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CARDIOVASCULAR

The Inducible Nitric-Oxide Synthase Modulates Endothelin-1-Dependent Release of Prostacyclin and Inhibition of Platelet Aggregation ex Vivo in the Mouse

Department of Pharmacology, Institut de Pharmacologie de Sherbrooke (E.C., I.B., P.D.-J.) and Division of Rheumatology (A.J.dB.-F.), School of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada

Received May 14, 2007; accepted September 19, 2007.

	Abstract

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Nitric oxide and other reactive oxygen species generated by nitric-oxide synthases (NOS) modulate, among several other cellular responses, the production of eicosanoids and platelet aggregation. The roles of specific NOS in these two phenomena remain to be determined. Thus, the present study assessed whether inducible NOS (iNOS) and endothelial NOS (eNOS) modulate in a similar manner the production of eicosanoids and platelet aggregation. Mice knocked out for eNOS (eNOS–/–) or iNOS (iNOS–/–) and their wild-type (WT) congeners were used to analyze agonist-induced increases in plasma levels of eicosanoids as well as inhibition of platelet aggregation ex vivo. Systemically administered endothelin-1 (ET-1) triggered an increase in plasma levels of 6-keto prostaglandin F₁ (6-keto PGF₁) in WT and eNOS–/– but not in iNOS–/– mice. ET-1 (0.01–1 nmol/kg) also induced a dose-dependent inhibition of platelet aggregation in WT and eNOS–/– but not in iNOS–/– mice. Another agonist, bradykinin (10 nmol/kg), triggered the release of 6-keto PGF₁ and inhibited platelet aggregation in all strains of mice studied. In addition, ADP-induced platelet aggregation in vitro was similarly reduced by iloprost (100 nM) in iNOS–/– mice and WT congeners. In another series of experiments, ET-1 (0.1 nmol/kg) significantly increased 8-isoprostane plasma levels in WT but not in iNOS–/– mice. Finally, a 3-week treatment with anti-oxidants inhibited the capacity of ET-1 to significantly increase plasma 6-keto PGF₁ in WT mice. We show for the first time that iNOS is involved in the control of ET-1-induced prostacyclin release and related inhibition of platelet aggregation in the murine model.

Another labile factor, NO, has also been shown to interfere with platelet function via cGMP-dependent mechanisms (Hogan et al., 1988). NO can be generated from several cellular sources, via one or the other of three nitric-oxide synthases [neuronal (nNOS), inducible (iNOS), endothelial (eNOS)].

A third endothelial-derived factor, endothelin-1 (ET-1) (Yanagisawa et al., 1988), when injected systemically triggers indomethacin-sensitive inhibition of platelet aggregation ex vivo in both the rabbit and canine model (Filep et al., 1991; McMurdo et al., 1993). More recently, in a mouse model, we reported that ET-1-induced antiplatelet properties are COX-2 and prostacyclin synthase-dependent, whereas the same effects afforded by bradykinin (BK) require the dual contributions of COX-2 and NOS (Labonté et al., 2001). Finally, the antiplatelet properties of ET-1 in the mouse model were found to be solely dependent on ET_B receptor activation (Labonté et al., 2001).

Interestingly, an increasing body of evidence supports a regulation of eicosanoid production by nitric oxide or NO-derived molecular species, such as peroxinitrites and superoxide anions. Marnett and coworkers reported that targeted deletion of iNOS in the mouse causes a decrease in the urinary levels of prostaglandin E₂ and F2-isoprostane (Marnett et al., 2000). The same study also reported that serum thromboxane B₂ (TxB₂) levels increased in iNOS–/– mice, suggesting that NO or other oxidant derivatives generated by the inducible isoform play significant roles in the genesis of AA metabolites. It has also been suggested that a constitutively expressed COX-2 in murine pulmonary tissues is required for protease-activated receptor-1 and protease-activated receptor-2-induced release of prostanoids (Kawabata et al., 2004). Finally, mouse pulmonary alveolar epithelial cells release basally prostaglandin E₂ mainly via COX-2 activity (Lama et al., 2002).

Thus, considering the above-mentioned studies, it is suggested that both physiologically functional iNOS and COX-2 are involved in the modulation of eicosanoid in the murine model. However, whether iNOS and eNOS act in a similar manner as modulators of AA metabolite production remains to be investigated. Therefore, the main goal of the present study was to compare the contribution of iNOS or eNOS in endothelin-1-induced production of PGI₂, as well as in the anti-platelet properties of the 21 amino acid peptide in the mouse model. Furthermore, we attempted to correlate the PGI₂ and reactive oxygen species-releasing properties of ET-1 by two approaches, namely, via an antioxidant regime and by monitoring the production of 8-isoprostane as a marker of oxidative stress (Morrow et al., 1992; Janssen, 2001). Overall, our results suggest a dynamic role for the inducible NOS in the prostacyclin-dependent modulation of platelet aggregation afforded by ET-1.

	Materials and Methods

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Animals
Wild-type (WT) mice (males and females, 16–19 weeks old) C57BL/6J were purchased from Charles River (Montreal, QC, Canada). Breeding couples of homozygous iNOS–/– (Laubach et al., 1995) or eNOS–/– (Shesely et al., 1996) mice, with the same genetic background, were obtained from The Jackson Laboratory (Bar Harbor, ME). All animals were kept at constant room temperature (±23°C) and humidity (78%) under a controlled light/dark cycle (6:00 AM–6:00 PM). Animal care and experiments were approved by the Ethic Committee on Animal Research of the Université de Sherbrooke.

Blood Platelet Counts and Hematocrit Measurement
The mouse was restrained, and a distal 1-cm segment of tail was subsequently cut with a scalpel blade. A capillary was then filled with whole blood and transferred to a Unopette micro collection system (Fisher Scientific, Nepean, ON, Canada) for red blood cell lysis. Afterward, the lysate was carefully charged in a Neubauer hemocytometer for blood platelet counts (400x magnification). The experiment was repeated at least twice for each animal.

For hematocrit monitoring, blood was collected in two separate heparinized microhematocrit capillary tubes and centrifuged in a microcapillary centrifuge model MB (International Equipment Co., Needham, MA).

Western Immunoblotting
WT, iNOS–/– or eNOS–/– mice were euthanized by cervical dislocation, and the heart, kidneys, lungs, and skeletal muscle of the left rear paw were removed.

Total proteins were extracted from organs and the protein concentration was assessed by the Bradford method. Samples were then applied on a 10% SDS-polyacrylamide gel (COX-1, 30 µg; COX-2, 40 µg; and PGI₂S, 30 µg) for protein separation. Proteins were then transferred on a polyvinylidene difluoride membrane (GE Healthcare, Baie D'Urfé, QC, Canada), which was blocked with 8% low fat dry milk. Thereafter, the membrane was incubated with the primary antibody and subsequently with horseradish-peroxidase-conjugated antibody. Primary antibodies for COX-1, COX-2, and PGI₂S were purchased from Cayman Chemical (Hornby, ON, Canada); those for actin were from Sigma (Oakville, ON, Canada); and secondary antibodies have been obtained from GE Healthcare. The visualization of the immunoreactive proteins was performed using the chemiluminescent detection reagent ECL Plus from GE Healthcare.

Ex Vivo Platelet Aggregation Study
The animals were anesthetized with a mixture of ketamine (87 mg/kg)/xylazine (13 mg/kg) injected intramuscularly. The left jugular vein and the right carotid artery were cannulated using a polyethylene-10 catheter, the former serving for i.v. injection and the latter for blood pressure monitoring and blood sampling. Changes in mean arterial blood pressure (MAP) (expressed in changes from basal values), under the anesthesia regime described above, were detected with a Statham pressure transducer (Statham Transducers Inc., Plato Rey, Puerto Rico) and recorded on a polygraph (model 79; Grass Instruments, Quincy, MA). After a 15-min stabilization, vehicle (phosphate-buffered saline; PBS), ET-1 (0.01–1 nmol/kg), or BK (10 nmol/kg) were administered i.v., and the responses were monitored for 5 min before blood sampling. The blood of two mice was pooled to obtain a volume of 1.5 ml in Eppendorf tubes with heparin (15 USP U/ml). The platelet-rich plasma and the platelet-poor plasma were obtained by centrifugation of the blood sample, and the platelet aggregation was monitored using an aggregometer model 490-2D (Chrono-Log Corporation, Havertown, PA), as described previously by our laboratory (Labonté et al., 2001). Induction of platelet aggregation was performed using ADP at a final concentration of 5 to 5.5 µM in WT (C57BL/6J), eNOS–/–, and iNOS–/– mice to attain approximately 50% platelet aggregation. Separate groups of iNOS–/– and WT mice were treated with diethylenetriamine/NO (DETA/NO) (Sigma) 6 h before injection of vehicle (PBS) or ET-1 (0.1 nmol/kg). Mice were injected intraperitoneally with a dose of 51.7 mg/kg DETA/NO dissolved in saline 0.9%. This dose has been reported to be efficient to induce release of nitric oxide in the same strains of mice used in the present study (Cerwinka et al., 2002). DETA/NO is a metabolically stable NO donor that scarcely increases plasma levels of oxidative product of NO (Yamamoto and Bing, 2000).

In Vitro Platelet Aggregation Study
Blood was collected from WT, iNOS–/–, or eNOS–/– mice according to the protocol described in the previous section, with the exception that heparin was replaced by sodium citrate 3.8% (w/v) at a ratio 9:1 as anticoagulant. Platelet-rich plasma and platelet-poor plasma were then obtained as described in the above-mentioned section. After a 1-min stabilization period in the aggregometer to allow baseline setting, 100 nM iloprost (Cayman Chemical), a stable analog of prostacyclin, or vehicle (saline 0.9%) was administered 2 min before the addition of 10 µM ADP.

Plasma Levels of Prostacyclin and Thromboxane A₂
Blood samples of 500 µl [sodium citrate 0.35% (w/v)] were collected 30 s after i.v. injection of vehicle (PBS), ET-1 (0.1 nmol/kg), or BK (10 nmol/kg), resulting in 200 µl of plasma postcentrifugation. Each sample was then purified and concentrated as described previously (Gratton et al., 1997) using Amprep ethyl C2 mini-columns (100 mg; GE Healthcare). Samples were eluted with 1.5 ml of a mixture of 60% acetonitrile and 0.1% trifluoroacetic acid, and they were dried (SC110A SpeedVac Plus; ThermoSavant, Holbrook, NY) for final reconstitution in 150 µl of enzyme immunoassay (EIA) kit buffer. The stable metabolites of either TxA₂ (TxB₂) or PGI₂ (6-keto prostaglandin F₁; 6-keto PGF₁) were quantified using respective EIA kits (Cayman Chemical). Unlike for TxB₂ monitoring, 6-keto PGF₁ was assayed without purification procedures.

Antioxidant Treatment in Wild-Type Mice
WT mice were treated with vehicle or a combination of a superoxide dismutase mimetic [tempol (4-hydroxy-tempo): 1 mM; Sigma] and a NADPH oxidase inhibitor [apocynin (4'-hydroxy-3'-methoxyacetophenone): 1.5 mM; Sigma] in drinking water for 3 weeks. These concentrations in drinking water have previously been shown to be efficient in reducing endothelin-1-induced superoxide anion production in rats (Elmarakby et al., 2005). Mice were subsequently tested for ET-1-induced increases of plasma level of proctacyclin as described above.

Monitoring of Total Plasma 8-Isoprostane
Plasma levels of total 8-isoprostane in WT or iNOS KO mice were assessed by an EIA kit from Cayman Chemical. The procedure for blood sampling was similar to that detailed above for prostacyclin and thromboxane A₂ monitoring following administration of PBS or ET-1 (0.1 nmol/kg). However, for the isoprostane protocol, 750-µl blood volumes were collected to obtain 300 µl of postcentrifugation plasma in which 0.005% (w/v) butylated hydroxytoluene was added before freezing. Sample purification was done following the recommendations of the manufacturer, using solid-phase extraction (SPE C18; 500 mg) cartridges (Waters, Mississauga, ON, Canada). Samples were eluted using 5 ml of ethyl acetate containing 1% methanol, evaporated under a gentle stream of nitrogen, and resuspended in 250 µl of EIA buffer to be thereafter assayed using the EIA kit.

Monitoring of Plasma Free Nitrotyrosines
Basal-free nitrotyrosines, an indicator of peroxynitrite levels, were determined in plasma applied directly on a 96-well EIA kit (Cayman Chemical) (Feng et al., 2001).

Monitoring of Nitrates and Nitrites
Total nitrates/nitrites were measured using nitrates/nitrites colorimetric assay kits based on the Griess reaction (Cayman Chemical), in accordance with the manufacturer's recommendations.

Chemicals
ADP was obtained either from Chrono-Log Corporation or Sigma, and it was reconstituted in sterile irrigation grade 0.9% saline. ET-1 (Peptides International Inc., Louisville, KY) and BK (synthesized in our laboratory) were reconstituted and diluted with PBS.

Statistical Analyses
Data are expressed as mean ± S.E.M. of n experiments. Statistical analyses were performed by Student's t test (***, p < 0.001; **, p < 0.01, and *, p < 0.05).

	Results

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Basal Parameters in WT, iNOS–/–, and eNOS–/– Mice. Table 1 summarizes several basal parameters measured from plasma and blood of WT, iNOS–/–, and eNOS–/– mice. No differences were found in hematocrit in the three strains of mice, whereas blood platelet counts were similar in WT and iNOS–/– mice, yet they were reduced in eNOS–/– mice.

In contrast, reductions in plasma levels of nitrates/nitrites and free nitrotyrosines were found in iNOS–/– mice, but not eNOS–/– mice compared with WT littermates.

Finally, the knockout of either iNOS or eNOS genes in mice induced a significant increase in the basal plasma levels of 6-keto PGF₁ and TxB₂, stable metabolites of PGI₂ and TxA₂ respectively, compared with WT mice.

Lack of Regulation of COX-1, COX-2, and PGI₂ Synthase Protein Levels by iNOS- and eNOS-Derived NO. In this series of experiments, we assessed whether an up-regulation in COX-1, COX-2, or PGI₂ synthase protein levels was responsible for the increase in plasma levels of 6-keto PGF₁ and TxB₂ observed in iNOS–/– and eNOS–/– mice. We performed Western immunoblotting for each enzyme in cardiac or pulmonary homogenates derived from the three mouse strains.

Interestingly, the COX-2 protein was present in all organs studied (Fig. 1) in the three strains of mice. In addition, our results show that the protein levels of COX-1, COX-2, or PGI₂ synthase were not affected by the knockout of iNOS or eNOS genes in the heart, the lungs (Fig. 1), the kidneys, and in the skeletal muscle (data not shown) of these mice. The Western immunoblots results also correlate with a lack of regulation of COX-1 and COX-2 mRNA expression in the four organs studied from iNOS–/– and eNOS–/– mice compared with WT congeners (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Protein levels of COX-1, COX-2, and PGI2S in the heart and lungs derived from WT or iNOS–/– mice (A) and WT or eNOS–/– mice (B) (results are representative of six different mice of each strain). Independent experiments have been performed to compare iNOS–/– versus WT mice and eNOS–/– versus WT mice.

1

ET-1 (0.1 nmol/kg i.v.) induced a significant increase in the release of 6-keto PGF₁ in eNOS–/– or WT mice, but interestingly, not in iNOS–/– mice (Fig. 2A). In addition, ET-1 (0.1 nmol/kg i.v.) induces an increase in plasma levels of TxB₂ in both knockout animals, but not in their WT congeners (Fig. 2A).

View larger version (35K): [in this window] [in a new window]   Fig. 2. Variation () in plasma concentrations (picograms per milliliter) of 6-keto PGF1 and TxB2 following intravenous administration of ET-1 (0.1 nmol/kg) (A) or BK (10 nmol/kg) (B) in WT, iNOS–/–, and eNOS–/– mice. n = 6 to 9 for each treatment in each type of mice. ***, p < 0.001; **, p < 0.01; *, p < 0.05 versus vehicle (PBS) treatment (basal levels illustrated in Table 1).

1

Endothelin-1-Induced Inhibition of ex Vivo Platelet Aggregation in WT, iNOS–/–, and eNOS–/– Mice. We have previously shown that ET-1-induced inhibition of platelet aggregation is COX-2-dependent (Labonté et al., 2001). In this series of experiments, the capacity of ET-1 to stimulate the release of prostacyclin (Fig. 2A) was correlated with the antiplatelet properties of the peptide in iNOS–/–, eNOS–/–, and WT mice.

Figure 3 represents a typical experiment showing the inability of ET-1 (0.1 nmol/kg) to reduce ADP-induced platelet aggregation ex vivo in iNOS–/– mice (Fig. 3C), in contrast to the inhibitory response afforded by the same peptide in WT congeners (Fig. 3B).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Typical tracings of ADP-induced platelet aggregation following i.v. administration of vehicle (PBS) (A) or ET-1 (0.1 nmol/kg) in WT (B) or in iNOS–/– (C) mice. These tracings are representative of seven or eight different experiments.

View larger version (47K): [in this window] [in a new window]   Fig. 4. A, effects of the intravenous administration of the vehicle (PBS), ET-1 (0.01–1 nmol/kg), and BK (10 nmol/kg) on platelet aggregation ex vivo in WT (), iNOS–/– (), and eNOS–/– () mice. Results are expressed in percentage of platelet aggregation (n = 5–8 for each treatment in each type of mice). B, maximum variation () of MAP (mm Hg) of PBS, ET-1, and BK in these same strains of mice (n = 10–16 for each treatment in each type of mice). Basal MAP averaged for each mouse strain (n = 62–77) under anesthesia (WT, 60.54 ± 0.75; iNOS–/–, 66.81 ± 0.93, p < 0.001; eNOS–/–, 90.42 ± 1.83 mm Hg, p < 0.001 versus WT mice). Each bar represents the mean ± S.E.M. ***, p < 0.001; **, p < 0.01; *, p < 0.05 versus vehicle treatment; , p < 0.01 versus the same dose in WT mice.

Effects of an Exogenously Administered NO Donor in iNOS–/– Mice. In this series of experiments, we assessed whether a nitric oxide donor (e.g., DETA/NO) could restore the ET-1-induced inhibition of platelet aggregation in iNOS–/– mice. Thus, platelet aggregation following i.v. injection of vehicle (PBS) or ET-1 (0.1 nmol/kg) was monitored in WT and iNOS–/– mice treated previously with DETA/NO. This NO donor did not modify the ET-1-induced inhibition of platelet aggregation observed in WT mice (DETA/NO + PBS, 52.8 ± 0.80%; DETA/NO + ET-1, 35 ± 2.06%; p < 0.001 versus PBS injection), nor did it restore the inhibitory properties of the same peptide in iNOS–/– mice (DETA/NO + PBS, 52.4 ± 3.88%; DETA/NO + ET-1, 49 ± 2.85%; p < 0.01 versus ET-1 in WT mice) (n = 4–5). Finally, DETA/NO did not alter basal MAP in iNOS–/– or WT mice used in the present study (results not shown), in accordance with Cerwinka et al. (2002).

ET-1-Dependent Release of Reactive Oxygen Species in the Mouse Model. In a separate series of experiments, ET-1 (0.1 nmol/kg) triggered a significant increase in 8-isoprostane plasma levels in WT mice (PBS, 179.11 ± 20.55; + ET-1, 267.63 ± 13.51 pg/ml plasma; p < 0.01 versus PBS; n = 6–7), but not in iNOS–/– mice (PBS, 277.85 ± 30.52; + ET-1, 351.31 ± 23.58 pg/ml plasma; n = 7). Worthy of notice, basal levels of 8-isoprostane were significantly higher in iNOS–/– mice compared with WT congeners (p < 0.05; n = 6–7).

Furthermore, a 3-week pretreatment with apocynin and tempol enhanced basal plasma levels of 6-keto PGF₁ in WT mice (220.28 ± 30.90 pg/ml plasma; p < 0.05, n = 6), compared with nontreated animals (Table 1). Interestingly, ET-1 (0.1 nmol/kg) did not significantly increase plasma levels of 6-keto PGF₁ (275.96 ± 19.14 pg/ml plasma; n = 6), in tempol/apocynin-treated animals compared with vehicle-administered mice subjected to the same antioxidant regime.

In Vitro Inhibition of Platelet Aggregation by a Prostacyclin Analog. In this last series of experiments, we compared the in vitro responsiveness of platelets derived from WT, eNOS–/–, and iNOS–/– mice to a prostacyclin analog iloprost (100 nM). Figure 5 shows that iloprost equally reduced ADP-induced platelet aggregation in vitro in iNOS–/– and WT mice, whereas the inhibitory properties of the prostacyclin analog were potentiated significantly in platelets derived from eNOS–/– mice compared with those from WT mice.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Percentage of inhibition of ADP-induced platelet aggregation, obtained following injection of iloprost (100 nM), an analog of prostacyclin, in vitro in the platelet-rich plasma of WT, iNOS–/–, or eNOS–/– mice. n = 5 for each type of mouse. *, p < 0.05 versus WT mice.

	Discussion

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The ET-1-specific intracellular mechanisms involved in the iNOS-PGI₂ cross talk, proposed in the present study, have yet to be fully elucidated. Interestingly, it has been reported that ET-1 stimulates the production of superoxide anions () in vascular cells as well as in carotid arteries in the rat model (Li et al., 2003; Pollock and Pollock, 2005). Furthermore, regulates COX-2 by increasing the expression of the enzyme (von Knethen et al., 1999; Martinez et al., 2000), whereas the iNOS-derived NO enhances the activity of the enzyme via cGMP-independent pathway mechanisms (Shinmura et al., 2002). Our results show that plasma levels of 8-isoprostane were significantly enhanced by ET-1 in WT, but not in iNOS–/– mice, irrespective of enhanced basal levels of the oxidated eicosanoid reported here in the latter strain, as also previously shown in eNOS–/– mice (Shi et al., 2002). Furthermore, an antioxidant regime with tempol and apocynin (Elmarakby et al., 2005) reduced the capacity of ET-1 to stimulate plasma increase of 6-keto PGF₁ in WT congeners. These two series of results suggest that ET-1 enhances the production of ROS in the wild-type mouse, as shown previously in the rat model (Elmarakby et al., 2005). Our data also highlight the contribution of ROS in the ET-1-induced production of PGI₂ in WT mice.

In the present study, protein levels of COX-2 and prostacyclin synthase were also shown to be unaltered by the repression of the inducible NOS gene. Considering that we have previously shown that ET-1-dependent inhibition of ADP-induced platelet aggregation is fully reversed by either COX-2 (NS-398) or prostacyclin synthase (tranylcypromine) inhibitors (Labonté et al., 2001), we suggest that the lack of PGI₂-releasing properties of ET-1 in iNOS–/– mice is caused by a reduction in COX-2 or PGI₂ synthase activity per se rather than by changes in protein levels of these two enzymes. This state of events would be due to a diminished capacity of the 21-amino acid peptide to generate ROS, such as in iNOS–/– mice. It is established that NO, generated by the inducible NOS, reacts with to produce peroxynitrites, a known signaling molecule (Faraci, 2006). The predominant role of ROS rather than NO itself was also confirmed by the fact that DETA/NO did not correct the repression of ET-1-induced inhibition of platelet aggregation ex vivo observed in iNOS–/– mice.

Finally, the prostacyclin receptor agonist iloprost (Armstrong et al., 1989; Costantini et al., 1990) reversed ADP-induced platelet aggregation in vitro in WT, iNOS–/–, and eNOS–/– mice. This last result suggests that cAMP-dependent inhibition of aggregation following direct PGI₂ receptor stimulation (Krause and Krais, 1986) in mouse platelets is left intact in iNOS–/– or eNOS–/– mice compared with WT congeners. This last series of experiments allows us to confirm that the repression of the ET-1-induced inhibition of platelet aggregation in iNOS–/– mice can clearly be associated with the lost capacity of the peptide to increase plasma PGI₂ rather than a modulation of platelet reactivity to the stimulation of prostacyclin receptors.

On a physiological point of view, several studies have recently confirmed the pivotal role of COX-2 in the production of PGI₂ in humans (Stichtenoth et al., 2005; Grosser, 2006). Likewise, the ex vivo platelet inhibition afforded by endothelin-1 in the murine model has been previously shown, by our group, to be also dependent on COX-2-derived prostacyclin synthesis (Labonté et al., 2001). Whether these COX-2-dependent antiplatelet responses occurring in the mouse model can be translated to human needs to be confirmed. A recent review (Flavahan, 2007), for example, exerts some caution on this latter issue by arguing for a predominant contribution of the COX-1 isoform in the production of prostacyclin in human subjects.

Worthy of notice, the potential role of the neuronal isoform of NOS in our ex vivo platelet aggregation model has not been investigated. Among other studies, the platelet-derived growth factor has been shown to up-regulate nNOS expression in isolated blood vessels of WT, eNOS–/–, and iNOS–/– but not nNOS–/– mice (Nakata et al., 2005). In addition, neuronal NOS-dependent compensatory mechanisms on coronary blood flow in eNOS–/– mice have also been reported (Huang et al., 2002). However, this type of nNOS-dependent compensation in iNOS–/– mice is, to our knowledge, yet to be explored.

In summary, our results support the concept that the inducible NOS plays a pivotal role in the prostacyclin-releasing and antiplatelet properties of endothelin-1 in the mouse. Furthermore, our study provides new insight on the putative cross talk between the inducible NOS and cyclooxygenase-dependent mechanisms in cardiovascular homeostasis.

	Acknowledgements

 
We gratefully acknowledge Helene Morin for secretarial assistance and Dr. Alain Cadieux for logistical support.

	Footnotes

 
This work was supported by Canadian Institutes for Health Research Grant MOP-57764. P.D.-J. and A.J.dB.-F. are National and Senior Scholars of the Fonds de la Recherche en Santé du Québec (FRSQ), respectively. E.C. is in receipt of a FRSQ Studentship.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.125690.

ABBREVIATIONS: AA, arachidonic acid; PGI₂, prostacyclin; TxA₂, thromboxane A₂; WT, wild-type; COX, cyclooxygenase; PGI₂S, prostacyclin synthase; NOS, nitric-oxide synthase(s); nNOS, neuronal nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; eNOS, endothelial nitricoxide synthase; ET-1, endothelin-1; MAP, mean arterial blood pressure; PBS, phosphate-buffered saline; BK, bradykinin; DET/NO, diethylenetriamine/nitric oxide; EIA, enzyme immunoassay; 6-keto PGF₁, 6-keto prostaglandin F₁; , superoxide anions.

Address correspondence to: Dr. Pedro D'Orléans-Juste, Department of Pharmacology, School of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, QC J1H 5N4, Canada. E-mail: labpdj{at}usherbrooke.ca

	References

Top Abstract Materials and Methods Results Discussion References

 

Armstrong RA, Lawrence RA, Jones RL, Wilson NH, and Collier A (1989) Functional and ligand binding studies suggest heterogeneity of platelet prostacyclin receptors. Br J Pharmacol 97: 657–668.[Medline]

Brandes RP, Schmitz-Winnenthal FH, Feletou M, Godecke A, Huang PL, Vanhoutte PM, Fleming I, and Busse R (2000) An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice. Proc Natl Acad SciUSA 97: 9747–9752.[Abstract/Free Full Text]

Cerwinka WH, Cooper D, Krieglstein CF, Feelisch M, and Granger DN (2002) Nitric oxide modulates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules. Am J Physiol 282: H1111–H1117.

Costantini V, Fuschiotti P, Giampietri A, Allegrucci M, Agnelli G, Nenci GG, and Fioretti MC (1990) Effects of a stable prostacyclin analogue on platelet activity and on host immunocompetence in mice. Prostaglandins 39: 581–599.[CrossRef][Medline]

Elmarakby AA, Loomis ED, Pollock JS, and Pollock DM (2005) NADPH oxidase inhibition attenuates oxidative stress but not hypertension produced by chronic ET-1. Hypertension 45: 283–287.[Abstract/Free Full Text]

Faraci FM (2006) Reactive oxygen species: influence on cerebral vascular tone. J Appl Physiol 100: 739–743.[Abstract/Free Full Text]

Feng Q, Lu X, Jones DL, Shen J, and Arnold JM (2001) Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice. Circulation 104: 700–704.[Abstract/Free Full Text]

Filep JG, Herman F, Battistini B, Chabrier PE, Braquet P, and Sirois P (1991) Antiaggregatory and hypotensive effects of endothelin-1 in beagle dogs: role for prostacyclin. J Cardiovasc Pharmacol 17 (Suppl 7): S216–S218.[Medline]

Flavahan NA (2007) Balancing prostanoid activity in the human vascular system. Trends Pharmacol Sci 28: 106–110.[CrossRef][Medline]

Gratton JP, Cournoyer G, Loffler BM, Sirois P, and D'Orleans-Juste P (1997) ET(B) receptor and nitric oxide synthase blockade induce BQ-123-sensitive pressor effects in the rabbit. Hypertension 30: 1204–1209.[Abstract/Free Full Text]

Grosser T (2006) The pharmacology of selective inhibition of COX-2. Thromb Hae-most 96: 393–400.[Medline]

Hamberg M, Svensson J, and Samuelsson B (1975) Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad SciUSA 72: 2994–2998.[Abstract/Free Full Text]

Hogan JC, Lewis MJ, and Henderson AH (1988) In vivo EDRF activity influences platelet function. Br J Pharmacol 94: 1020–1022.[Medline]

Huang A, Sun D, Shesely EG, Levee EM, Koller A, and Kaley G (2002) Neuronal NOS-dependent dilation to flow in coronary arteries of male eNOS-KO mice. Am J Physiol 282: H429–H436.

Janssen LJ (2001) Isoprostanes: an overview and putative roles in pulmonary pathophysiology. Am J Physiol 280: L1067–L1082.

Kawabata A, Kubo S, Ishiki T, Kawao N, Sekiguchi F, Kuroda R, Hollenberg MD, Kanke T, and Saito N (2004) Proteinase-activated receptor-2-mediated relaxation in mouse tracheal and bronchial smooth muscle: signal transduction mechanisms and distinct agonist sensitivity. J Pharmacol Exp Ther 311: 402–410.[Abstract/Free Full Text]

Krause W and Krais T (1986) Pharmacokinetics and pharmacodynamics of the prostacyclin analogue iloprost in man. Eur J Clin Pharmacol 30: 61–68.[CrossRef][Medline]

Labonté J, Brochu I, Honore JC, and D'Orleans-Juste P (2001) Role of ET_B and B₂ receptors in the ex vivo platelet inhibitory properties of endothelin and bradykinin in the mouse. Br J Pharmacol 132: 934–940.[CrossRef][Medline]

Lama V, Moore BB, Christensen P, Toews GB, and Peters-Golden M (2002) Prostaglandin E₂ synthesis and suppression of fibroblast proliferation by alveolar epithelial cells is cyclooxygenase-2-dependent. Am J Respir Cell Mol Biol 27: 752–758.[Abstract/Free Full Text]

Laubach VE, Shesely EG, Smithies O, and Sherman PA (1995) Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad SciUSA 92: 10688–10692.[Abstract/Free Full Text]

Li L, Fink GD, Watts SW, Northcott CA, Galligan JJ, Pagano PJ, and Chen AF (2003) Endothelin-1 increases vascular superoxide via endothelin(A)-NADPH oxidase pathway in low-renin hypertension. Circulation 107: 1053–1058.[Abstract/Free Full Text]

Marnett LJ, Wright TL, Crews BC, Tannenbaum SR, and Morrow JD (2000) Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase. J Biol Chem 275: 13427–13430.[Abstract/Free Full Text]

Martinez J, Sanchez T, and Moreno JJ (2000) Regulation of prostaglandin E₂ production by the superoxide radical and nitric oxide in mouse peritoneal macrophages. Free Radic Res 32: 303–311.[CrossRef][Medline]

Mattera GG, Catalioto RM, Criscuoli M, and Subissi A (1994) Endothelins induce prostacyclin release in both vascular and non-vascular tissue. Naunyn Schmiedebergs Arch Pharmacol 350: 410–415.[Medline]

McMurdo L, Lidbury PS, Thiemermann C, and Vane JR (1993) Mediation of endothelin-1-induced inhibition of platelet aggregation via the ET_B receptor. Br J Pharmacol 109: 530–534.[Medline]

Moncada S, Higgs EA, and Vane JR (1977) Human arterial and venous tissues generate prostacyclin (prostaglandin x), a potent inhibitor of platelet aggregation. Lancet 1: 18–20.[CrossRef][Medline]

Morrow JD, Awad JA, Boss HJ, Blair IA, and Roberts LJ 2nd (1992) Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids. Proc Natl Acad SciUSA 89: 10721–10725.[Abstract/Free Full Text]

Nakata S, Tsutsui M, Shimokawa H, Tamura M, Tasaki H, Morishita T, Suda O, Ueno S, Toyohira Y, Nakashima Y, et al. (2005) Vascular neuronal NO synthase is selectively upregulated by platelet-derived growth factor: involvement of the MEK/ERK pathway. Arterioscler Thromb Vasc Biol 25: 2502–2508.[Abstract/Free Full Text]

Norata GD, Callegari E, Inoue H, and Catapano AL (2004) HDL3 induces cyclooxygenase-2 expression and prostacyclin release in human endothelial cells via a p38 MAPK/CRE-dependent pathway: effects on COX-2/PGI-synthase coupling. Arterioscler Thromb Vasc Biol 24: 871–877.[Abstract/Free Full Text]

Pollock DM and Pollock JS (2005) Endothelin and oxidative stress in the vascular system. Curr Vasc Pharmacol 3: 365–367.[CrossRef][Medline]

Shen RF and Tai HH (1998) Thromboxanes: synthase and receptors. J Biomed Sci 5: 153–172.[CrossRef][Medline]

Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, and Smithies O (1996) Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc Natl Acad SciUSA 93: 13176–13181.[Abstract/Free Full Text]

Shi W, Wang X, Shih DM, Laubach VE, Navab M, and Lusis AJ (2002) Paradoxical reduction of fatty streak formation in mice lacking endothelial nitric oxide synthase. Circulation 105: 2078–2082.[Abstract/Free Full Text]

Shinmura K, Xuan YT, Tang XL, Kodani E, Han H, Zhu Y, and Bolli R (2002) Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning. Circ Res 90: 602–608.[Abstract/Free Full Text]

Stichtenoth DO, Marhauer V, Tsikas D, Gutzki FM, and Frolich JC (2005) Effects of specific COX-2-inhibition on renin release and renal and systemic prostanoid synthesis in healthy volunteers. Kidney Int 68: 2197–2207.[CrossRef][Medline]

von Knethen A, Callsen D, and Brune B (1999) Superoxide attenuates macrophage apoptosis by NF-kappa B and AP-1 activation that promotes cyclooxygenase-2 expression. J Immunol 163: 2858–2866.[Abstract/Free Full Text]

Yamamoto T and Bing RJ (2000) Nitric oxide donors. Proc Soc Exp Biol Med 225: 200–206.[Abstract/Free Full Text]

Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411–415.[CrossRef][Medline]

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