Nitric Oxide Produced by Endothelial Cells Increases Production of Eicosanoids Through Activation of Prostaglandin H Synthase
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.
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.
Materials and Methods
αMEM, 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 PGF1α 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 αMEM culture media at a concentration of 10 mmol/L. A stock solution of 2 μg/mL hemoglobin was prepared in αMEM. A stock solution of 10 mmol/L of A23187 was prepared in DMSO and further diluted to 0.1 mmol/L in αMEM. 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 αMEM 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 PGF1α immediately before the experiment.
Total protein for cells was measured by the Bradford method,25 with bovine serum albumin used as a standard.
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 PGF1α and TxB2, respectively, by using enzyme immunoassay kits. The lower limit of detection for 6-keto PGF1α 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 PGF1α, 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 PGF1α 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 PGF1α.
To determine the effect of nitrites on 6-keto PGF1α 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 PGF1α.
Time Course of NO and 6-Keto PGF1α Production
This experiment was performed to identify the time course of nitrite and 6-keto PGF1α 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 PGF1α 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 PGF1α 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 PGF1α 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 PGF1α. In preliminary experiments, 100 μmol/L arachidonic acid resulted in maximal 6-keto PGF1α 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 PGF1α.
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 PGF1α.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 PGF1α.
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 2× 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 PGF1α to determine whether new enzyme synthesis was necessary for 6-keto PGF1α 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 PGF1α.
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.
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 PGF1α 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 PGF1α 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.
Endothelial cells stimulated with A23187 released significantly more nitrite, 6-keto PGF1α, and TxB2 than the control cells. The release of nitrite as well as 6-keto PGF1α 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 PGF1α, 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 PGF1α, 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 PGF1α and TxB2 production (Table 1⇑). These data indicate that the effect of L-NAME on 6-keto PGF1α 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 PGF1α 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 PGF1α 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 PGF1α 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 PGF1α 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.
Fig 1⇑, right, depicts the accumulation 6-keto PGF1α 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 PGF1α 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 PGF1α, 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 PGF1α at each time point. The basal production of 6-keto PGF1α was not significantly affected by L-NAME.
Exogenous NO Increased 6-Keto PGF1α 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 PGF1α 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 PGF1α production (data not shown).
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 PGF1α 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 PGF1α 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 PGF1α 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.
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 PGF1α 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.
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.
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.
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 PGF1α 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 PGF1α 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 PGF1α 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 PGF1α 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 PGF1α 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 PGF1α in a manner similar to that of cells exposed to only A23187 (3893±723 versus 4316±562 pg/106 cells for 24 hours).
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 PGF1α 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 PGF1α 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 PGF1α 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 PGF1α and TxB2 production was due to NO. In addition, exogenous NO donors that act by spontaneous release of NO36 also stimulated 6-keto PGF1α production from the endothelial cells. This stimulated 6-keto PGF1α production by exogenous NO persisted in the presence of L-NAME, which further indicates that L-NAME does not affect 6-keto PGF1α production through non–NO-mediated mechanisms.
Furthermore, protecting NO from degradation by superoxide anions increased 6-keto PGF1α production. Conversely, the NO scavenger hemoglobin reduced 6-keto PGF1α 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 PGF1α 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 PGF1α production even in the presence of excess arachidonic acid. Furthermore, L-NAME completely inhibited the additional increase of 6-keto PGF1α 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 PGF1α production, and there was a rapid elevation of 6-keto PGF1α after NO stimulation. Furthermore, acetylating and inactivating a preexisting enzyme with acetylsalicylic acid prevented NO-stimulated 6-keto PGF1α 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 PGF1α production. Nitrite production followed a time course similar to that of 6-keto PGF1α production from 0 to 8 hours. However, from 8 to 24 hours nitrite accumulation continued, whereas 6-keto PGF1α 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 PGF1α 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 PGF1α production. In contrast, LY83583 had no effect on 6-keto PGF1α 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 PGF1α 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 PGF1α 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
|6-keto PGF1α||=||6-ketoprostaglandin F1α|
|L-NAME||=||N-nitro-l-arginine methyl ester|
|PGHS-1||=||constitutive PGH synthase|
|PGHS-2||=||inducible PGH synthase|
|TTBS||=||TBS plus 1% Tween-20|
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.
- © 1995 American Heart Association, Inc.
Vanhoutte PM. Role of calcium and endothelium in hypertension, cardiovascular disease, and subsequent vascular events. J Cardiovasc Pharmacol. 1992;19:S6-S10.
Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994;298:249-258.
Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992;6:3051-3064.
Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci U S A. 1992;89:7384-7388.
Hemler ME, Lands WEM. Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis. J Biol Chem. 1980;255:6253-6261.
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.
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.
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.
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.
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.
Davidge ST, Hubel CA, McLaughlin MK. Cyclooxygenase-dependent vasoconstrictor alters vascular function in the vitamin E–deprived rat. Circ Res. 1993;73:79-88.
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.
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.
Raz A, Wyche A, Siegel N, Needleman P. Regulation of fibroblast cyclooxygenase synthesis by interleukin-1. J Biol Chem. 1988;263:3022-3028.
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.
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.
Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;277:680-685.
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.
Rao GN, Berk BC. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ Res. 1992;70:593-599.
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.
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.