© 1995 American Heart Association, Inc.
Articles |
Nitric Oxide Produced by Endothelial Cells Increases Production of Eicosanoids Through Activation of Prostaglandin H Synthase
From the Department of Obstetrics, Gynecology and Reproductive Sciences and Magee-Womens Research Institute, University of Pittsburgh (Pa).
Correspondence to Sandra T. Davidge, Magee-Womens Research Institute, Rm 620, 204 Craft Ave, Pittsburgh, PA 15213.
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Abstract |
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Abstract The endothelium serves many functional roles, including the modulation of vascular smooth muscle tone through the release of vasoactive agents such as nitric oxide (NO) and the eicosanoids. We proposed that NO produced by endothelial cells would increase the production of eicosanoids through enhanced expression and/or activation of prostaglandin H synthase. NO and eicosanoid synthesis were stimulated in a bovine coronary microvessel endothelial cell line with the calcium ionophore A23187 (1 µmol/L). Our data demonstrated the following: (1) A23187 stimulated NO synthesis along with prostacyclin and thromboxane production. (2) Inhibition of NO synthesis with NG-nitro-L-arginine methyl ester (0.1 mmol/L) significantly diminished both prostacyclin and thromboxane production. (3) Cells incubated with hemoglobin (2 µg/mL), which inactivates NO, decreased A23187-stimulated prostacyclin production, whereas cells incubated with superoxide dismutase (20 U/mL), which protects NO from superoxide anions, enhanced prostacyclin production. (4) Exogenous NO stimulated prostacyclin production. (5) The interaction of NO with prostacyclin persisted in the presence of excess exogenous arachidonic acid (100 µmol/L). (6) Cyclooxygenase activity in cell lysates increased in the first hour of NO stimulation. (7) NO stimulation of prostacyclin occurred within 1 hour and continued for 8 hours. (8) Neither constitutive nor inducible prostaglandin H synthase enzyme expression was altered by NO. (9) Cycloheximide (10 µmol/L) had no effect on A23187 stimulation of prostacyclin production. (10) Exogenous cGMP (10 µmol/L) or a phosphodiesterase inhibitor (1 mmol/L) did not affect prostacyclin production. These data indicate that stimulating synthesis of endogenous NO in cultured endothelial cells increased eicosanoid production through activation of prostaglandin H synthase.
Key Words: endothelium • nitric oxide • eicosanoids • prostacyclin • prostaglandin H synthase
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Introduction |
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Endothelial cells that line the lumen of all blood vessels contribute to normal vascular physiology. Impaired endothelial cell function has been implicated as a mediator of many cardiovascular diseases, including atherosclerosis, ischemic injury, hypertension, and the pregnancy disorder preeclampsia.1 2 3 4 The many functional roles of endothelial cells include the modulation of vascular smooth muscle tone through release of vasoactive agents such as NO and the eicosanoids, prostacyclin and thromboxane. NO and the eicosanoids have actions especially relevant to cardiovascular morbidity. NO and prostacyclin are potent vasorelaxants and inhibitors of platelet aggregation that counterbalance the vasoconstrictor and platelet-aggregating properties of thromboxane.5
NO is formed from L-arginine by the enzyme NO synthase.6 7 8 Several isoforms of the NO synthase have been characterized and purified from different cell types and tissues.6 A constitutive NO synthase isoform predominates in endothelial cells. Increased intracellular calcium activates the endothelial constitutive NO synthase to produce NO. NO binds to the heme component of soluble guanylate cyclase of the adjacent smooth muscle cells, which increases cGMP production, resulting in vasorelaxation.7 8
Eicosanoid production is regulated by the expression and activity of PGH synthase once arachidonic acid is released from phospholipid stores.9 There are at least two PGH synthase isozymes: a constitutive and an inducible form (PGHS-1 and PGHS-2, respectively). PGHS-1 regulates steady state levels of prostaglandin synthesis, and the increased synthesis of prostaglandins appears to result from the increased transcription of the PGH synthase gene.10 Another type of regulation involves the acute induction of PGHS-2 in cells that contain little or no enzyme.11 Synthesis of prostaglandins in these cells is dependent on enhanced PGHS-2 expression. The expression of both PGH synthase isozymes has been reported in endothelial cells.9 12
PGH synthase has both a cyclooxygenase activity that incorporates two molecules of oxygen into arachidonic acid to form prostaglandin endoperoxide G2 and a peroxidase activity that catalyzes a two-electron reduction of prostaglandin endoperoxide G2 to prostaglandin endoperoxide H2.9 Activation of PGH synthase is not completely understood. However, it is established that there is a requirement for a hydroperoxide initiator13 and that the cyclooxygenase component of PGH synthase requires oxidative conversion to a form that is capable of abstracting the 13-pro-S hydrogen from arachidonate.9
Although the NO- and eicosanoid-producing pathways have been studied extensively, little is known about potential interactions between the two pathways. NO is an oxidizing radical14 that has been reported to induce transcription factors15 and interact with heme-containing proteins.8 PGH synthase is a heme-containing enzyme9 that can be regulated at the level of expression and/or by oxidative activation, raising the possibility that NO could regulate PGH synthase expression and/or activity. Information regarding such a potential interaction using several cell types, tissues, and isolated enzyme is limited and conflicting. It has been reported that NO inhibited the formation of PGH synthase products in lipopolysaccharide-treated liver macrophages.16 In contrast, NO increased prostaglandin E2 production in a lipopolysaccharide-treated macrophage cell line.17 In addition, NO appears to stimulate prostaglandin E2 from norepinephrine-stimulated hypothalamic fragments18 and cytokine-stimulated islets of Langerhans,19 whereas basal NO and exogenously administered NO increased eicosanoid synthesis in the rat uterus.20 However, a recent study found no evidence for a direct effect of NO on PGH synthase activity in a purified enzyme preparation.21
In endothelial cells, there is evidence for a corelease of NO and the eicosanoids22 23 ; however, a direct interaction of NO with eicosanoid production in endothelial cells has not been reported. The present study tested the effect of NO, which was produced by the stimulation of endothelial constitutive NO synthase, upon the expression and activity of PGH synthase in endothelial cells. We report that NO produced by endothelial cells increases the production of eicosanoids through activation of PGH synthase.
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Materials and Methods |
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Reagents
MEM, horse serum, L-glutamine,
gentamicin, kanamycin, and nystatin were obtained from GIBCO.
L-Arginine, D-arginine, L-NAME, A23187,
hemoglobin, superoxide dismutase, cycloheximide, sodium nitroprusside,
arachidonic acid, methylene blue, IBMX, and
8-bromo-cGMP were purchased from Sigma Chemical Co. Sodium hydrosulfite
(dithionite) was purchased from Aldrich Chemical Co. A
guanylate cyclase inhibitor, LY83583, was
obtained from Lilly Co. Polyclonal anti–PGHS-2 (C-terminus) and
chicken recombinant mitogen-inducible PGHS-2 standard were purchased
from Oxford Biomedical Research, Inc. Meclofenamate, NONOate,
polyclonal rabbit anti-ram seminal vesicle PGH synthase antibody,
purified ram seminal vesicle PGH synthase standard, and enzyme
immunoassay kits for 6-keto PGF1 and TxB2
were obtained from Cayman Chemical Co.
Stock solutions of L-arginine, D-arginine,
L-NAME, cycloheximide, sodium nitroprusside, methylene blue, IBMX,
8-bromo-cGMP, and LY83583 were prepared in MEM culture media at a
concentration of 10 mmol/L. A stock solution of 2 µg/mL hemoglobin
was prepared in MEM. A stock solution of 10 mmol/L of A23187 was
prepared in DMSO and further diluted to 0.1 mmol/L in MEM. A stock
solution of arachidonic acid was prepared in ethanol at
a concentration of 10 mmol/L.
Endothelial Cell Culture
A bovine coronary microvascular
endothelial cell line was obtained from Gensia, Inc.
Since the establishment of this cell line, the phenotype of
these endothelial cells has been maintained for over
180 passages. The cellular characteristics include growth as a
monolayer, a cobblestone morphology at confluence, positive
immunostaining for von Willebrand
factor–related antigen, the presence of receptors for
acetylated low-density lipoproteins, and secretion of
tissue-type plasminogen
activator.24 In addition, these cells express
the mRNA and protein for the constitutive endothelial
NO synthase, demonstrate NO synthase activity, secrete nitrites (a
stable end product of NO), and also produce
prostaglandins (authors' unpublished data, 1994).
Cells were grown at 37°C in a humidified atmosphere of 5%
CO2/95% air with MEM containing 0.6 mmol/L
L-arginine, 10% horse serum, 2 mmol/L
L-glutamine, gentamicin (5 µg/mL), kanamycin (20
µg/mL), and nystatin (10 U/mL).
Cells were plated at confluence in six-well tissue culture plates at
106 cells per milliliter (1 mL per well). After
attaching to plates, cells were quiesced in serum-free medium
containing 0.05% bovine serum albumin for 24 hours before
experimental stimulation. The medium was not changed again immediately
before the experiment so as not to mechanically disturb the cells. This
results in a measurable basal production of nitrites and 6-keto
PGF1 immediately before the experiment.
Total protein for cells was measured by the Bradford method,25 with bovine serum albumin used as a standard.
Nitrite Assay
Nitrite, a stable end product of NO, was measured in the
culture media by using the spectrophotometric Greiss reaction (Green et
al26 ). An aliquot of medium (180 µL) from each culture
well was mixed with 40 µL of Greiss reagent (1% sulfanilamide and
0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid).
The mixture was incubated for 10 minutes at room temperature, and the
absorbance (optical density, 550 nm) was measured in a Vmax kinetic
microplate reader (Molecular Devices). Concentrations were determined
by comparison with a sodium nitrite standard. The lower limit of
detection was 0.2 µmol/L.
Prostacyclin and Thromboxane A2
Assays
Prostacyclin and thromboxane A2 were
measured in the cell culture media as their stable metabolites, 6-keto
PGF1 and TxB2, respectively, by using
enzyme immunoassay kits. The lower limit of detection for 6-keto
PGF1 was 3.9 pg/mL; the lower limit of detection for
TxB2 was 7.8 pg/mL. All assays contained media controls for
the various agents used in the present study to determine that
there was no effect of the agents on the assays.
Endogenous Stimulation of NO and
Eicosanoids
The general protocol was to stimulate endogenous NO
synthesis and then measure the simultaneous generation of
PGH synthase products. NO production was then specifically
inhibited to determine whether NO synthesis was responsible for
eicosanoid production. The constitutive
endothelial NO synthase was activated by increasing
cytoplasmic calcium with the calcium ionophore A23187 (1 µmol/L).
Some cells were preincubated for 30 minutes with the NO synthesis
inhibitor L-NAME (0.1 mmol/L) before exposure of cells to
A23187. Since L-NAME is a competitive inhibitor of
L-arginine, the substrate for NO synthesis, experiments
were repeated in the presence or absence of excess
L-arginine (6.0 mmol/L) or the inactive isomer
D-arginine (6.0 mmol/L) to determine the specificity of
L-NAME inhibition. All experiments contained control cells that were
exposed to L-NAME, L-arginine, or
D-arginine in the absence of A23187. In addition, each
experiment contained control cells with medium alone or medium in the
presence of dilute DMSO, the vehicle for A23187. Medium was collected
24 hours later for measurements of nitrites, 6-keto
PGF1, and TxB2.
Another series of experiments was conducted to determine whether NO
stabilizing (with superoxide dismutase) or sequestering (with
hemoglobin) would enhance or diminish 6-keto PGF1
production. Cells were preincubated for 10 minutes with
superoxide dismutase (20 U/mL) before stimulation with (or without)
A23187. Before use, hemoglobin was reduced with dithionite, as
previously described.27 Hemoglobin (2 µg/mL) was added
to the cells immediately before stimulation with (or without) A23187.
Medium was collected 1 and 24 hours later for measurements of nitrites
and 6-keto PGF1.
To determine the effect of nitrites on 6-keto PGF1
production, cells were incubated with 1 to 100 µmol/L sodium
nitrite. Medium was collected 24 hours later for measurements of
nitrites and 6-keto PGF1.
Time Course of NO and 6-Keto PGF1
Production
This experiment was performed to identify the time course of
nitrite and 6-keto PGF1 production from these
cells. Medium was removed from cells incubated in the absence or
presence of L-NAME and/or A23187 after 1, 4, 8, and 24 hours.
Effect of Exogenous NO on 6-Keto PGF1
Production
To further evaluate whether NO increases prostacyclin
production, exogenous NO donors SNAP (1 µmol/L), NONOate (1
µmol/L), or SNP (1 µmol/L) were added to cells in culture. After 1
hour, nitrite and 6-keto PGF1 levels were measured from
the culture media.
In addition, to further evaluate the specificity of L-NAME inhibition,
SNAP, NONOate, or SNP was added to cells in the presence of L-NAME.
After 1 hour, nitrite and 6-keto PGF1 levels were
measured from the culture media.
Effect of Exogenous Arachidonic Acid
The following experiments were performed to determine whether
PGH synthase activation was, in part, responsible for the increase in
eicosanoid synthesis. Exogenous arachidonic acid (100
µmol/L) was preincubated with cells 30 minutes before the stimulation
with (or without) A23187 in the absence or presence of L-NAME. Medium
was collected at 1, 4, 8, and 24 hours from cells and assayed for
6-keto PGF1. In preliminary experiments, 100
µmol/L arachidonic acid resulted in maximal 6-keto
PGF1 production.
To determine whether the effect of exogenous NO persisted in the
presence of excess arachidonic acid, experiments were
performed with exogenous arachidonic acid (100
µmol/L) in the absence or presence of exogenous NO donors SNAP (1
µmol/L), NONOate (1 µmol/L), or SNP (1 µmol/L).
Arachidonic acid was preincubated with cells 30 minutes
before the stimulation with the NO donors. Medium was collected at 1
hour from the cells and assayed for 6-keto
PGF1.
Cyclooxygenase Activity of PGH
Synthase
Cyclooxygenase activity was performed on
cells that in culture were exposed for 1 hour or 24 hours to control or
A23187 in the absence or presence of L-NAME. In addition,
cyclooxygenase activity experiments were also
conducted on cells that were cultured for 1 hour with the exogenous NO
donor NONOate (1 µmol/L) or were exposed to NONOate after cell
lysis.
Endothelial cells were harvested from the six-well
plates with 500 µL of cold PBS containing 1 mmol/L
N-ethylmaleimide and 0.3 mmol/L phenylmethylsulfonyl
fluoride.28 After sonication, the cells were
assayed for cyclooxygenase activity by measuring
the conversion of arachidonic acid to 6-keto
PGF1.29 Arachidonic
acid was added at a final concentration of 100 µmol/L. The reaction
proceeded for 15 minutes at 37°C and was stopped by adding
meclofenamate to a final concentration of 10 µmol/L, followed by
rapid freezing in liquid nitrogen. Aliquots of the reaction mixture
were assayed for 6-keto PGF1.
Effect of Endogenous NO Stimulation on PGH
Synthase Expression
PGH synthase expression was determined by Western immunoblot, as
previously described.23 Cells were collected 2 and 24
hours after stimulation with A23187. In addition, as a positive
control, some cells were exposed to 20% serum and collected after 2
and 24 hours of incubation. These time points were chosen as optimal
measurements for both PGHS-1 and PGHS-2. Previous studies have reported
that in human umbilical vein endothelial cells,
interleukin-1 stimulation caused a steady increase in PGHS-1 protein
levels for 15 hours, which remained elevated up to 24
hours.30 Maximal expression of PGHS-2 levels has been
reported to occur at 24 hours in a cytokine-stimulated
macrophage cell line (RAW 264.7),31 but serum
caused a maximal expression of PGHS-2 within 2 hours in 3T3
cells.32
Cells were washed with PBS and solubilized with lysis buffer (25 mmol/L
Tris-HCL [pH 6.8] with 0.5% Triton X-100). For gel electrophoresis,
samples were diluted by addition of an equal volume of 2x gel sample
buffer (40 mmol/L Tris-Cl [pH 6.8], 2% SDS, 10% 2-mercaptoethanol,
20% glycerol, and 0.02% bromphenol blue). Samples were boiled for 5
minutes and centrifuged at 10 000g for 2 minutes.
Aliquots (10 µL) were loaded into individual wells formed within the
stacking gel (5% acrylamide in stacking gel buffer and 25
mmol/L Tris-HCl [pH 6.8]) overlayed on 8% to 23%
acrylamide gradient gels in Tris-HCl (pH 8.8) and separated
by electrophoresis at 100 to 300 V for 
1 hour in a Daiichi mini-gel
apparatus according to the method of
Laemmli.33
After separation, samples were transferred to a membrane (Nylon NT, Magnagraph, Inc) in a wet tank (Hoeffer). Prestained standards (high and low molecular weight, Bio-Rad) were included in separate lanes in each gel for identification of the approximate molecular weight of unknowns. After transfer, the membranes were incubated with TBS (50 mmol/L Tris-HCl [pH 7.5] and 500 mmol/L NaCl) containing 5% nonfat dry milk (1 hour) and TTBS (two changes, 5 minutes each). Immunoblots for the constitutive PGHS-1 used a polyclonal rabbit anti-ram seminal vesicle PGH synthase antibody with each immunoblot containing a standard of 10 fmol/10 µL of purified ram seminal vesicle PGH synthase. For the inducible PGHS-2, a polyclonal rabbit antibody was prepared against a synthetic peptide corresponding to a distinct C-terminal region of the inducible PGHS-2, with each immunoblot containing a standard of 100 ng of a mitogen-inducible PGH synthase from a chicken recombinant protein. Antibodies were incubated for 12 to 18 hours and rinsed with TTBS (three changes, 20 minutes each), goat anti-rabbit IgG conjugated to alkaline phosphatase (Promega) diluted 1:10 000 in TBS (2 hours), and TTBS (three changes, 20 minutes each). Immunoreactive bands were visualized by the addition of substrate (100 µg/mL nitro blue tetrazolium and 50 µg/mL 5-bromo-4-chloro-3-indoyl phosphate) in developing buffer (50 mmol/L Tris-Cl [pH 10.0], 100 mmol/L NaCl, and 3 mmol/L MgCl2).
In addition to measuring enzyme mass, additional experiments were
designed to determine whether new PGH synthase enzyme synthesis
contributed to the cyclooxygenase activity. Cells
were preincubated for 10 minutes with cycloheximide (10 µmol/L) to
inhibit translation before stimulation with (or without) A23187.
Another set of cells used acetylsalicylic acid (0.2
mmol/L) to inhibit preexisting cyclooxygenase
activity. Cells were incubated with acetysalicylic acid for 30 minutes,
washed, and then stimulated with A23187. Cell culture medium was
assayed for 6-keto PGF1 to determine whether new enzyme
synthesis was necessary for 6-keto PGF1
production.
Effect of cGMP
To determine whether the interaction of NO with
prostaglandins was mediated through cGMP,
endothelial cells were stimulated with (and without)
A23187 in the absence or presence of guanylate cyclase
inhibitors methylene blue (50 µmol/L) and LY83583 (10
µmol/L) with or without IBMX (1 mmol/L) to inhibit phosphodiesterase
activity. In addition, cells (not stimulated with A23187) were exposed
to 8-bromo-cGMP (10 µmol/L) in the absence or presence of IBMX.
Medium was collected 2 and 24 hours later for measurement of 6-keto
PGF1.
Data Analysis
Data are summarized as mean±SEM of at least three
individual experiments containing three replicates per condition. A
one-way ANOVA was used to determine statistical differences among three
or more groups. Pairwise comparison was then conducted with a post hoc
analysis using the Bonferroni test. Differences among means
were considered significant at P<.01.
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All data are expressed per 106 cells. Each well contained 106 cells, as measured by a hemocytometer, when a confluent monolayer had been established. Total protein in each well (
20 µg of protein per well) did not change significantly with
stimulation by different agents.
Stimulation of Endogenous Synthesis of NO With A23187
Increased the Production of 6-keto PGF1
and TxB2
The first series of experiments was designed to determine
whether NO produced by endothelial cells would increase
the production of eicosanoids. Table 1
depicts
endothelial production of nitrites 6-keto
PGF1 and TxB2 after stimulation for
24 hours with A23187 in the absence or presence of L-NAME,
L-arginine, or D-arginine. The effect of
L-NAME and its reversal by L-arginine but not
D-arginine were also present if cells were assayed at
1 hour.
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Endothelial cells stimulated with A23187 released
significantly more nitrite, 6-keto PGF1, and
TxB2 than the control cells. The release of nitrite as well
as 6-keto PGF1 and TxB2 was significantly
inhibited by coincubation with the NO synthesis inhibitor
L-NAME (Table 1
). The presence of L-NAME did not have a significant
effect on basal production of nitrite, 6-keto
PGF1, and TxB2 (Table 1). These data
indicate that a portion of the stimulated release of prostacyclin and
thromboxane was due to NO.
To determine that the effect of L-NAME to alter eicosanoid
production was due to the inhibition of NO formation, cells
were coincubated with either excess of the biologically active
stereoisomer L-arginine or the inactive isomer
D-arginine. There was no effect of excess
L-arginine or D-arginine on nitrite, 6-keto
PGF1, or TxB2 production in
the absence of A23187 (Table 1). Excess L-arginine but
not D-arginine prevented L-NAME inhibition of
A23187-stimulated nitrite as well as 6-keto PGF1 and
TxB2 production (Table 1). These data indicate that
the effect of L-NAME on 6-keto PGF1 and TxB2
production is due specifically to
L-arginine–dependent NO synthesis.
Superoxide dismutase (20 U/mL), which protects NO from superoxide
anions, significantly enhanced A23187-stimulated 6-keto
PGF1 production at 1 hour (4069±215 versus
3483±95 pg/106 cells, P<.05) and 24
hours (6355±459 versus 4944±144 pg/106 cells,
P<.05). Hemoglobin (2 µg/mL), which inactivates NO,
significantly decreased A23187-stimulated 6-keto PGF1
production (2618±268 versus 3467±81 pg/106
cells for 1 hour, P<.05) and 24 hours (3988±289 versus
4944±144 pg/106 cells for 24 hours,
P<.05). There was no significant effect of either
hemoglobin or superoxide dismutase on cells not stimulated with
A23187.
Exogenous sodium nitrite (1, 10, or 100 µmol/L) had no
significant effect on cell culture pH (which remained constant at
7.3) or 6-keto PGF1 production (850±35,
910±96, or 791±58 pg/106 cells for 24 hours),
respectively.
Collectively, these data indicate that A23187 stimulated endogenous NO production, which subsequently increased eicosanoid production from the endothelial cells.
Time Course of Nitrite and 6-Keto PGF1
Production
Fig 1, left, depicts the accumulation of nitrites
from the cell culture media for 1, 4, 8, and 24 hours after stimulation
with (or without) A23187 in the absence or presence of L-NAME. This
time course demonstrated a rapid elevation of nitrites in response to
A23187 stimulation for the first hour that continued for 8 hours. From
8 to 24 hours after A23187 stimulation, nitrite accumulation continued
to increase gradually. Adding L-NAME (0.1 mmol/L) at the beginning of
the incubation effectively inhibited nitrite production for the
24 hours. The basal production of nitrites is low from these
endothelial cells. L-NAME alone did not significantly
affect basal nitrite levels.
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), control cells in the
presence of the NO synthesis inhibitor L-NAME (0.01 mmol/L,
), and cells stimulated with the calcium ionophore A23187 (1
µmol/L) in the absence (
) or presence of L-NAME (
). Cells
were quiesced 24 hours before stimulation, resulting in a basal
production of nitrites depicted at time 0. Nitrite accumulation
was greater (P<.01) with A23187 at all time points. No
other responses were different. Stimulated nitrite production
was significantly inhibited in the presence of L-NAME at each time
point, and the difference persisted throughout the incubation. Each
point represents the mean±SEM of at least three individual
experiments containing three replicates per condition. Right, Time
course of 6-keto PGF1
Fig 1, right, depicts the accumulation 6-keto PGF1 from
the cell culture media for 1, 4, 8, and 24 hours after stimulation with
(or without) A23187 in the absence or presence of L-NAME. The time
course of 6-keto PGF1 was in parallel with that of the
nitrites, which demonstrated a rapid elevation for the first hour that
continued for 8 hours. However, for 6-keto PGF1,
there was no further increase in accumulation from 8 to 24 hours after
A23187 stimulation. L-NAME (0.1 mmol/L) added at the beginning of the
incubation significantly inhibited stimulated 6-keto
PGF1 at each time point. The basal production of
6-keto PGF1 was not significantly affected by
L-NAME.
Exogenous NO Increased 6-Keto PGF1
Production
Table 2 presents results from
endothelial cells stimulated for 1 hour with either
SNAP (1 µmol/L), SNP (1 µmol/L), or NONOate (1 µmol/L). Release
of 6-keto PGF1 was significantly increased with all
three exogenous NO-producing agents. The presence of L-NAME had no
significant effect on exogenous NO-stimulated 6-keto
PGF1 production (data not shown).
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The next set of experiments was conducted to determine whether PGH synthase activation was in part responsible for increased eicosanoid production. The presence of excess substrate, arachidonic acid, would allow PGH synthase to be the rate-limiting enzyme.
NO Stimulation of 6-Keto PGF1 Persisted in the
Presence of Exogenous Arachidonic Acid in Intact
Cultured Cells
Fig 2 depicts the time-course response to
exogenous arachidonic acid alone or with A23187 in the
absence or presence of L-NAME. A concentration of 100 µmol/L of
arachidonic acid was determined as maximally effective
in preliminary experiments. At each time point (1, 4, 8, or 24 hours),
6-keto PGF1 production was increased in cells
stimulated with arachidonic acid and A23187 compared
with cells stimulated with arachidonic acid alone.
L-NAME did not affect the response of arachidonic acid
alone. However, L-NAME completely inhibited the additional response due
to A23187. These findings indicate that A23187, even in the presence of
exogenous arachidonic acid, stimulated 6-keto
PGF1 production through an elevation of NO. With
this experimental design, arachidonic acid was not rate
limiting; therefore, these data suggest that at least part of the
effect of NO was to activate the cyclooxygenase
component of PGH synthase.
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Fig 3 depicts the effect of exogenous NO in the presence
of exogenous arachidonic acid (100 µmol/L). Each NO
donor, SNAP, NONOate, or SNP, significantly increased 6-keto
PGF1 production even in the presence of excess
arachidonic acid. These data further suggest that
activation of PGH synthase is at least partly involved in the increase
in eicosanoid synthesis produced by NO.
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Endogenous or Exogenous NO Stimulation Increased
Cyclooxygenase Activity in Cell
Lysates
Cell lysates prepared after stimulating
endothelial cells for 1 hour with A23187 demonstrated a
significant increase in cyclooxygenase activity
(Fig 4, left). However, after a 24-hour stimulation with
A23187, cyclooxygenase activity was significantly
decreased (Fig 4, right). Coincubation with L-NAME had no effect on the
cyclooxygenase activity of untreated cells (data
not shown) but significantly decreased
cyclooxygenase activity at 1 hour (Fig 4, left). At
24 hours, cyclooxygenase activity was significantly
greater when L-NAME was included with A23187 than with A23187 alone
(Fig 4, right). These findings indicate that
cyclooxygenase activation by NO occurs by 1 hour
and that the enzyme is almost completely inactive by 24 hours.
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Cell lysates prepared after stimulating endothelial cells in culture for 1 hour with NONOate also significantly increased cyclooxygenase activity (2562±274 versus 4479±255 pg/106 cells for 15 minutes, P<.01). After cell lysis, the effect of NONOate increased cyclooxygenase activity, but these data did not reach significance (4843±319 versus 7180±1607 pg/106 cells for 15 minutes, P<.06).
Cyclooxygenase activation could be due to increased activity of preexisting enzyme and/or increased expression of PGH synthase.
PGH Synthase Expression
Fig 5, left, is a representative
immunoblot of six blots performed for the constitutive PGHS-1. A major
immunoreactive protein band was observed at 70 kD that was identical
to the standard ram seminal vesicle PGH synthase (lane 5). There was no
difference between control and A23187-stimulated cells in the absence
or presence of L-NAME at either 2 or 24 hours of stimulation. These
data indicate no obvious regulation of the expression of the
constitutive PGHS-1 by increased NO.
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Fig 5, right, depicts a representative immunoblot of
six blots performed for the inducible PGHS-2. A major immunoreactive
protein band was observed with the positive control
(endothelial cells stimulated with 20% serum) and the
inducible PGHS-2 standard (lanes 5 and 6, respectively). There was no
measurable induction of the inducible PGHS-2 for the control or
A21387-stimulated cells in the absence or presence of L-NAME at either
2 or 24 hours of stimulation.
In cells that were preincubated with the irreversible
cyclooxygenase inhibitor
acetylsalicylic acid (0.2 mmol/L) and then washed,
the subsequent stimulation with A23187 did not increase 6-keto
PGF1 production (control, 227±36
pg/106 cells for 24 hours; A23187, 108±28.9
pg/106 cells for 24 hours). These data suggest that
new synthesis of PGH synthase did not occur. Furthermore, preincubating
cells with cycloheximide (10 µmol/L) to block translation of new
protein had no significant effect on A23187 stimulation of 6-keto
PGF1 production from these
endothelial cells (control, 976±119.6
pg/106 cells for 24 hours;
control+cycloheximide, 807±101.1 pg/106 cells
for 24 hours; A23187, 3534±158 pg/106 cells for 24
hours; and A23187+cycloheximide, 3229±146
pg/106 cells for 24 hours). Collectively, these data
indicate that A23187-stimulated NO production activated
preexisting PGH synthase enzyme.
Effect of cGMP on 6-Keto PGF1
Production
We tested whether cGMP mediated the NO stimulation of eicosanoid
synthesis. 8-Bromo-cGMP (10 µmol/L) in the absence or presence of the
phosphodiesterase inhibitor IBMX (1 mmol/L) had no
significant effect on 6-keto PGF1 production
after either 2 hours (data not shown) or 24 hours of stimulation (Table 3).
|
Cells stimulated with A23187 in the presence of methylene blue (50
µmol/L) demonstrated significantly lower 6-keto PGF1
production compared with cells stimulated with A23187 alone
(2620±908 versus 4939±792 pg/106 cells for 24
hours, P<.01). However, cells stimulated with A23187 in the
presence of the guanylate cyclase inhibitor
LY83583 (10 µmol/L) produced 6-keto PGF1 in a manner
similar to that of cells exposed to only A23187 (3893±723 versus
4316±562 pg/106 cells for 24 hours).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have demonstrated that stimulating the synthesis of endogenous NO in cultured endothelial cells increased eicosanoid production through the activation of PGH synthase. The calcium ionophore A23187 stimulated NO synthesis, which resulted in increase in 6-keto PGF1
and TxB2
production. A23187, acting by a non–receptor-mediated
mechanism allows a flux of calcium into the cells.34 An
increase in intracellular calcium activates the constitutive NO
synthase, resulting in NO production.7 8 However,
increased cytoplasmic calcium activates many cellular responses in
addition to NO synthesis. Of relevance to the present study, A23187
activates phospholipase A2 to stimulate the release of
arachidonic acid, which would subsequently lead to an
increase in prostaglandin
production.35 Nonetheless, we demonstrated that in
the present study, a significant amount of 6-keto
PGF1 and TxB2 production by the
endothelial cells was due to NO. L-NAME, an
inhibitor of NO synthesis, reduced NO as well as 6-keto
PGF1 and TxB2 production stimulated
by A23187. This inhibition was reversed by the excess biologically
active substrate, L-arginine, but not the inactive
stereoisomer, D-arginine. The effect of L-NAME was thus
specific for NO inhibition, indicating that a portion of the 6-keto
PGF1 and TxB2 production was due to
NO. In addition, exogenous NO donors that act by spontaneous release of
NO36 also stimulated 6-keto PGF1
production from the endothelial cells. This
stimulated 6-keto PGF1 production by exogenous
NO persisted in the presence of L-NAME, which further indicates that
L-NAME does not affect 6-keto PGF1 production
through non–NO-mediated mechanisms.
Furthermore, protecting NO from degradation by superoxide anions
increased 6-keto PGF1 production. Conversely,
the NO scavenger hemoglobin reduced 6-keto PGF1
production. Hemoglobin binds and inactivates NO by oxidizing it
to NO2 and NO3. Hemoglobin remains
extracellular, suggesting that part of the endogenous NO
produced exits the cell and acts in a paracrine manner to stimulate
eicosanoid production from the same or adjacent cells. However,
it is also possible that the avid affinity of NO for hemoglobin and the
free diffusibility of NO through plasma membranes prevent accumulation
of NO intracellularly to concentrations sufficient to activate
cyclooxygenase.
Both 6-keto PGF1 and TxB2 were stimulated by
NO, indicating an action at a common precursor enzyme. The relevant
precursor enzymes are phospholipases, which release
arachidonic acid from membrane phospholipid, and PGH
synthase, which metabolizes arachidonic acid to
PGH2. We found that NO (from either endogenous
or exogenous sources) stimulated 6-keto PGF1
production even in the presence of excess
arachidonic acid. Furthermore, L-NAME completely
inhibited the additional increase of 6-keto PGF1 by
A23187 in the presence of exogenous arachidonic acid.
Since the substrate, arachidonic acid, was not rate
limiting, these data indicate that NO activation is at the level of PGH
synthase. However, these findings do not exclude an additional
interaction of NO with phospholipase A2.
In cell lysates, the effect of exogenous NO on PGH synthase cyclooxygenase activity after cell lysis did not reach significance; this was most likely due to the high variability in cyclooxygenase activity in the NO-stimulated cells. However, cyclooxygenase activity was increased by 1 hour of exogenous NO and by NO stimulation with A23187 in cells in culture. These data are in agreement with those of Salvemini et al,17 who demonstrated that NO activates cyclooxygenase enzymes.
Our data indicate that NO increased the activity of PGH synthase. Expression of PGH synthase is known to be important in the regulation of prostaglandin synthesis.9 NO could be regulating eicosanoid production through the transcription or translation of a new enzyme. A recent report demonstrated a colocalized expression of NO synthase and the immediate-early gene transcription factors, c-jun and c-fos, in spinal cord neurons and that NO induced the immediate-early gene, c-fos.15 In addition, many transcription factors are regulated by oxygen radicals.37 38 39 NO as an oxidizing radical could activate an early-immediate gene transcription factor, leading to new synthesis of PGH synthase. PGH synthase is also an immediate-early gene that has characteristics similar to the transcription factors.11 Alternatively, NO could directly activate the transcription of the PGH synthase enzyme. However, we found no evidence for the induction of PGH synthase. The enzyme mass for the constitutive enzyme did not change, and there was no evidence for the induction of the inducible PGH synthase at either 2 or 24 hours after stimulation.
Additional evidence supports the concept that NO did not affect PGH
synthase expression. Cycloheximide, which would block translation, had
no effect on NO-stimulated 6-keto PGF1
production, and there was a rapid elevation of 6-keto
PGF1 after NO stimulation. Furthermore, acetylating and
inactivating a preexisting enzyme with
acetylsalicylic acid prevented NO-stimulated 6-keto
PGF1 production. Finally,
cyclooxygenase activity was increased at 1 hour but
was significantly reduced by 24 hours.
Cyclooxygenase has a suicide-inactivation mechanism
that appears to be an intrinsic property of the catalytic activity.
Each mole of purified enzyme can form 1300 mol of
prostaglandin G2 before the
cyclooxygenase activity disappears.9
Therefore, reduced cyclooxygenase activity at 24
hours would suggest that new synthesis of the enzyme did not occur.
These data indicate that NO did not induce synthesis of a new enzyme
but rather activated the cyclooxygenase component
of preexisting PGH synthase.
These data are further supported by comparing the time course of
nitrite production with 6-keto PGF1
production. Nitrite production followed a time course
similar to that of 6-keto PGF1 production from 0
to 8 hours. However, from 8 to 24 hours nitrite accumulation continued,
whereas 6-keto PGF1 production was maximal at 8
hours. This is not surprising, since PGH synthase has a
suicide-inactivation mechanism, and indeed, we demonstrated decreased
cyclooxygenase activation by 24 hours. Therefore NO
can still be accumulating in the bath without affecting 6-keto
PGF1 production.
PGH synthase activation is not completely understood. One proposed mechanism requires a hydroperoxide initiator to generate an oxidized enzyme intermediate that forms a free radical coincident with cyclooxygenase catalysis.9 This model proposes that hydroperoxide initiators are reduced by the ferric enzyme that produces higher oxidation states of the heme group. A tyrosyl radical is then formed by electron transfer from tyrosine to the oxidized heme, and this tyrosyl radical is involved in the hydrogen abstraction step of the cyclooxygenase reaction. NO, as an oxidizing radical, could potentially activate PGH synthase through its oxidative capacity to modify the heme or the cyclooxygenase component of the enzyme. Alternatively, NO could activate PGH synthase through its heme-binding properties in a manner similar to its activation of other heme-containing enzymes, such as guanylate cyclase. However, depending on the hemeprotein itself, the consequence of the heme-binding effect of NO may vary and may not be associated with a catalytic function.40
A recent study by Tsai et al21 found no evidence for a direct effect of NO on PGH synthase activity in purified or partially purified preparations of isolated ovine PGH synthase. In contrast, our data along with that of others16 17 18 19 20 indicate that there is an effect of NO on PGH synthase activation in intact tissues and cells. However, in light of the observations of Tsai et al, this activation may not be "direct" but may occur through intermediary pathways. One interesting possibility is that NO reacts with superoxide anions to form peroxynitrite formation along with hydroxyl radicals. The result may be an increase in lipid peroxidation, which would enhance PGH synthase activity. This "indirect" effect would not be present with the purified enzyme. Nonetheless, the interaction of NO and PGH synthase pathways, either direct or indirect, may represent an important mechanism for regulation in vascular function.
An increase in superoxide anion production by NO synthase is also a potential activator of cyclooxygenase. The overall NO synthase reaction is an electron oxidation of L-arginine, in which molecular oxygen, NADPH, flavine adenine dinucleotide, flavine mononucleotide, and tetrahydrobiopterin are required.6 Superoxide anion and NO are simultaneously formed through the reduction of molecular oxygen by NO synthase.41 However, there is a greater generation of superoxide anions in the absence of L-arginine, and the addition of L-arginine (1.0 mmol/L) reduced superoxide formation concomitant with enhanced production of NO.41 In the present study, L-arginine was in the media (0.6 mmol/L), and the addition of exogenous L-arginine (6.0 mmol/L) did not increase NO production by the endothelial cells. Therefore, L-arginine was not a limiting substrate in our conditions and most likely did not allow for a substantial generation of superoxide.
NO acts by cGMP-independent and -dependent mechanisms. The cytotoxic
effect of macrophage-derived NO to inactivate iron and
iron-containing enzymes is independent of cGMP.7
Conversely, relaxation of smooth muscle and inhibition of platelet
aggregation are secondary to an elevation of cGMP.8 Since
vascular NO effects are secondary to cGMP, we tested whether NO acts by
cGMP in these vascular endothelial cells to activate
PGH synthase. Since there is not one direct approach to determine a
cGMP role, we addressed this issue with four different approaches using
exogenous cGMP, a phosphodiesterase inhibitor, and two
different guanylate cyclase inhibitors,
LY8358342 and methylene blue. However, there are
limitations to the specificity of these reagents. It is not clear that
either LY83583 or methylene blue is a specific inhibitor of
guanylate cyclase. Indeed, although methylene blue has been
used extensively as an inhibitor of soluble
guanylate cyclase, it also acts as a direct
inhibitor of NO synthase.43 Furthermore, the
phosphodiesterase inhibitor IBMX is also reported to
inhibit phospholipase activity.44 With these caveats, we
observed in the present study that exogenous 8-bromo-cGMP or a
phosphodiesterase inhibitor did not increase eicosanoid
production by the endothelial cells. However,
methylene blue significantly diminished A23187-stimulated 6-keto
PGF1 production. In contrast, LY83583 had no
effect on 6-keto PGF1 production. In light of
our other data, the effect of methylene blue (which may act as a direct
inhibitor of NO synthase43 ) on 6-keto
PGF1 production from the
endothelial cells is more likely due to diminished NO
production than to decreased cGMP. Overall, our data suggest
that NO stimulation of 6-keto PGF1 production is
independent of cGMP.
Our study design was to activate the constitutive NO synthase to stimulate the endogenous production of NO by endothelial cells. Previous studies of NO and cyclooxygenase interactions have not focused on the endothelium but have concentrated primarily on cells with a role in inflammation.16 17 19 However, the results from these previous studies are conflicting. In hepatic macrophages, the effect of increasing NO was to reduce cyclooxygenase activity.16 However, in a macrophage cell line (RAW 264.7)17 and cytokine-stimulated islets of Langerhans,19 increased NO was associated with increased eicosanoid production. The differences in these studies are difficult to reconcile. At present, the best explanation is perhaps cell-specific responses. It is tempting to invoke a dose-response relation, with the low levels of NO produced by the constitutive NO synthase in endothelial cells resulting in stimulation of cyclooxygenase by oxidative activation and the higher concentrations of NO produced by the inducible NO synthase in inflammatory cells inhibiting cyclooxygenase activity, perhaps by quenching tyrosyl radicals.45 However, the two studies with macrophage cells noted above produced opposite results at what appear to be similar NO concentrations; these results argue against a dose-dependent phenomenon for NO. Further research is needed to delineate the mechanisms by which NO activates or inhibits PGH synthase.
In summary, the present data indicate that NO increases the production of eicosanoids through activation of PGH synthase in these endothelial cells. A link between NO and the regulation of eicosanoid synthesis in vascular endothelial cells could represent an important mechanism for vascular control in physiological as well as pathological states.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grant 1PO1 HD-30367-01 and the Irene McLenehan Young Investigator's Research Fund of the Magee-Women's Health Foundation (Dr Davidge). Dr Baker is a Wellbeing (UK)/American College of Obstetrics and Gynecology Exchange Research Fellow. We gratefully acknowledge the help of Dr Jerzy Barankiewicz, Gensia, Inc. This manuscript is dedicated to the late Dr Zbigniew Rymaszewski, Department of Endocrinology, University of Cincinnati (Ohio), whose collaborations made this study possible.
Received September 16, 1994; accepted April 18, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Hennig B, Chow CK. Lipid peroxidation and endothelial cell injury: implications in atherosclerosis. Free Radic Biol Med. 1988;4:99-106. [Medline] [Order article via Infotrieve]
2. Kamata K, Miyata N, Kasuya Y. Impairment of endothelium-dependent relaxation and changes in levels of cyclic GMP in aorta from streptozotacin-induced diabetic rats. Br J Pharmacol. 1989;97:614-618. [Medline] [Order article via Infotrieve]
3. Vanhoutte PM. Role of calcium and endothelium in hypertension, cardiovascular disease, and subsequent vascular events. J Cardiovasc Pharmacol. 1992;19:S6-S10.
4. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol. 1989;161:1200-1204. [Medline] [Order article via Infotrieve]
5. Vane JR, Anffard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med. 1990;323:27-36. [Medline] [Order article via Infotrieve]
6. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994;298:249-258.
7. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992;6:3051-3064. [Abstract]
8.
Lowenstein CJ, Dinerman JL, Snyder SH. Nitric
oxide: a physiologic messenger. Ann Intern Med. 1994;120:227-237.
9. Smith WL, Marnett LJ. Prostaglandin endoperoxide synthase: structure and catalysis. Biochim Biophys Acta. 1991;1083:1-17. [Medline] [Order article via Infotrieve]
10. DeWitt DL. Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochim Biophys Acta. 1991;1083:121-134.[Medline] [Order article via Infotrieve]
11. Xie W, Robertson DL, Simmons DL. Mitogen-inducible prostaglandin G/H synthase: a new target for nonsteroidal antiinflammatory drugs. Drug Dev Res. 1992;25:249-265.
12.
Hla T, Neilson K. Human
cyclooxygenase-2 cDNA. Proc Natl Acad
Sci U S A. 1992;89:7384-7388.
13.
Hemler ME, Lands WEM. Evidence for a
peroxide-initiated free radical mechanism of prostaglandin
biosynthesis. J Biol Chem. 1980;255:6253-6261.
14. Lei SZ, Pan Z-H, Aggarwal SK, Chen H-SV, Hartman J, Sucher NJ, Lipton SA. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron. 1992;8:1087-1099. [Medline] [Order article via Infotrieve]
15. Herdegen T, Rüdiger S, Mayer B, Bravo R, Zimmermann M. Expression of nitric oxide synthase and colocalisation with Jun, Fos and Krox transcription factors in spinal cord neurons following noxious stimulation of the rat hindpaw. Mol Brain Res. 1994;22:245-258. [Medline] [Order article via Infotrieve]
16. Stadler J, Harbrecht BG, Di Silvio M, Curran RD, Jordan ML, Simmons RL, Billiar TR. Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells. J Leukoc Biol. 1993;53:165-172. [Abstract]
17.
Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie
MG, Needleman P. Nitric oxide activates
cyclooxygenase enzymes. Proc Natl
Acad Sci U S A. 1993;90:7240-7244.
18.
Rettori V, Gimeno M, Lyson K, McCann SM. Nitric
oxide mediates norepinephrine-induced
prostaglandin E2 release from the
hypothalamus. Proc Natl Acad Sci U S A. 1992;89:11543-11546.
19. Corbett JA, Kwon G, Turk J, McDaniel ML. IL-1ß induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of Langerhans: activation of cyclooxygenase by nitric oxide. Biochemistry. 1993;32:13767-13770. [Medline] [Order article via Infotrieve]
20.
Franchi AM, Chaud M, Rettori V, Suburo A, McCann SM,
Gimeno M. Role of nitric oxide in eicosanoid synthesis and
uterine motility in estrogen-treated rat uteri. Proc Natl
Acad Sci U S A. 1994;91:539-543.
21. Tsai A-l, Wei C, Kulmacz R. Interaction between nitric oxide and prostaglandin H synthase. Arch Biochem Biophys. 1994;313:367-372. [Medline] [Order article via Infotrieve]
22.
de Nucci G, Gryglewski RJ, Warner TD, Vane JR.
Receptor-mediated release of endothelium-derived
relaxing factor and prostacyclin from bovine aortic
endothelial cells is coupled. Proc Natl
Acad Sci U S A. 1988;85:2334-2338.
23. Davidge ST, Hubel CA, McLaughlin MK. Cyclooxygenase-dependent vasoconstrictor alters vascular function in the vitamin E–deprived rat. Circ Res. 1993;73:79-88. [Abstract]
24. Rymaszewski Z, Kessel BJ, Bai S, Abplanalp WA, Cohen RM, Barankiewicz J. Angiotensin II stimulates the secretion of plasminogen activator and inhibits the release of plasminogen activator inhibitor by capillary endothelial cells. J Vasc Res. 1992;29:191-192.
25. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
26. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Anaysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal Biochem. 1982;126:131-138. [Medline] [Order article via Infotrieve]
27. Ignarro LJ, Barry BK, Gruetter DY, Ohlstein EH, Gruetter CA, Kadowitz PJ, Baricos WH. Selective alterations in responsiveness of guanylate cyclase to activation by nitroso compounds during enzyme purification. Biochim Biophys Acta. 1981;673:394-407. [Medline] [Order article via Infotrieve]
28.
Smith MM, Schlesinger S, Lindstrom J, Merlie JP.
The effects of inhibiting oligosaccharide trimming by
1-deoxynojirimycin on the nicotinic acetylcholine receptor.
J Biol Chem. 1986;261:14825-14832.
29.
Raz A, Wyche A, Siegel N, Needleman P.
Regulation of fibroblast cyclooxygenase
synthesis by interleukin-1. J Biol Chem. 1988;263:3022-3028.
30.
Maier JAM, Hla T, Maciag T.
Cyclooxygenase is an immediate-early gene
induced by interleukin-1 in human endothelial
cells. J Biol Chem. 1990;265:10805-10808.
31. Riese J, Hoff T, Nordhoff A, DeWitt DL, Resch K, Kaever V. Transient expression of prostaglandin endoperoxide synthase-2 during mouse macrophage activation. J Leukoc Biol. 1994;55:476-482. [Abstract]
32. DeWitt DL, Meade EA. Serum and glucocorticoid regulation of gene transcription and expression of the prostaglandin H synthase-1 and prostaglandin synthase-2 isozymes. Arch Biochem Biophys. 1993;306:94-102. [Medline] [Order article via Infotrieve]
33. Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;277:680-685.
34. Pressman BC. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501-530. [Medline] [Order article via Infotrieve]
35. Rosenthal MD, Jones JE. Release of arachidonic acid from vascular endothelial cells: fatty acyl specificity is observed with receptor-mediated agonists and with the calcium ionophore A23187 but not with melittin. J Cell Physiol. 1988;136:333-340. [Medline] [Order article via Infotrieve]
36. Butler AR, Williams DLH. The physiological role of nitric oxide. Chem Soc Rev. 1993;22:233-241.
37. Crawford D, Zbinden I, Amstad P, Cerutti P. Oxidant stress induces the proto-oncogenes c-fos and c-myc in mouse epidermal cells. Oncogene. 1988;3:27-32.
38. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparantly widely used messengers in the activation of the NF-KB transcription factor and HIV-1. EMBO J. 1991;10:2247-2258. [Medline] [Order article via Infotrieve]
39.
Rao GN, Berk BC. Active oxygen species stimulate
vascular smooth muscle cell growth and proto-oncogene
expression. Circ Res. 1992;70:593-599.
40. Tsai A-l. How does NO activate hemeproteins? FEBS Lett. 1994;341:141-145. [Medline] [Order article via Infotrieve]
41.
Pou S, Surichamorn W, Bredt DS, Snyder SH, Rosen GM.
Generation of superoxide by purified brain nitric oxide
synthase. J Biol Chem. 1992;267:24173-24176.
42. Brandt MA, Conrad KP. In vivo and in vitro studies of a putative inhibitor of cyclid guanosine 3',5'-monophosphate production. Proc Soc Exp Biol Med. 1991;196:30-35. [Medline] [Order article via Infotrieve]
43. Mayer B, Brunner F, Schmidt K. Inhibition of nitric oxide synthesis by methylene blue. Biochem Pharmacol. 1993;45:367-374. [Medline] [Order article via Infotrieve]
44. Hong SL. Inhibition of prostacyclin synthesis in endothelial cells by methylisobutylxanthine is not mediated through elevated cAMP level. Biochim Biophys Acta. 1983;754:258-263. [Medline] [Order article via Infotrieve]
45. Lepoivre M, Fieschi F, Coves J, Thelander L, Fontecave M. Inactivation of ribonucleotide reductase by nitric oxide. Biochem Biophys Res Commun. 1991;179:442-448. [Medline] [Order article via Infotrieve]
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 R. Bolli, K. Shinmura, X.-L. Tang, E. Kodani, Y.-T. Xuan, Y. Guo, and B. Dawn Discovery of a new function of cyclooxygenase (COX)-2: COX-2 is a cardioprotective protein that alleviates ischemia/reperfusion injury and mediates the late phase of preconditioning Cardiovasc Res, August 15, 2002; 55(3): 506 - 519. [Abstract] [Full Text] [PDF]
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 R. Maas, E. Schwedhelm, J. Albsmeier, and R. H Boger The pathophysiology of erectile dysfunction related to endothelial dysfunction and mediators of vascular function Vascular Medicine, August 1, 2002; 7(3): 213 - 225. [Abstract] [PDF]
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F. C Blumberg, C. Lorenz, K. Wolf, P. Sandner, G. A.J Riegger, and M. Pfeifer Increased pulmonary prostacyclin synthesis in rats with chronic hypoxic pulmonary hypertension Cardiovasc Res, July 1, 2002; 55(1): 171 - 177. [Abstract] [Full Text] [PDF]
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 N. Ahmad, L. C. Chen, M. A. Gordon, J. D. Laskin, and D. L. Laskin Regulation of cyclooxygenase-2 by nitric oxide in activated hepatic macrophages during acute endotoxemia J. Leukoc. Biol., June 1, 2002; 71(6): 1005 - 1011. [Abstract] [Full Text] [PDF]
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K. Shinmura, Y.-T. Xuan, X.-L. Tang, E. Kodani, H. Han, Y. Zhu, and R. Bolli Inducible Nitric Oxide Synthase Modulates Cyclooxygenase-2 Activity in the Heart of Conscious Rabbits During the Late Phase of Ischemic Preconditioning Circ. Res., March 22, 2002; 90(5): 602 - 608. [Abstract] [Full Text] [PDF]
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Y. Zhang and C. W. Leffler Compensatory role of NO in cerebral circulation of piglets chronically treated with indomethacin Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2002; 282(2): R400 - R410. [Abstract] [Full Text] [PDF]
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 D. S.A. Majid and A. Nishiyama Nitric Oxide Blockade Enhances Renal Responses to Superoxide Dismutase Inhibition in Dogs Hypertension, February 1, 2002; 39(2): 293 - 297. [Abstract] [Full Text] [PDF]
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 A. Tsuda, K. A. Tanaka, C. Huraux, F. Szlam, N. Sato, K. Yamaguchi, and J. H. Levy The In Vitro Reversal of Histamine-Induced Vasodilation in the Human Internal Mammary Artery Anesth. Analg., December 1, 2001; 93(6): 1453 - 1459. [Abstract] [Full Text] [PDF]
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 J. P. Zenge, R. L. Rairigh, T. R. Grover, L. Storme, T. A. Parker, J. P. Kinsella, and S. H. Abman NO and prostaglandin interactions during hemodynamic stress in the fetal ovine pulmonary circulation Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1157 - L1163. [Abstract] [Full Text] [PDF]
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 R. J. Bing, M. Miyataka, K. A. Rich, N. Hanson, X. Wang, H. D. Slosser, and S.-R. Shi Nitric Oxide, Prostanoids, Cyclooxygenase, and Angiogenesis in Colon and Breast Cancer Clin. Cancer Res., November 1, 2001; 7(11): 3385 - 3392. [Abstract] [Full Text] [PDF]
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S. T. Davidge Prostaglandin H Synthase and Vascular Function Circ. Res., October 12, 2001; 89(8): 650 - 660. [Abstract] [Full Text] [PDF]
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 Y. Devaux, C. Seguin, S. Grosjean, N. de Talance, V. Camaeti, A. Burlet, F. Zannad, C. Meistelman, P.-M. Mertes, and D. Longrois Lipopolysaccharide-Induced Increase of Prostaglandin E2 Is Mediated by Inducible Nitric Oxide Synthase Activation of the Constitutive Cyclooxygenase and Induction of Membrane-Associated Prostaglandin E Synthase J. Immunol., October 1, 2001; 167(7): 3962 - 3971. [Abstract] [Full Text] [PDF]
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D. S. Sangha, S. Han, and R. E. Purdy Simulated microgravity upregulates an endothelial vasoconstrictor prostaglandin J Appl Physiol, August 1, 2001; 91(2): 789 - 796. [Abstract] [Full Text] [PDF]
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R. J Bing Myocardial ischemia and infarction: growth of ideas Cardiovasc Res, July 1, 2001; 51(1): 13 - 20. [Abstract] [Full Text] [PDF]
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J.-X. Chen, L. C. Berry Jr., B. W. Christman, M. Tanner, P. R. Myers, and B. O. Meyrick NO regulates LPS-stimulated cyclooxygenase gene expression and activity in pulmonary artery endothelium Am J Physiol Lung Cell Mol Physiol, March 1, 2001; 280(3): L450 - L457. [Abstract] [Full Text] [PDF]
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D. J. Berg, J. Zhang, D. M. Lauricella, and S. A. Moore IL-10 Is a Central Regulator of Cyclooxygenase-2 Expression and Prostaglandin Production J. Immunol., February 15, 2001; 166(4): 2674 - 2680. [Abstract] [Full Text] [PDF]
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V. B. O'Donnell and B. A. Freeman Interactions Between Nitric Oxide and Lipid Oxidation Pathways : Implications for Vascular Disease Circ. Res., January 19, 2001; 88(1): 12 - 21. [Abstract] [Full Text] [PDF]
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 Z.-G. Liu, X.-C. Liu, A. P.C. Yim, and G.-W. He Direct measurement of nitric oxide release from saphenous vein: abolishment by surgical preparation Ann. Thorac. Surg., January 1, 2001; 71(1): 133 - 137. [Abstract] [Full Text] [PDF]
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 T. Yamamoto and R. J. Bing Nitric Oxide Donors Experimental Biology and Medicine, December 1, 2000; 225(3): 200 - 206. [Abstract] [Full Text]
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 A. Lemancewicz, H. Laudanska, T. Laudanski, A. Karpiuk, and S. Batra Permeability of fetal membranes to calcium and magnesium: possible role in preterm labour Hum. Reprod., September 1, 2000; 15(9): 2018 - 2022. [Abstract] [Full Text] [PDF]
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 A. Bloodsworth, V. B. O'Donnell, and B. A. Freeman Nitric Oxide Regulation of Free Radical- and Enzyme-Mediated Lipid and Lipoprotein Oxidation Arterioscler Thromb Vasc Biol, July 1, 2000; 20(7): 1707 - 1715. [Abstract] [Full Text] [PDF]
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L. J. Marnett, T. L. Wright, B. C. Crews, S. R. Tannenbaum, and J. D. Morrow Regulation of Prostaglandin Biosynthesis by Nitric Oxide Is Revealed by Targeted Deletion of Inducible Nitric-oxide Synthase J. Biol. Chem., April 28, 2000; 275(18): 13427 - 13430. [Abstract] [Full Text] [PDF]
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 M. KAKUYAMA, A. AHLUWALIA, J. RODRIGO, and P. VALLANCE Cholinergic Contraction Is Altered in nNOS Knockouts . Cooperative Modulation of Neural Bronchoconstriction by nNOS and COX Am. J. Respir. Crit. Care Med., December 1, 1999; 160(6): 2072 - 2078. [Abstract] [Full Text]
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 K. G. Stewart, Y. Zhang, and S. T. Davidge Estrogen Decreases Prostaglandin H Synthase Products From Endothelial Cells Reproductive Sciences, November 1, 1999; 6(6): 322 - 327. [Abstract] [PDF]
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H. He, V. J. Venema, X. Gu, R. C. Venema, M. B. Marrero, and R. B. Caldwell Vascular Endothelial Growth Factor Signals Endothelial Cell Production of Nitric Oxide and Prostacyclin through Flk-1/KDR Activation of c-Src J. Biol. Chem., August 27, 1999; 274(35): 25130 - 25135. [Abstract] [Full Text] [PDF]
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 D. C. GOODWIN, L. M. LANDINO, and L. J. MARNETT Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis FASEB J, July 1, 1999; 13(10): 1121 - 1136. [Abstract] [Full Text]
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R. J Bing, T. Yamamoto, M. Yamamoto, R. Kakar, and A. Cohen New look at myocardial infarction: toward a better aspirin Cardiovasc Res, July 1, 1999; 43(1): 25 - 31. [Abstract] [Full Text] [PDF]
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M. LEEMAN, V. Z. de BEYL, D. BIARENT, M. MAGGIORINI, C. MELOT, and R. NAEIJE Inhibition of Cyclooxygenase and Nitric Oxide Synthase in Hypoxic Vasoconstriction and Oleic Acid-Induced Lung Injury Am. J. Respir. Crit. Care Med., May 1, 1999; 159(5): 1383 - 1390. [Abstract] [Full Text] [PDF]
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 M. DÍAZ-CAZORLA, D. PÉREZ-SALA, and S. LAMAS Dual Effect of Nitric Oxide Donors on Cyclooxygenase-2 Expression in Human Mesangial Cells J. Am. Soc. Nephrol., May 1, 1999; 10(5): 943 - 952. [Abstract] [Full Text]
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 W. J. Calvo, G. Hajduczok, J. A. Russell, and S. L. Diamond Inhibition of Nitric Oxide but Not Prostacyclin Prevents Poststenotic Dilatation in Rabbit Femoral Artery Circulation, March 2, 1999; 99(8): 1069 - 1076. [Abstract] [Full Text] [PDF]
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F. J. Hughes, L. D. K. Buttery, M. V. J. Hukkanen, A. O'Donnell, J. Maclouf, and J. M. Polak Cytokine-induced Prostaglandin E2 Synthesis and Cyclooxygenase-2 Activity Are Regulated Both by a Nitric Oxide-dependent and -independent Mechanism in Rat Osteoblasts in Vitro J. Biol. Chem., January 15, 1999; 274(3): 1776 - 1782. [Abstract] [Full Text] [PDF]
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R. Hayman, J. Brockelsby, L. Kenny, and P. Baker Preeclampsia: The Endothelium, Circulating Factor(s) and Vascular Endothelial Growth Factor Reproductive Sciences, January 1, 1999; 6(1): 3 - 10. [Abstract] [PDF]
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P. Hardy, D. Abran, X. Hou, I. Lahaie, K. G. Peri, P. Asselin, D. R. Varma, and S. Chemtob A Major Role for Prostacyclin in Nitric Oxide–Induced Ocular Vasorelaxation in the Piglet Circ. Res., October 5, 1998; 83(7): 721 - 729. [Abstract] [Full Text] [PDF]
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A. Ichihara, J. D. Imig, E. W. Inscho, and L. G. Navar Cyclooxygenase-2 participates in tubular flow-dependent afferent arteriolar tone: interaction with neuronal NOS Am J Physiol Renal Physiol, October 1, 1998; 275(4): F605 - F612. [Abstract] [Full Text] [PDF]
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 S. Nogawa, C. Forster, F. Zhang, M. Nagayama, M. E. Ross, and C. Iadecola Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia PNAS, September 1, 1998; 95(18): 10966 - 10971. [Abstract] [Full Text] [PDF]
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S. T. Davidge and Y. Zhang Estrogen Replacement Suppresses a Prostaglandin H Synthase–Dependent Vasoconstrictor in Rat Mesenteric Arteries Circ. Res., August 24, 1998; 83(4): 388 - 395. [Abstract] [Full Text] [PDF]
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 H. Okamoto, O. Ito, R. J. Roman, A. G. Hudetz, and R. M. Bryan Jr Role of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 in Endotoxin-Induced Cerebral Hyperemia • Editorial Comment Stroke, June 1, 1998; 29(6): 1209 - 1218. [Abstract] [Full Text] [PDF]
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E. N.T.P. Bakker and P. Sipkema Permissive effect of nitric oxide in arachidonic acid induced dilation in isolated rat arterioles Cardiovasc Res, June 1, 1998; 38(3): 782 - 787. [Abstract] [Full Text] [PDF]
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D. B. Pearse, T. E. Dahms, and E. M. Wagner Microsphere-induced bronchial artery vasodilation: role of adenosine, prostacyclin, and nitric oxide Am J Physiol Heart Circ Physiol, March 1, 1998; 274(3): H760 - H768. [Abstract] [Full Text] [PDF]
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S. T. Davidge, J. Ojimba, and M. K. McLaughlin Vascular Function in the Vitamin E–Deprived Rat : An Interaction Between Nitric Oxide and Superoxide Anions Hypertension, March 1, 1998; 31(3): 830 - 835. [Abstract] [Full Text] [PDF]
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M. de Lorgeril Dietary arginine and the prevention of cardiovascular diseases Cardiovasc Res, March 1, 1998; 37(3): 560 - 563. [Full Text] [PDF]
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 A. Dellipizzi, M. L. Pucci, A. Y. Mosny, K. Deseyn, and A. Nasjletti Contribution of Constrictor Prostanoids to the Calcium-Dependent Basal Tone in the Aorta from Rats with Aortic Coarctation-Induced Hypertension: Relationship to Nitric Oxide J. Pharmacol. Exp. Ther., October 1, 1997; 283(1): 75 - 81. [Abstract] [Full Text]
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W. Steudel, F. Ichinose, P. L. Huang, W. E. Hurford, R. C. Jones, J. A. Bevan, M. C. Fishman, and W. M. Zapol Pulmonary Vasoconstriction and Hypertension in Mice With Targeted Disruption of the Endothelial Nitric Oxide Synthase (NOS 3) Gene Circ. Res., July 19, 1997; 81(1): 34 - 41. [Abstract] [Full Text]
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HarryM. Lander, DavidP. Hajjar, BarbaraL. Hempstead, UroojA. Mirza, BrianT. Chait, S. Campbell, and LawrenceA. Quilliam A Molecular Redox Switch on p21ras. STRUCTURAL BASIS FOR THE NITRIC OXIDE-p21ras INTERACTION J. Biol. Chem., February 14, 1997; 272(7): 4323 - 4326. [Abstract] [Full Text] [PDF]
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L.A. Danielson and K.P. Conrad Prostaglandins Maintain Renal Vasodilation and Hyperfiltration During Chronic Nitric Oxide Synthase Blockade in Conscious Pregnant Rats Circ. Res., December 1, 1996; 79(6): 1161 - 1166. [Abstract] [Full Text]
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L. Puybasset, M.-L. Bea, B. Ghaleh, J.-F. Giudicelli, and A. Berdeaux Coronary and Systemic Hemodynamic Effects of Sustained Inhibition of Nitric Oxide Synthesis in Conscious Dogs: Evidence for Cross Talk Between Nitric Oxide and Cyclooxygenase in Coronary Vessels Circ. Res., August 1, 1996; 79(2): 343 - 357. [Abstract] [Full Text]
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P. N. Baker, S. T. Davidge, J. Barankiewicz, and J. M. Roberts Plasma of Preeclamptic Women Stimulates and Then Inhibits Endothelial Prostacyclin Hypertension, January 1, 1996; 27(1): 56 - 61. [Abstract] [Full Text]
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K. Shinmura, Y.-T. Xuan, X.-L. Tang, E. Kodani, H. Han, Y. Zhu, and R. Bolli Inducible Nitric Oxide Synthase Modulates Cyclooxygenase-2 Activity in the Heart of Conscious Rabbits During the Late Phase of Ischemic Preconditioning Circ. Res., March 22, 2002; 90(5): 602 - 608. [Abstract] [Full Text] [PDF]
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