
cPLA2 and PGHS isoforms is notable. It seems
likely that cPLA2 protein is generated by an
induction of cPLA2 mRNA before PGHS and provides
PGHS with AA and that PGHS, which is synthesized belatedly for
cPLA2, in turn synthesizes
PGI2 through the PGHS pathway. The newly
synthesized cPLA2 and PGHS proteins apparently
act mainly in the later phase (after 180 minutes of BK stimulation) and
maintain the sustained phase of PGI2 generation.
Their time course appears to be a reasonable reaction; however, we
could not quantify the protein of cPLA2 and PGHS.
Further examinations to determine the relation of mRNA expression and
protein synthesis are necessary.
To investigate the mechanism of intracellular signal transduction in
BK-stimulated HUVEC, we evaluated the effects of cycloheximide and EGTA
on mRNA expression. As speculated, BK stimulation under the condition
of cycloheximide pretreatment remarkably increased the level of
cPLA2 and PGHS-2 mRNA expression in HUVEC. The
cycloheximide-induced superinduction of cPLA2
mRNA was previously reported in rat mesangial cells
incubated with epidermal growth factor.16 Cytosolic
phospholipase A2 mRNA, similar to immediate early
genes, contains an AU-rich sequence in its 3'-untranslated region, and
cycloheximide superinduces immediate early genes by altering the
stability of mRNA and inhibiting the synthesis of AU-binding factor,
which increases the instability of mRNA. In the case of PGHS-2,
cycloheximide also superinduces mRNA expression to about 90-fold that
of the control level. Because PGHS-2 mRNA also contains an AU-rich
sequence in its 3'-untranslated region,14 mechanisms
similar to those of cPLA2 are thought to be
involved in its cycloheximide-induced superinduction. Moreover,
Srivastava et al17 detected a new protein induced by
interleukin-1ß in rat mesangial cells and reported that
this protein stabilizes PGHS-2 mRNA by binding its AU-rich region.
Interestingly, the finding was completely different for PGHS-1 mRNA
expression. As its expression is almost completely inhibited by
cycloheximide, we speculate that there exists an enhancer protein that
is indispensable for the transcription of PGHS-1 mRNA. In our
present study, EGTA pretreatment inhibited the
Ca2+ influx by chelating extracellular
Ca2+, resulting in a decrease in the BK-induced
increase of [Ca2+]i by
50%. Although the importance of intracellular
Ca2+ as a second messenger in intracellular
signal transduction has been recognized, a detailed account of the
relation between the second messenger, including intracellular
Ca2+, and mRNA has not been provided. EGTA
pretreatment increased cPLA2 mRNA expression to
the same extent as pretreatment with cycloheximide but decreased PGHS-1
mRNA expression to almost zero. The finding that PGHS-2 mRNA expression
was not affected by EGTA pretreatment is unexpected because protein
phosphorylation by PKC18 or protein
tyrosine kinase activation is causally linked to expression of
mRNAs19 and because some kinds of PKC isoforms are
Ca2+-sensitive. The report that the
Ca2+ ionophore A23187 induces PGHS-2 mRNA
expression in IL-1ß–stimulated chondrocytes20 shows the
important role of intracellular Ca2+ in mRNA
expression. The elucidation of these phenomena must await further
investigations of the intracellular
Ca2+–dependent signaling mechanisms involved
with cPLA2 and PGHS mRNA expression.
This is the first report to investigate the effects of BK on the regulatory mechanisms of PGI2 generation by cPLA2 activity and the mRNA expression of cPLA2 and PGHS isoforms in human vascular endothelial cells. We determined that the elevation of cPLA2 activity caused by the increase of intracellular Ca2+ concentration is an important event in the early phase of BK-induced PGI2 synthesis, and we conclude that the mechanisms for the regulation of cPLA2 and PGHS isoforms in endothelial cell are different. However, because we could not directly measure the protein and PGHS activity, further evaluations are needed to elucidate the regulatory mechanisms of BK-induced PGI2 synthesis in HUVEC.
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Footnotes |
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S. Yamasaki and S. Komatsu contributed equally to this work.
Received May 4, 1999; first decision June 8, 1999; accepted March 1, 2000.
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References |
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