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Am J Physiol Heart Circ Physiol 295: H343-H351, 2008. First published May 9, 2008; doi:10.1152/ajpheart.01350.2007
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The role of eNOS, iNOS, and NF-{kappa}B in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin

Yumei Ye,1,2 Juan D. Martinez,1 Regino J. Perez-Polo,3 Yu Lin,1,2 Barry F. Uretsky,1,2 and Yochai Birnbaum1,2,3

1Department of Internal Medicine, 2Division of Cardiology, and 3Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas

Submitted 18 November 2007 ; accepted in final form 6 May 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS–/– and iNOS–/– mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-{kappa}B dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS–/– and iNOS–/– mice received ATV (10 mg·kg–1·day–1; ATV+) or water alone (ATV–) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-{kappa}B in WT mice. It also increased myocardial COX2 activity. In eNOS–/– mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-{kappa}B was not activated by ATV in the eNOS–/– mice. In the iNOS–/– mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-{kappa}B was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-{kappa}B, dependent. Activation of COX2 is dependent on iNOS.

endothelial nitric oxide synthase; inducible nitric oxide synthase; nuclear factor-{kappa}B

THE 3-HYDROXY-3-METHYLGLUTARYL coenzyme A (HMG-CoA) reductase inhibitors (statins) protect against ischemia-reperfusion injury and, when administered before ischemia (2, 5, 7, 25, 26, 34–36, 43, 44, 46–48, 52, 53) or immediately upon reperfusion (4, 17, 47), limit myocardial infarct size (IS) in various animal models. Several investigators have shown that the activation of endothelial nitric oxide synthase (eNOS) is essential for this protective effect, since nonspecific nitric oxide synthase (NOS) inhibitors blunt the IS-limiting effect of statins (5, 48) and since statins do not reduce IS in eNOS–/– mice (1, 4, 18, 25, 52). However, most of these studies used a particular eNOS–/– line (Harvard). As reported by Sharp et al. (37), there are two distinct lines of eNOS–/– mice: the Harvard line lacks compensatory increases in inducible NOS (iNOS) and has an IS bigger than the corresponding wild-type (WT) mice, and the University of North Carolina line, which has compensatory increases in iNOS expression and an IS smaller than the corresponding WT mice. It has been suggested that iNOS can be protective and compensate for the lack in eNOS and that eNOS may compensate for a lack of iNOS in adenosine-triggered preconditioning (3). Others have suggested that neuronal NOS (nNOS) is upregulated in the University of North Carolina eNOS–/– mice and can restore NOS-dependent vasodilation (13, 30). It has not been demonstrated whether statins limit IS in the University of North Carolina eNOS–/– mouse. Moreover, although there is abundant literature on the pathways of activation of eNOS by statins (21, 28), less is known about the signaling pathways of protection downstream from eNOS. It has been suggested that statins activate the reperfusion injury salvage kinase pathway (4, 17), which appears to be independent of NOS activation (22, 23). On the other hand, we have shown that statins also activate iNOS (2, 7, 34, 53), cytosolic phospholipase A2 (cPLA2) (7, 53), cyclooxygenase 2 (COX2) (2, 7, 53), and the specific prostaglandin synthases, PGI2 synthase (7), PGE2 synthase (7), and PGD2 synthase (54). Blocking either iNOS (7, 34) or COX2 (2, 7) abrogates the IS-limiting effect of atorvastatin (ATV). In the rat, iNOS activates COX2 by S-nitrosylation (2). However, it is unclear whether iNOS is needed to upregulate COX2 expression, since pioglitazone, a peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) agonist, increases COX2 expression and activity without upregulating iNOS (53). It has been shown that NF-{kappa}B mediates the upregulation of iNOS and/or COX2 in delayed ischemic preconditioning (10, 40). However, several studies (14, 15, 32, 33, 41, 49) have suggested that statins suppress NF-{kappa}B activation. Therefore, we asked whether 3-day ATV pretreatment limits IS in the University of North Carolina line of eNOS–/– and iNOS–/– mice. We also asked whether eNOS, iNOS, and NF-{kappa}B are needed for the upregulation and/or activation of COX2 by ATV.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Male C57BL/6 WT, University of North Carolina eNOS–/–, and iNOS–/– mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). The protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch.

Treatment. Mice received a 3-day pretreatment with ATV (10 mg·kg–1·day–1) dissolved in water or water alone, administered by oral gavage once daily. On the fourth day mice underwent coronary artery ligation for 30 min followed by 4 h of reperfusion (IS protocol) (n = 10 in each group), or the mice were euthanized under anesthesia and their hearts were explanted without being subjected to ischemia, rinsed in cold PBS (pH 7.4) containing 0.16 mg/ml heparin to remove red blood cells and clots, frozen in liquid nitrogen, and stored at –80°C for further analyses [immunoblotting, calcium-dependent and -independent NOS activity, 6-keto-PGF1{alpha} levels, COX2 activity, electrophoretic mobility shift assay (EMSA) for NF-{kappa}B, and immunofluorescence for NF-{kappa}B]. There were 4 mice in each group.

Infarct size. On the fourth day mice were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (6 mg/kg), intubated and ventilated (fraction of inspired oxygen = 30%). The rectal temperature was monitored, and body temperature was maintained between 36.7° and 37.3°C throughout the experiment. The chest was opened and the left coronary artery was encircled with a suture and ligated for 30 min. Ischemia was verified by the regional dysfunction and a discoloration of the ischemic zone. Isoflurane (1–2.5% titrated to effect) was added after the beginning of ischemia to maintain anesthesia. At 30 min of ischemia, the snare was released and myocardial reperfusion was verified by the change in the color of the myocardium. Subcutaneous 0.1 mg/kg buprenorphine was administered, the chest was closed, and the mice were recovered from anesthesia. Four hours after reperfusion, the mice were reanesthetized, the coronary artery was reoccluded, Evans blue dye (3%) was injected into the right ventricle, and the mice was euthanized under deep anesthesia (2, 7, 43, 53).

The prespecified exclusion criteria were lack of signs of ischemia during coronary artery ligation, lack of signs of reperfusion after release of the snare, prolonged ventricular arrhythmia with hypotension, and area at risk (AR) ≤10% of the left ventricular weight.

Determination of AR and IS. Hearts were excised and the left ventricle was sliced transversely into six sections. Slices were incubated for 10 min at 37°C in 1% buffered (pH = 7.4) 2,3,5-triphenyltetrazolium chloride (TTC), fixed in a 10% formaldehyde, and photographed to identify the AR (uncolored by the blue dye), the IS (unstained by TTC), and the nonischemic zones (colored by blue dye). The area of AR and IS in each slice was determined by planimetry, converted into percentages of the whole for each slice, and multiplied by the weight of the slice and the results summed to obtain the weight of the myocardial AR and IS (2, 7, 43, 53).

The effect of NF-{kappa}B and JAK inhibitors on the induction of iNOS and COX2 by ATV. WT mice were treated with intraperitoneal ATV (5 mg/kg), vehicle alone (DMSO, 5%), ATV + AG-490 (JAK inhibitor, 40 µg/kg), or ATV + SN50 (NF-{kappa}B inhibitor, 400 µg/kg; n = 4 in each group). Hearts were harvested 8 h later and assessed for iNOS, COX2, I{kappa}B, and β-actin expression in the whole cell lysate and phosphorylated (p)-STAT-1 and lamin B in the nuclear fraction.

Immunoblotting. Myocardial samples from the left ventricular wall were homogenized in radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology) and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant was collected and the total protein concentration was determined using the Lowry protein assay. The protein samples (50 µg) with loading buffer were run in 4–20% Tris·HCl Ready Gel at a 100 V for 2 h until the desired molecular weight bands were separated. After electrophoresis, the gel was equilibrated in transfer buffer containing 25 mM Tris, 193 mM glycine, 0.1% SDS, and 10% methanol, and the proteins were transferred to nitrocellulose membrane. The protein signals were quantified by an image-scanning densitometer, and the strength of each protein signal was normalized to the corresponding β-actin stain signal. Signals for p-STAT-1 in the nuclear fraction were normalized to the corresponding lamin B signal. Data are expressed as a ratio between the protein and the corresponding β-actin or lamin B signal density.

NOS activity. Myocardial samples were homogenized in a buffer containing 25 mM Tris·HCl (PH 7.4), 1 mM EDTA, and 1 mM EGTA, and centrifuged at 10,000 g for 15 min. The supernatant, containing the soluble enzyme iNOS, and the pellet, containing the membrane-bound eNOS and nNOS [calcium dependent NOS (cNOS)], were separated. The pellet was resuspended in homogenization buffer. NOS activity was determined by measuring the conversion of L-[14C]arginine to L-[14C]citrulline using a commercial kit (Cayman Chemical, Ann Arbor, MI). For assessing calcium-dependent NOS (cNOS) activity, CaCl2 was added to the samples. For assessing calcium-independent (ciNOS) activity, CaCl2 was omitted from the solution. NOS activity was defined as counts per minute (cpm) (34).

6-Keto-PGF1{alpha} and PLA2 activity. Myocardial samples of the anterior wall of the left ventricle were perfused with a PBS solution (pH 7.4) containing 0.16 mg/ml heparin to remove red blood cells and clots, homogenized in cold PBS (pH 7.4), and centrifuged. The supernatants were collected and stored on ice. Measurement of 6-keto-PGF1{alpha}, the stable metabolite of prostacyclin, and PLA2 activity were made using immunoassay kits.

COX activity. Myocardial samples of the anterior wall of the left ventricle were perfused and rinsed with 0.05 M Tris buffer (pH 7.4) containing 0.16 mg/ml heparin to remove any red blood cells and clots. Samples were homogenized in 5–10 ml of cold buffer [containing 0.1 M Tris·HCl (pH 7.8) containing 1 mM EDTA] per gram tissue and centrifuged at 10,000 g for 15 min at 4°C, and the supernatant was collected and stored on ice. The COX activity assay kit measures the peroxidase activity of COX, assayed colorimetrically by monitoring the appearance of oxidized N,N,N',N'-tetramethyl-p-phenylenediamaine at 590 nm. Each myocardial sample was tested in triplicate [the first without an inhibitor; the second with DuP-697 (0.286 µM), a specific COX2 inhibitor; and the third with Sc560 (0.314 µM), a specific COX1 inhibitor]. COX1 activity was calculated as the difference between total COX activity in the sample without an inhibitor and the sample with Sc560, and COX2 activity as the difference between total COX activity in the sample without an inhibitor and the sample with DuP-697.

Nuclear extraction. Myocardial samples (0.25 g) were homogenized, mixed with Buffer A Mix [containing 10 mM HEPES (pH 7.9), 10 mM KCl, 10 mM EDTA, 100 mM DTT, protease inhibitor cocktail, and 10% Igepal (Sigma, St. Louis, MO)], homogenized again, incubated for 15 min on ice, and centrifuged at 850 g for 10 min at 4°C. The supernatants were discharged, Buffer A Mix was added again, and the samples were incubated for an additional 15 min on ice and centrifuged at 15,000 g for 3 min at 4°C. The supernatants were discharged, and the pellets were resuspended in 150 µl of Buffer B Mix [containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 10% glycerol, protease inhibitor cocktail, and 10% Igepal]. The tubes were shaken on ice at 200 rpm for 2 h and centrifuged at 15,000 g for 5 min at 4°C, and the supernatants were collected as the nuclear fraction and stored at –80°C until used [modified from Dignam et al. (16)].

EMSA. EMSA was carried out as described elsewhere with some modifications (19). Briefly, the EMSA gel is made with 30% polyacrylamide, 10x Tris borate-EDTA (TBE), 10% ammonium persulfate, H2O, and N,N,N',N'-tetramethylethylenediamine to yield 7.5% gel. Regarding the NF-{kappa}B probes, oligonucleotides encompassing the IgG-{kappa}B enhancer sequence (GGGACTTTCC) were 5' labeled with a 32P-ATP and T4 polynucleotide kinase. Binding reactions with 40 µg of nuclear extracts were performed in a 20 µl volume containing 20,000 cpm of probe, 2 µg of polydeoxyinosinic-deoxycytidylic acid (poly-dI-dC), 10 µl of TK100 buffer [containing 25 mM HEPES (pH 7.9), 20% glycerol, 1 mM EDTA, 100 mM KCl, 2 mM MgCl2, 2 mM DTT, and 2 mM PMSF], and competitor. Nuclear extracts were incubated with the poly-dI-dC on ice for 10 min, and the buffer and probe were then added. Incubation was continued for 20 min at room temperature, after which the reaction mixtures were loaded on the gel in 0.25x TBE buffer (pH 7.2). Gels were dried and exposed to X-ray film. When antibodies were used in EMSA for the immunodepletion/supershift study, nuclear extracts were incubated with the different antibodies for 30 min at 4°C before the addition of the poly-dI-dC.

Immunofluorescence. Myocardial sections were incubated in a blocking solution (containing 1–3% normal serum/5% BSA/0.1% Triton X-100) for 30 min at room temperature and rinsed in PBS. The primary anti-NF-{kappa}B p65 antibodies were centrifuged at 12,000 rpm at 4°C for 2 min and diluted in PBS. The primary antibodies (50–100 µl) were added to each section, and the samples were incubated for 1–24 h at 4°C in a humidified box and washed three times in PBS. The Alexa-secondary antibodies [goat anti-mouse IgG with Alexa 488 (488-GAM) for monoclonal primary antibodies (A-11029, Molecular Probe), and goat anti-rabbit IgG with Alexa 594 (594-GAR) for rabbit polyclonal primary antibodies (A-11037, Molecular Probe)] were centrifuged at 12,000 rpm at 4°C for 2 min and diluted 1:200–1:400 in PBS. The sections were incubated with the Alexa-secondary antibodies in the dark at room temperature for 1 h, washed three times in PBS, incubated in 1 µg/ml 4,6-diamidino-2-phenylindole (1:1,000 dilution) at room temperature for 5 min in the dark, and rinsed with PBS. The sections were dried, Fluoromount-G was added onto the slides, and the slides were covered with coverslips. Slices were stored at 4°C until examined and photographed under the microscope.

Biotin switch assay. S-nitrosylation of COX2 was determined with the biotin switch method, as has been previously described (2). Myocardial samples were homogenized with HEN buffer [25 mM HEPES (pH 7.7)-0.1 mM EDTA-0.01 mM necuproine]. The supernatant containing membrane fragments and the cytosolic protein was recovered. The samples were incubated for 30 min at 4°C with blocking solution containing HEN buffer, 0.1% SDS, and 20 mM N-ethylmaleimide to block free thiols. Lysates were centrifuged at 16,000 g for 10 min at 4°C. Cold acetone was added to precipitate the proteins. The pellets were resuspended in HEN buffer with 1% SDS, with 20 mM sodium ascorbate added to decompose the S-nitrosothiol. The resulting free thiols in the sample were reacted with 0.05 mM biotinylating agent biocytin (MPB) for 30 min at room temperature. The excess MPB was removed by additional protein precipitation in cold acetone. COX2 was immunoprecipitated with anti-COX2 polyclonal antibody. Immunoprecipitates were washed three times with HEN buffer and resuspended in 50 µl of HEN containing Laemmli sample buffer, boiled at 95°C for 5 min, loaded on 10% acrylamide gels, and transferred to nitrocellulose. The biotinylated COX2 protein was detected with horseradish peroxidase-linked streptavidin. All procedures up to biotinylation were performed in the dark. The membranes were stripped with a stripping buffer and blotted again with anti-COX2 antibodies. The signal densities of the biotinylated COX2 and total COX2 were quantified by an image-scanning densitometer, and the ratio of the densities was calculated for each animal.

Materials. ATV was purchased from Pfizer Pharmaceuticals (New York, NY). NOS-activity kit, ELISA kit for 6-keto-PGF1{alpha}, and COX2 activity were purchased from Cayman Chemical. Polyclonal anti-iNOS antibodies were purchased from Cayman Chemical, polyclonal anti-Ser-1177 p-eNOS antibodies from Cell Signaling (Beverly, MA), monoclonal anti-eNOS antibodies and monoclonal anti-COX2 antibodies from BD Bioscience (San Jose, CA), and monoclonal anti-β-actin antibody from Sigma. Anti-NF-{kappa}B antibodies (NF-{kappa}B p65, NF-{kappa}B p50) were purchased from Millipore (Billerica, MA). Anti-lamin B, anti-I{kappa}B, and anti-p-STAT-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). N-(3-malemidylpropionyl) biocytin was purchased from Molecular Probes (Eugene, OR). ImmunoPure streptavidin, horseradish peroxidase conjugated was purchased from Pierce Biotechnology, (Rockford, IL). AG-490 was purchased from Sigma, and SN50 was from Calbiochem (San Diego, CA).

Statistical analysis. Data are presented as means ± SE. The significance level {alpha} is 0.05. Body weight, left ventricular weight, and the size of the AR were compared using ANOVA. Data on IS (as a percentage of the AR) and enzyme expression and activity were compared between the ATV-treated and not-treated groups using t-test or Mann-Whitney rank sum test when appropriate. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Infarct size. Ten mice were included in each group. None of the mice died. Two WT mice treated with ATV were excluded due to lack of signs of ischemia during coronary artery ligation. Body weight, left ventricular weight, and the AR size were comparable between the ATV+ (10 mg·kg–1·day–1 ATV) and ATV– (water alone) mice (Table 1). Oral ATV decreased IS in WT but not in eNOS–/– or iNOS–/– mice (Fig. 1). IS was comparable in the ATV– eNOS–/– and the ATV– WT mice (P = 0.256).


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  		Table 1. Protocol 1: body weight, LV weight, and AR

 

Figure 1
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  		Fig. 1. The effect of atorvastatin (ATV) on infarct size (IS) in the wild-type (WT), endothelial nitric oxide synthase (eNOS)–/– and inducible nitric oxide synthase (iNOS)–/– mice. ATV limited IS in the WT but not in the eNOS–/– and iNOS–/– mice. There were 8 mice in the WT ATV+ (mice that received 10 mg·kg–1·day–1 ATV) group and 10 mice in each of the other groups. ATV–, mice receiving water alone; NS, not significant.

 
Immunoblotting. All immunoblotting analyses were done in hearts that were explanted on the fourth day of the experiment without being subjected to regional ischemia.

In the WT and iNOS–/– mice, ATV increased the expression of both total eNOS (Fig. 2A) and p-eNOS (Fig. 2B). As expected, there was no expression of eNOS and p-eNOS in the eNOS–/– mice. ATV upregulated iNOS expression only in the WT mice but not in the eNOS–/– or iNOS–/– mice (Fig. 2C). On the other hand, COX2 expression was upregulated by ATV only in the WT and eNOS–/– but not the iNOS–/– mice (Fig. 2D).


Figure 2
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  		Fig. 2. Samples of immunoblots and densitometric analyses of myocardial expression of total eNOS (A), Ser-1177 phosphorylated (p)-eNOS (B), iNOS (C), and cyclooxygenase 2 (COX2; D) in WT, eNOS–/–, and iNOS–/– mice with or without ATV pretreatment. Total eNOS and Ser-1177 p-eNOS levels were increased by ATV in the WT and iNOS–/– mice. There was no expression of eNOS in the eNOS–/– mice. ATV augmented iNOS levels only in the WT mice. On the other hand, ATV increased COX2 levels in the WT and eNOS–/– mice but not in the iNOS–/– mice. There were 4 mice in each group.

 
NOS activity. All enzyme activity analyses were done in hearts that were explanted on the fourth day of the experiment without being subjected to regional ischemia. In agreement with the p-eNOS immunoblotting results, ATV augmented cNOS activity in the WT and iNOS–/– mice but not in the eNOS–/– mice (Fig. 3A). ciNOS activity was augmented by ATV only in the WT mice (Fig. 3B).


Figure 3
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  		Fig. 3. Myocardial calcium-dependent (c)NOS activity (A), calcium-independent (ci)NOS activity (B), 6-keto-PGF1{alpha} levels (C), and COX2 activity (D) in WT, eNOS–/–, and iNOS–/– mice with or without ATV pretreatment. ATV augmented cNOS activity in the WT and iNOS–/– mice. In contrast, ciNOS activity was increased by ATV only in the WT mice. Similarly, 6-keto-PGF1{alpha} levels and COX2 activity were augmented by ATV only in the WT mice. There were 4 mice in each group. cpm, Counts/min.

 
Myocardial 6-keto-PGF1{alpha} levels and COX activity. ATV significantly increased myocardial 6-keto-PGF1{alpha} levels only in the WT mice (Fig. 3C). ATV did not affect COX1 activity in all three strains (data not shown). COX2 activity was increased by ATV only in the WT mice but not in the eNOS–/– and iNOS–/– mice (Fig. 3D). Thus the upregulated COX2 protein in the eNOS–/– mice treated with ATV was inactive.

NF-{kappa}B activation. EMSA was done in hearts that were explanted on the fourth day of the experiment without being subjected to regional ischemia. EMSA showed that NF-{kappa}B was activated in the WT and iNOS–/– mice but not in the eNOS–/– mice (Fig. 4A). Immunoblotting of I{kappa}B in the cytosol confirmed the EMSA results, showing decreased I{kappa}B levels in the ATV-treated WT and iNOS–/– mice (Fig. 4B). Immunofluorescent staining of myocardial tissue of WT mice showed increased staining of NF-{kappa}B in the cell nuclei of mice treated with ATV (Fig. 5).


Figure 4
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  		Fig. 4. A: electrophoretic mobility shift assay showing activation of NF-{kappa}B (p50 and p65) in the WT and iNOS–/– mice but not in the eNOS–/– mice. The NF-{kappa}B and the supershift bands are marked with arrows. B: a sample of immunoblot and densitometric analysis of cytosolic I{kappa}B showing a decrease in IµB levels in the WT and iNOS–/– mice but not in the eNOS–/– mice. There were 4 mice in each group.

 

Figure 5
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  		Fig. 5. Immunofluorescent staining of NF-{kappa}B in heart tissue of WT mice not receiving (ATV–) or receiving (ATV+) ATV. The nuclei are stained in blue 4,6-diamidino-2-phenylindole (DAPI) and NF-{kappa}B p65 in red. Translocation of NF-{kappa}B into the nuclei is seen in the ATV-treated mice.

 
The effect of NF-{kappa}B and JAK inhibitors on the induction of iNOS and COX2 by ATV. SN50, an NF-{kappa}B inhibitor, blocked the ATV-induced decrease in I{kappa}B levels, indicating blocking NF-{kappa}B activation. AG49 blocked the ATV-induced increase in p-STAT-1 levels in the nuclear fraction, indicating the blockade of the JAK-STAT activation. SN50 completely blocked the induction of iNOS, whereas AG490, a JAK inhibitor, had no effect (Fig. 6). In contrast, AG490 and SN50 alone did not block the induction of COX2 by ATV.


Figure 6
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  		Fig. 6. Myocardial COX2, iNOS, I{kappa}B, and β-actin levels and nuclear p-STAT-1 and lamin B levels in mice treated with intraperitoneal ATV alone or with AG490 (a JAK inhibitor) or SN50 (an NF-{kappa}B inhibitor). A: samples of immunoblots. B: densitometric analysis of myocardial COX2 levels. C: densitometric analysis of myocardial iNOS levels. D: densitometric analysis of myocardial I{kappa}B levels. E: densitometric analysis of myocardial p-STAT-1 levels. *P < 0.001 vs. controls (Cont); #P < 0.001 vs. ATV. AG490 blocked the ATV-induced increase in p-STAT-1 levels in the nucleus, indicating inhibition of the JAK-STAT pathway. SN50 prevented the ATV-induced decrease in I{kappa}B levels, indicating inhibition of the NF-{kappa}B pathway. ATV augmented COX2 and iNOS levels. SN50 and AG490 did not block the induction of COX2 by ATV. SN50 blunted ATV induction of iNOS, suggesting that iNOS expression is dependent on NF-{kappa}B. There were 4 mice in each group.

 
S-nitrosylation of COX2. ATV induced S-nitrosylation of COX2 only in the WT mice. COX2, induced by ATV in the eNOS–/– mice, was not S-nitrosylated, in agreement with its being inactive (Fig. 7).


Figure 7
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  		Fig. 7. A: sample of the "biotin switch" assay showing S-nitrosylation of COX2 in the ATV-treated WT but not in the ATV–-treated WT or the eNOS–/– and iNOS–/– mice. B: immunoblotting with COX2 after stripping the membranes, showing the precipitates in the ATV-treated WT and eNOS–/– but not the ATV-treated iNOS–/– mice. There were 4 mice in each group.

 
A summary of the findings is presented in Table 2.


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  		Table 2. Summary of the effects of ATV in wild-type, eNOS–/–, and iNOS–/– mice

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our main finding is that ATV did not limit IS in the University of North Carolina eNOS–/– and iNOS–/– mice, as reported for the Harvard eNOS–/– mice (1, 18, 25, 52). The induction of COX2 by ATV was eNOS and NF-{kappa}B independent, since ATV increased COX2 expression in eNOS–/– mice and since, in these mice, NF-{kappa}B was not activated by ATV. On the other hand, in the iNOS–/– mice, NF-{kappa}B was activated by ATV; however, COX2 expression was not changed. Moreover, SN50, an NF-{kappa}B inhibitor, did not block COX2 expression, although it blocked iNOS expression. These data are in agreement with the literature on delayed ischemic preconditioning that NF-{kappa}B activation is downstream to eNOS (10). On the other hand, COX2 activity was increased by ATV only in the WT mice. In the eNOS–/– mice, ATV increased COX2 expression, but the COX2 was inactive. Of note, the upregulated COX2 in the eNOS–/– mice was not S-nitrosylated. Previously, we have shown that myocardial COX2, upregulated by ATV, is inactivated by an iNOS inhibitor that decreased its S-nitrosylation (2) (Table 2).

We have previously shown that the 3-day ATV treatment does not upregulate nNOS levels in the rat myocardium (7). Since the activities of both eNOS and nNOS are calcium dependent, cNOS activity reflects the activity of both enzymes. In the present study, we did not observe an increase in cNOS activity in the eNOS–/– mice, suggesting that ATV did not induce a significant upregulation of nNOS in these mice. Moreover, ATV did not decrease IS in the University of North Carolina eNOS–/– mice, suggesting that, in contrast to the effect on vascular relaxation (13, 30), nNOS does not compensate for the lack of eNOS in our model.

iNOS. Our findings are in disagreement with those of Sharp et al. (37) that iNOS expression is upregulated in the University of North Carolina eNOS–/– mice. Myocardial levels of iNOS were very low in the eNOS–/– mice and were not upregulated by ATV in these mice. Accordingly, ciNOS activity was not increased in the eNOS–/– mice compared with the WT mice. The present study confirms our previous results that iNOS upregulation is essential for the IS-limiting effect of ATV. Scalia et al. (36) have also shown that simvastatin does not limit IS in iNOS–/– mice. We have previously shown that 1400W, a selective iNOS inhibitor, abrogates the IS-limiting effect of 3-day pretreatment with ATV by inhibiting the activation of COX2 via S-nitrosylation (2). In that study, 1400W did not affect total and Ser-1177-phosphorylated eNOS levels and cNOS activity, although it blocked the ATV induction of ciNOS activity. In the present study, iNOS expression and ciNOS activity were not upregulated in the eNOS–/– mice, suggesting that iNOS is downstream of eNOS, as has been described for the delayed form of ischemic preconditioning (10, 23). It has been suggested that eNOS activation leads to the activation of soluble guanylate cyclase, protein kinase C-{varepsilon} (PKC-{varepsilon}), NF-{kappa}B, and JAK-STAT pathways, leading to activation of iNOS and/or COX2 (3, 9–11, 23). In contrast, in other models eNOS has been reported to inhibit NF-{kappa}B activation (45). This may reflect that NF-{kappa}B activation involves both inflammatory (p65/p50) and anti-inflammatory signaling (cRel/p52), as well as other noncanonical pathways. It is probable that the upregulation of iNOS by ATV is NF-{kappa}B dependent (Fig. 6). Indeed, both NF-{kappa}B and iNOS expression and activity were not increased by ATV in the eNOS–/– mice.

Our data suggest that the expression of COX2 is dependent on the presence of intact iNOS, since COX2 expression was not increased in the iNOS–/– mice despite the activation of NF-{kappa}B. Previously, it has been shown that, 24 h after ischemic preconditioning stimulus, COX2 expression was increased in both WT and iNOS–/– mice, suggesting that with a more robust stimulus than statin pretreatment, such as preconditioning, iNOS is not essential for COX2 upregulation (50). Although iNOS expression and ciNOS activity were not increased by ATV in the eNOS–/– mice, the presence of the intact iNOS gene may enable COX2 upregulation. Alternatively, a gene adjacent to iNOS on chromosome 10 that is responsible for upregulating COX2 expression could have been erroneously deleted in the iNOS–/– mice. However, as pioglitazone, a PPAR-{gamma} agonist, augments COX2 expression in the same iNOS–/– mice strain (8). This is probably not the explanation.

The overexpressed COX2 in the ATV-treated eNOS–/– mice was not activated and not S-nitrosylated. Although we cannot exclude the possibility that eNOS S-nitrosylates COX2 in the present study, previously we have shown that 1400W, a selective iNOS inhibitor, prevents COX2 S-nitrosylation by ATV in the heart without affecting cNOS activity (2). Kim et al. (27) have shown that S-nitrosylation by iNOS is essential for COX2 activation in inflammatory cells. Moreover, Xuan et al. (50) have shown the although ischemic preconditioning increased COX expression in iNOS–/– mice, the COX2 was inactive. Thus iNOS is needed for both the expression and activation of myocardial COX2 by ATV. eNOS is a membrane-bound enzyme, whereas iNOS is a soluble enzyme; thus the localization of these two enzymes in the cells is different, and although COX2 is a membrane-bound enzyme (42), it seems that iNOS, but not eNOS, is responsible for COX2 S-nitrosylation.

Our laboratory (2) and others (27) have used the biotin switch assay to assess S-nitrosylation. Recently, it has been suggested that artifacts may interfere with the interpretation of the test (24). However, currently, there are no reliable alternative assays to assess for S-nitrosylation. It is plausible that other mediators may activate COX2 independent of S-nitrosylation, especially in inflammatory models. However, it seems that when COX2 is upregulated by ATV without an inflammatory stimulus, iNOS-induced S-nitrosylation is essential for COX2 activation in the rat and mouse myocardium. It may be that nitrosylation of COX2 is not a general prerequisite but rather is restricted to the myocardium.

Of note, ciNOS activity in the iNOS–/– mice was comparable with that of the WT control group and not nil. This represents background noise of the method and not residual activity, as has also been shown by Guo et al. (20).

COX2. Previously, we have shown that ATV upregulates COX2 expression and activity (2, 7, 53). Inhibiting COX2, but not COX1, abrogated the IS-limiting effect of ATV (2, 7). COX2 is essential for mediating the protective effect of delayed ischemic preconditioning, since COX2 inhibition abrogates IS limitation by preconditioning (10, 12, 38, 39). It is well established that NF-{kappa}B affects COX2 expression (10, 29, 40, 45). However, other pathways, such as the JAK-STAT signaling pathway (10, 45, 50, 51), phosphatidylinositol 3-kinase through CCAAT enhancer-binding protein, or ERK via cAMP response element-binding protein, can also upregulate COX2 expression independent of NF-{kappa}B (45). Our data suggest that the upregulation of COX2 expression by oral ATV in the heart is independent of NF-{kappa}B, since COX2 expression was increased by ATV in eNOS–/– mice, despite the fact that NF-{kappa}B was not activated. Moreover, in the iNOS–/– mice, COX2 expression was not increased by ATV despite activation of NF-{kappa}B. Furthermore, SN50 did not block the ATV-induction of COX2 expression, although it blocked iNOS induction, supporting the fact that the induction of iNOS, but not COX2, is NF-{kappa}B dependent (Fig. 6). In addition, AG490 also did not affect COX2 induction by intraperitoneal ATV, suggesting that COX2 induction may be independent of JAK-STAT, in contrast to the findings in ischemic preconditioning (10). Further studies are needed to clarify the role of JAK-STAT in mediating statin-induced myocardial protection.

The dose of ATV used in the present study (10 mg·kg–1·day–1) may be considered high. Indeed, several investigators have shown the reduction of IS with lower doses of statin; however, in all these studies statins were administered intraperitoneally, subcutaneously, or intravenously. In a dose-ranging study, we have shown that the 3-day oral pretreatment with ATV at 10 and 75 mg·kg–1·day–1 reduces IS in the rat (5), whereas at a dose of 1 to 2 mg·kg–1·day–1 oral ATV has no effect (5, 34). Moreover, we have shown that in the rat, blood levels of ATV 16 h after a third dose of 10 mg·kg–1·day–1 are comparable with those seen in humans treated with ATV at 80 mg/day (6). Other investigators have also used equivalent or even higher doses of oral statins to show myocardial protection (31, 48).

In conclusion, we have shown that both iNOS and eNOS are essential for mediating the IS-limiting effect of ATV. Induction of eNOS leads to NF-{kappa}B-dependent iNOS upregulation. On the other hand, the induction of COX2 expression by ATV is eNOS and probably NF-{kappa}B independent. However, iNOS is needed for both the increased expression and activation of COX2.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
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This study was supported by The Edward D. and Sally M. Futch Endowment of the Division of Cardiology, University of Texas Medical Branch.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Birnbaum, Div. of Cardiology, Univ. of Texas Medical Branch, 5106 John Sealy Annex, 301 Univ. Blvd., Galveston, TX 77555-0553 (e-mail: yobirnba{at}utmb.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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