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Published as doi: 10.1096/fj.08-108159.
 (The FASEB Journal. 2008;22:3154-3164.)
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Cyclooxygenases 1 and 2 contribute to peroxynitrite-mediated inflammatory pain hypersensitivity

Michael M. Ndengele*,1, Salvatore Cuzzocrea{dagger},{ddagger}, Emanuela Esposito{ddagger}, Emanuela Mazzon{ddagger}, Rosanna Di Paola{ddagger}, George M. Matuschak* and Daniela Salvemini*,2

* Department of Internal Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Saint Louis University School of Medicine, St. Louis, Missouri, USA;

{dagger} Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Messina, Italy; and

{ddagger} IRCCS Centro Neurolesi "Bonino-Pulejo" Messina, Messina, Italy

2Correspondence: Department of Internal Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, 1402 S. Grand Blvd., Deslodge Towers, 7th Floor, Saint Louis University School of Medicine, St. Louis, MO 63104-1028, USA. E-mail: salvemd{at}slu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxynitrite (ONOO–), the reaction product of the interaction between superoxide (O2·–) and nitric oxide (·NO), is a potent proinflammatory and cytotoxic nitrooxidative species. Its role as a mediator of hyperalgesia (clinically defined as an augmented sensitivity to painful stimuli) is not known. In light of the known proinflammatory properties of ONOO–, our study addressed its potential involvement in the development of hyperalgesia associated with tissue damage and inflammation. Intraplantar injection in rats of the ONOO– precursor O2·– (1 µM) led to the development of thermal hyperalgesia associated with a profound localized inflammatory response. Both events were blocked by L-NAME (NG-nitro-L-arginine methyl ester, 3–30 mg/kg), a nitric oxide synthase inhibitor, or by FeTM-4-PyP5+ [Fe(III)5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, 3–30 mg/kg], an ONOO– decomposition catalyst. These results suggested that locally synthesized ONOO– produced in situ by O2·– and ·NO is key in the development of inflammatory hyperalgesia. The direct link between ONOO– and hyperalgesia was further supported by demonstrating that intraplantar injection of soluble ONOO– itself (1 µM) similarly led to inflammatory hyperalgesia. ONOO– generated by the interaction between exogenous administration of O2·– and endogenous ·NO, or provided by direct injection of ONOO–, activated the transcription factor NF-{kappa}B in paw tissues, enhancing expression of the inducible but not the constitutive cyclooxygenase enzyme (COX-2 and COX-1, respectively). ONOO–-mediated hyperalgesia was blocked in a dose-dependent manner by intraperitoneal injections of indomethacin (10 mg/kg), a nonselective COX-1/COX-2 inhibitor, or NS398 [N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; 10 mg/kg] a selective COX-2 inhibitor, as well as by an anti-prostaglandin (PG) E2 antibody (200 µg). In another established model of inflammation-related hyperalgesia by intraplantar injection of carrageenan in rats, inhibition of ONOO– with FeTM-4-PyP5+ (3–30 mg/kg) inhibited the development of hyperalgesia and the release of PGE2 in paw tissue exudates. Furthermore, FeTM-4-PyP5+ synergized with indomethacin and NS397 (1–10 mg/kg) to block both hyperalgesia and edema. Taken together, these data show for the first time that ONOO– is a potent mediator of inflammation-derived hyperalgesia operating via the COX-to-PGE2 pathway. These results provide a pharmacological rationale for the development of inhibitors of peroxynitrite biosynthesis as novel nonnarcotic analgesics. The broad implications of our study are that dual inhibition of both ONOO– formation and COX activity may provide an alternative therapeutic approach to the management of pain: effective analgesia with reduced side-effects typically associated with the use of COX inhibitors.—Ndengele, M. N., Cuzzocrea, S., Esposito, E., Mazzon, E., Di Paola, R., Matuschak, G. M., Salvemini, D. Cyclooxygenases 1 and 2 contribute to peroxynitrite-mediated inflammatory pain hypersensitivity.


Key Words: superoxide • nitric oxide • PGE2 • COX-1 • COX-2 • hyperalgesia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PEROXYNITRITE (ONOO–), the reaction product of the interaction between superoxide (O2·–) and nitric oxide (·NO), is a potent proinflammatory nitroxide implicated in acute and chronic inflammatory conditions of many etiologies (1 2 3 4) . Tissue injury and inflammation often accompany rapid development of hypersensitivities to noxious and nonnoxious stimuli (hyperalgesia and allodynia, respectively). Both peripheral mechanisms at the site of injury and central processes, particularly in the spinal cord, contribute to this phenomenon (5) via the release of nitrooxidative species. Specifically, ·NO, O2·– and ONOO–, in addition to cyclooxygenase (COX)-derived prostaglandins (PGs), play an important role in the development of such peripheral and central sensitization (6 , 7) . We recently reported that O2·–, in addition to its well-established role of O2·– in inflammation, is also involved in hyperalgesia. Notably, the direct intraplantar injection of O2·– in rats evokes potent thermal hyperalgesia (8) . Its removal with superoxide dismutase mimetics (SODm) blocks hyperalgesia associated with inflammation (8 , 9) or following chronic morphine administration (morphine induced antinociceptive tolerance) (10) , and in response to spinal activation of the N-methyl-D-aspartate (NMDA) receptor (11) . Although the relative contributions of ONOO– to hyperalgesia are incompletely understood, abrogation of its biological activity with ONOO– decomposition catalysts such FeTM-4-PyP5+ (Fe(III)5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin) (4 , 12) inhibited the development of morphine-induced hyperalgesia and antinociceptive tolerance (10 , 13) .

One potential molecular pathway by which nitrooxidative stress (herein defined as stress caused by the presence of O2·–, ·NO, and ONOO–) may influence inflammatory events associated with the development of altered pain sensitivity is through modulation of cyclooxygenase (COX) enzymes (14 , 15) . As originally reported by our group (16) and subsequently extended by others (15 , 17 , 18) , the COX enzymes (constitutive COX-1 and inducible COX-2) are "receptor targets" for the multifaceted action of ·NO and as such are regulated in its presence. Although the mechanisms by which ·NO activates COX enzymes remain undefined, we now know that ONOO– is involved in this activation through the oxidative inactivation or modification of key amino acids residues in the COX polypetide backbone (19 , 20) . Other possibilities in this complex reaction biochemistry have been raised and discussed in detail (21) . Furthermore, nitrooxidative species activate transcription factors such as AP-1 and NF-{kappa}B, as well as mitogen-activated protein kinases (MAPKs) such as p38 MAP kinase, which is known to induce COX-2 protein expression during inflammation (22 23 24 25) . Substantial evidence supports the conclusion that the activation or induction of COX enzymes by nitrooxidative stress augments the production of proinflammatory and pronociceptive prostaglandin E2 (PGE2) at sites of inflammation (15 , 16) . Because O2·– and ·NO are often coproduced during tissue injury and inflammation, we hypothesized that ONOO– formed by their interaction is the likely key determinant of hyperalgesia driven by nitrooxidative stress.

Accordingly, the goals of our study were 1) to determine whether ONOO– is a hyperalgesic mediator; and 2) if so, to determine whether it acts through the COX-to-PGE2 pathway. Results of this study support the proposition that therapeutic manipulations of ONOO– and its downstream consequences are mechanistically grounded targets for the development of novel nonnarcotic analgesics. In light of the synergism shown herein between the nonsteroidal anti-inflammatory drugs (NSAIDs) indomethacin and NS398 in relation to FeTM-4-PyP5+, our results suggest that combining NSAIDs and ONOO– decomposition catalysts may provide an improved therapeutic strategy to NSAIDs alone for inflammatory pain management.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Sprague-Dawley rats (male, 175–200 g; Harlan, Indianapolis IN, USA) were used throughout these studies and housed and cared for using protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Saint Louis University Medical Center and were in accordance with the U.S. National Institutes of Health Guidelines on Laboratory Animal Welfare. Animal use at the University of Messina was likewise in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with European Economic Community regulations.

Drug administration
All drugs were given by intraperitoneal injection (1 ml/kg) 30 min before intraplantar injection of O2·–, ONOO–, or carrageenan. The PGE2 monoclonal antibody (lyophilized powder EIA; Cayman Chemicals, Milan, Italy) was given by intraperitoneal injection at 200 µg/kg, 18 h prior to intraplantar injection of PGE2, as described previously (26) . FeTM-4-PyP5+ was purchased from Alexis (Milan, Italy) and NS398 [N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide]from Cayman Chemicals. Decomposed superoxide was prepared using a dimethyl sulfoxide solution of potassium superoxide exposed to air for 30 min, and decomposed peroxynitrite was purchased from Upstate. All other drugs were purchased from Sigma (St. Louis, MO, USA). Ionic charges on FeTM-4-PyP5+ have been omitted from all figures for clarity.

Induction of thermal hyperalgesia and edema by O2·–, ONOO–, or carrageenan
Lightly anesthetized rats [CO2 (80%)/O2 (20%)] received a subplantar injection of O2·–, ONOO–, or their vehicle (decomposed O2·– or ONOO–, respectively, in a total volume of 50 µl) or carrageenan (0.1 ml of a 1% suspension in 0.85% NaCl) into the right hindpaw. Hyperalgesic responses to heat were determined at specified time points as described by Hargreaves (27) ; a cutoff latency of 20 s was used to prevent tissue damage. Rats were individually confined to plexiglass chambers. A mobile unit consisting of a high-intensity projector bulb was positioned to deliver a thermal stimulus directly to an individual hindpaw from beneath the chamber. The withdrawal latency period of injected and contralateral paws was determined to the nearest 0.1 s with an electronic clock circuit and thermocouple. Each point represented the change (s) in withdrawal latency (calculated from the withdrawal latency of the contralateral paw minus the withdrawal latency of the injected paw) at each time point. Results are expressed as Paw-withdrawal latency change (s). Development of paw edema was assessed by measuring paw-volume changes before and after O2·–, ONOO–, or carrageenan with a plethysmometer (Ugo-Basile, Varese, Italy) as described previously (28) . Results are expressed as Paw-volume change (ml).

Determination of PGE2 release in carrageenan-injected rat paws
PGE2 released in the paw exudates was measured as described previously (28) . Briefly, at 6 h following the intraplantar injection of carrageenan, rats in each group were sacrificed, and each paw was excised at the level of the calcaneus bone. Paws were gently centrifuged at 250 g for 20 min to recover a sample of the edematous fluid, and the volume of fluid recovered from each paw was measured. PGE2 levels were measured by ELISA using commercially available kits (Cayman), and results were expressed in pg/paw, normalizing values to the amount of exudate recovered from each paw. All determinations were performed in duplicate.

Western blot analysis for I{kappa}B-{alpha}, the p65 subunit of NF-{kappa}B, COX-1, and COX-2
Cytosolic and nuclear extracts were prepared as described previously (29) , with slight modifications. Briefly, paw tissues from each rat were suspended in extraction buffer A, containing 0.2 mM PMSF, 0.15 µM pepstatin A, 20 µM leupeptin, and 1 mM sodium orthovanadate, homogenized at the highest setting for 2 min, and centrifuged at 1000 g for 10 min at 4°C. Supernatants represented the cytosolic fraction. The pellets, containing enriched nuclei, were resuspended in buffer B, containing 1% Triton X-100, 150 mM NaCl, 10 mM TRIS-HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.2 mM PMSF, 20 µm leupeptin, and 0.2 mM sodium orthovanadate. After centrifugation at 15,000 g for 30 min at 4°C, the supernatants containing the nuclear protein were stored at –80°C for further analysis. The levels of I{kappa}B-{alpha}, COX-1, and COX-2 were quantified in the cytosolic fraction from paw tissue collected 60 min after O2·– administration; levels of the NF-{kappa}B subunit p65 were quantified in the nuclear fraction. The filters were blocked with 1x PBS containing 5% (w/v) nonfat dried milk (PM) for 40 min at room temperature and subsequently probed with specific antibodies to I{kappa}B-{alpha} (1:1000; Santa Cruz Biotechnology, Milan, Italy), anti-iNOS (1:2000; Transduction Laboratories, Milan Italy), anti-eNOS (1:1000; Transduction Laboratories), anti-COX-2, (1:1500; Cayman Chemicals), anti-COX-1 (1:1000; Cayman Chemicals), or anti-p65 (1:500; Santa Cruz Biotechnology) in 1x PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20 (PMT) at 4°C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse immunoglobulin G (IgG) secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of total protein, they were also incubated in the presence of the antibody against {alpha}-tubulin protein (1:10,000; Sigma-Aldrich Corp., Milan Italy). The relative expression of the protein bands for I{kappa}B-{alpha} (~37 kDa), NF-{kappa}B p65 (65 kDa), iNOS (~130 kDa), eNOS (~155 kDa), COX-2 (~72 kDa), and COX-1 (~70 kDa) was quantified by densitometric scanning of the X-ray films with an imaging densitometer (GS-700; Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst; IBM, Armonk, NY, USA), and standardized for densitometric analysis to {alpha}-tubulin levels.

Histological examination of the O2·–-inflamed rat hindpaw
For histopathological examination, biopsies of paws were taken 60 min after intraplantar injection of O2·–. Tissue from the pads of rat hindpaws was removed with a scalpel. Tissue samples were fixed in 10% (w/v) PBS-buffered formaldehyde for 1 wk at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Deland, FL, USA). Tissue sections (thickness 7 µm) were deparaffinized with xylene, stained with hematoxylin/eosin, and studied using light microscopy (Leitz Dialux 22; Leica Microsystems, Wetzlar, Germany). All the histological studies were performed in a masked fashion.

Immunohistochemical localization of nitrotyrosine
Sixty minutes after intraplantar injection of O2·–, tissue localizations for the formation of nitrotyrosine, a specific marker of nitrosative stress, were measured by immunohistochemical analysis in the paw tissue sections. Briefly, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde, and 8 µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody (Upstate, Lake Placid, NY, USA; 1:500 in PBS, v/v). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin-peroxidase complex (DBA). To verify the binding specificity for nitrotyrosine, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreactions were positive in all the experiments performed.

Statistical evaluation
All values in the figures and text are expressed as mean ± SE for groups of n animals. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different days. The results were analyzed by 1-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Peroxynitrite is a potent hyperalgesic mediator
As reported in our previous studies (8) , intraplantar injection of potassium O2·– (1 µM, n=6) evoked a hyperalgesic response of rapid onset (within 5 min) to noxious heat that reached a peak by 60–90 min (not shown). All subsequent observations were made and samples were collected at 60 min. The development of thermal hyperalgesia after O2·– (Fig. 1 ) coincided with protein nitration in paw tissues (Fig. 2 B, B1) and tissue damage and inflammation as evidenced histologically by edema and a marked inflammatory cell infiltration (Fig. 2E ). The hyperalgesic responses to O2·– were attenuated in a dose-dependent manner by pretreatment with the nonselective NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester; ref. 30 ) (3–30 mg/kg, n=6), (Fig. 1) . Because ·NO reacts with O2·– at a diffusion-limited rate to form ONOO– (1) , these results with L-NAME together with our previous findings with superoxide dismutase mimetics (8) suggest that ONOO– from these two reactive species is the common denominator in the molecular and biochemical pathways leading to O2·–-mediated hyperalgesia, a role confirmed by the well-characterized ONOO–decomposition catalyst FeTM-4-PyP5+ (4) . Like L-NAME, FeTM-4-PyP5+ also inhibited O2·-mediated hyperalgesia in a dose-dependent manner (3–30 mg/kg, n=6) (Fig. 1) . Furthermore, FeTM-4-PyP5+ (30 mg/kg, n=6) blocked protein nitration as detected by immunohistochemistry (Fig. 2C ) and led to a significant improvement in tissue damage and inflammation, characterized by inhibition of edema and inflammatory cell infiltration (Fig. 2F ). Because L-NAME is a nonselective NOS inhibitor, it is unclear whether the ONOO– is generated from O2·– interacting with ·NO derived from the constitutive or inducible NOS enzymes, or both; this interesting question was not part of the overall objectives of the present study. The role of ONOO– as a mediator of hyperalgesia was underscored by the finding that intraplantar injection of ONOO– (0.1–3 µM, n=6) evoked a dose-dependent hyperalgesic response to noxious heat that peaked by 60–90 min (Fig. 3 A). The time course of ONOO–-induced hyperalgesia was similar to that using O2·– (8) . The dose-dependent development of ONOO–-induced hyperalgesia also was inhibited by FeTM-4-PyP5+ (3–30 mg/kg, n=6; Fig. 3B ). The intraplantar injection of O2·– and ONOO– led to paw edema, which was also inhibited as anticipated by FeTM-4-PyP5+ (not shown).


Figure 1
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  		Figure 1. Direct hyperalgesic effects of superoxide (O2·–). The hyperalgesic responses obtained at 60 min (time of peak hyperalgesia) after intraplantar injection of O2·– (1 µM) are blocked in a dose-dependent manner by L-NAME (3–30 mg/kg) and FeTM-4-PyP5+ (3–30 mg/kg). Drugs were given i.p. 30 min before O2·–. Results are expressed as mean ± SE for 6 rats. *P < 0.001 vs. vehicle; {dagger}P < 0.001 vs. O2·– alone.


Figure 2
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  		Figure 2. No staining for nitrotyrosine is observed in paw tissues obtained from vehicle-treated rats (A). On the contrary, 60 min after intraplantar injection of O2·– (1 µM), paw tissue sections reveal a positive staining (B) in paw tissues from carrageenan-treated rats, which is primarily localized in inflammatory cells (B1, arrows). A marked reduction in nitrotyrosine staining (C) is found in the paw tissues of the O2·–-injected rats treated with FeTM-4-PyP5+. Similarly, at histological examination, the paws reveal inflammatory changes demonstrated by the presence of edema and marked inflammatory cell infiltration (E). Treatment with FeTM-4-PyP5+ significantly reduces the pathological changes and prevents the inflammatory cells infiltration (F). No histological alteration is observed in paw tissues obtained from sham-treated rats (D). Figures are representative of at least three experiments performed on different experimental days.


Figure 3
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  		Figure 3. Hyperalgesic responses to ONOO– are blocked by FeTM-4-PyP5+. Intraplantar injection of ONOO– (0.1–3 µM) leads to time-dependent hyperalgesic responses that maximize within 60 min (A). ONOO– (0.1–1 µM) mediated hyperalgesia at peak time is blocked by FeTM-4-PyP5+ in a dose-dependent manner (3–30 mg/kg) (B). FeTM-4-PyP5+ was given i.p. 30 min before the intraplantar injection of ONOO–. *P < 0.01 vs. preinjection values; {dagger}P < 0.01 vs. responses in the absence of FeTM-4-PyP5+. Results are expressed as mean ± SE for 6 rats.

The COX-to-PGE2 pathway plays a critical role in peroxynitrite-mediated hyperalgesia
Because the results obtained with ONOO– produced locally in paw tissues from endogenous ·NO and exogenous O2·– resembled results obtained with exogenous ONOO–, only results with the former approach are presented in the figures for simplicity. When compared to vehicle, Western blot analysis revealed that ONOO– activated NF-{kappa}B by augmented I{kappa}B-{alpha} degradation (Fig. 4 A, A1) and increased total NF-{kappa}B p65 nuclear localization (Fig. 4B, B1 ). As shown in Fig. 5 A, A1 and as reported by others (31 , 32) , a low basal level of COX-2 protein was detected in cytosolic extracts from tissues of vehicle-treated rats. Injection of ONOO– led to a significant increase in COX-2 protein accumulation (Fig. 5A, A1 ). Notably, levels of constitutive COX-1 were not increased by ONOO– (Fig. 5B, B1 ). The nonselective COX-1/COX-2 inhibitor, indomethacin (10 mg/kg, n=6) or the more COX-2 selective inhibitor (33 34 35) NS398 (10 mg/kg, n=6) given intraperitoneally 30 min prior to the intraplantar injection of O2·– or ONOO– (1 µM) partially but significantly reduced hyperalgesia (Fig. 6 ). NS398 was used at doses previously shown to display in vivo selectivity for COX-2 vs. COX-1 (33 34 35) . Furthermore, as can be seen in Fig. 6 , intraperitoneal injection of an anti-PGE2 antibody (200 µg/kg) 18 h before the intraplantar injection of O2·– or ONOO– (1 µM) blocked thermal hyperalgesia (Fig. 6) . The anti-PGE2 antibody was used at a dose previously shown to inhibit the development of carrageenan- and PGE2-induced hyperalgesia (26) . These results suggest that hyperalgesic responses to ONOO– are mediated at least in part through COX-1 and COX-2 and that PGE2 is an important signaling eicosanoid involved in this response.


Figure 4
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  		Figure 4. Activation of paw tissue NF-{kappa}B by ONOO–. As measured at 60 min, intraplantar injection of O2·– (1 µM) or ONOO– (1 µM, not shown) activates NF-{kappa}B in paw tissues, as evidenced by I{kappa}B-{alpha} degradation (A, A1) and increased total NF-{kappa}B p65 nuclear expression (B, B1). Intraperitoneal injection of FeTM-4-PyP5+ (30 mg/kg) blocks NF-{kappa}B activation. Gels are representative of results from 6 animals (A, B); densitometric analyses of all animals per group are reported (A1, B1). Results are expressed as mean ± SE for 6 rats. *P < 0.01 vs. preinjection values; {dagger}P < 0.01 vs. responses in the absence of FeTM-4-PyP5+. {alpha}-Tubulin shows equal loading.


Figure 5
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  		Figure 5. Induction of COX-2 in paw tissues by ONOO–. As measured at 60 min, intraplantar injection of O2·– (1 µM) or ONOO– (1 µM, not shown) increases COX-2 protein expression in paw tissues (A, A1) but not COX-1 (B, B1). Intraperitoneal injection of FeTM-4-PyP5+ (30 mg/kg) blocks COX-2 induction (A, A1). Gels are representative of results from 6 animals (A, B); densitometric analyses of all animals per group are reported (A1, B1). Results are expressed as mean ± SE for 6 rats. *P < 0.01 vs. preinjection values; {dagger}P < 0.01 vs. responses in the absence of FeTM-4-PyP5+. {alpha}-Tubulin shows equal loading.


Figure 6
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  		Figure 6. The cyclooxygenase pathway contributes to ONOO– mediated hyperalgesia. The hyperalgesic responses obtained at time of peak hyperalgesia (60 min) after intraplantar injection of O2·– or ONOO– (1 µM) are blocked by indomethacin (10 mg/kg), NS398 (10 mg/kg), or an anti-PGE2 antibody (200 µg/kg). Drugs were given i.p. 30 min or 18 h (for the anti-PGE2 antibody) before O2·– or ONOO–. Results are expressed as mean ± SE for 6 rats. *P < 0.01 vs. vehicle; {dagger}P < 0.01 vs. responses in the absence of drugs.

The peroxynitrite decomposition catalyst FeTM-4-PyP5+ blocks carrageenan-induced hyperalgesia
Having established that ONOO– is a potent hyperalgesic mediator operating via the COX-to-PGE2 pathway, we next extended and confirmed our findings in a well-established model of hyperalgesia associated with inflammation (27) . The development of thermal hyperalgesia and inflammation following the intraplantar injection of carrageenan is mediated by the production of O2· and ·NO (8 , 28 , 36) and COX-1-/COX-2-derived PGE2 at peripheral and spinal sites (33 , 35 , 37 38 39 40 41) . Here we show that removal of ONOO– with FeTM-4-PyP5+ (3–30 mg/kg, n=6) given by intraperitoneal injection 30 min before carrageenan caused a dose-dependent inhibition of thermal hyperalgesia at all time points tested (Fig. 7 A), edema, and PGE2 release in paw tissue fluids (n=6, Fig. 7C ) The antihyperalgesic effects of FeTM-4-PyP5+ (30 mg/kg) were not affected by naloxone (given at 1 mg/kg s.c., 30 min prior to FeTM-4-PyP5+, n=6; not shown), excluding the potential involvement of an opiate pathway.


Figure 7
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  		Figure 7. Inhibition of carrageenan-induced hyperalgesia by FeTM-4-PyP5+. The development of thermal hyperalgesia following intraplantar injection of carrageenan is blocked in a dose-dependent manner by FeTM-4-PyP5+ (3–30 mg/kg) (A). As measured at the 6 h time point after carrageenan, inhibition of hyperalgesia by FeTM-4-PyP5+ is associated with a dose-dependent inhibition of PGE2 in paw tissue fluids (C). As expected, treatment blocks edema formation at all time points (B). FeTM-4-PyP5+ was given i.p. 30 min before carrageenan. *P < 0.01 vs. preinjection values; {dagger}P < 0.01 vs. responses in the absence of FeTM-4-PyP5+. Results are expressed as mean ± SE for 6 rats.

Synergistic inhibition of carrageenan-induced hyperalgesia by combinations of COX inhibitors and ONOO– decomposition catalysts
We next examined whether dual administration of an ONOO– decomposition catalyst and a COX inhibitor act in concert to additively block hyperalgesia. As reported by others (33 34 35) , intraperitoneal administration of NS398 (1–10 mg/kg, n=6) attenuated carrageenan-induced hyperalgesia and edema in a dose-dependent manner (Fig. 8 A, B). The combination of a low dose of FeTM-4-PyP5+ (3 mg/kg, n=6) and a low dose of NS398 (1 mg/kg, n=6) led to a significant attenuation of hyperalgesia and edema (Fig. 9 A, B), suggesting a synergistic interactions between these two pharmacological agents. Similar results were obtained with FeTM-4-PyP5+ and indomethacin (not shown).


Figure 8
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  		Figure 8. Inhibition of carrageenan-induced hyperalgesia and edema by NS398. The development of thermal hyperalgesia (A) and edema (B) following intraplantar injection of carrageenan is blocked in a dose-dependent manner by NS398 (1–10 mg/kg). Drugs were given i.p. 30 min before carrageenan. *P < 0.01 vs. preinjection values; {dagger}P < 0.01 vs. responses in the absence of NS398. Results are expressed as mean ± SE for 6 rats.


Figure 9
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  		Figure 9. Inhibition of carrageenan-induced hyperalgesia and edema by subthreshold doses of NS398 and FeTM-4-PyP5+. The combination of a low-threshold dose of NS398 (1 mg/kg) when combined with a low-threshold dose of FeTM-4-PyP5+ (3 mg/kg) results in profound inhibition of hyperalgesia (A) and edema (B). The degree of inhibition reached with the combination of NS398 and FeTM-4-PyP5+ is similar to that reached with the maximal dose of FeTM-4-PyP5+ (30 mg/kg) and NS398 (10 mg/kg) when given as a single entity. Drugs were given i.p. 30 min before carrageenan. *P < 0.01 vs. preinjection values; {dagger}P < 0.01 vs. responses in the absence of FeTM-4-PyP5+ or NS398. Results are expressed as mean ± SE for 6 rats.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ONOO– is a well-established mediator of acute and chronic inflammation, but its role in nociception is not well understood. Here we show that ONOO–, as generated by the interaction between exogenously supplied O2·– and endogenous ·NO, or when provided by direct injection of ONOO–, led to the development of thermal hyperalgesia associated with inflammation. Moreover, both the nociceptive and inflammatory responses were blocked with FeTM-4-PyP5+. To better define the molecular pathways involved in ONOO–-induced hyperalgesia, we focused our initial efforts on the COX pathway because of its regulation by nitrooxidative stress (14 , 15) . Even so, other pathways and mechanisms may also be involved, including the ability of ONOO– to release proinflammatory and pronociceptive cytokines such as TNF-{alpha}, IL-1β, and IL-6 (23 , 24 , 42 43 44) and to cause post-translational nitration and modification of key proteins involved in central and peripheral sensitization (8 , 10 , 11) . Still, inhibiting synthesis or release of PGE2, one of the most thoroughly studied hyperalgesic PGs, contributes to the antihyperalgesic action of NOS inhibitors and to the hyperalgesic action of nitric oxide donors, respectively. Indeed, attenuation of hyperalgesia during tissue injury with NOS inhibitors (45) , or in iNOS-deficient mice (46) , has been associated with a profound attenuation of peripheral and spinal release of PGE2. Similarly, the hyperalgesia resulting from exogenous administration of nitric oxide donors such as nitroglycerin is mediated at least in part by release of PGs and is blocked by NSAIDs such as indomethacin and nimesulide (47 48 49) . Our results identify the COX-to-PGE2 pathway as a key determinant in the hyperalgesic responses of ONOO–. Thus, the hyperalgesic responses to ONOO– were inhibited by the nonselective COX-1 and COX-2 inhibitor indomethacin, by the selective COX-2 inhibitor NS398 (33 34 35) , and by an anti-PGE2 antibody. When administered exogenously, PGE2 causes mechanical hyperalgesia in humans and experimental animals (50 51 52 53 54) and sensitizes primary afferent nociceptors (55 56 57 58) . These pronociceptive effects of PGE2 are mediated via adenyl cyclase-cAMP-protein kinase A, since agents that inhibit this kinase as well as those that inhibit adenyl cyclase attenuate PGE2-induced hyperalgesia (59 60 61) . Moreover, inhibition of COX-1 and COX-2 elicits antihyperalgesic effects associated with reduction of PGE2 formation (33 , 39 , 50 , 62 63 64 65) , and anti-PGE2 antibodies block hyperalgesia associated with tissue damage and inflammation (26 , 41) .

Our findings strongly support the notion that nitrooxidative stress can modulate the COX pathway through both enzymatic activation and enzymatic induction (14 , 15) . The mechanisms by which ·NO activate COX enzymes remain undefined but involve ONOO–-mediated oxidative inactivation or modification of key amino acids residues in the polypetide backbone (19 , 20) . In addition to effects on COX-2 enzyme activity, ·NO and ONOO– increase the production of PGs from macrophages by acting post-transcriptionally or translationally to increase COX-2 protein levels or to increase its mRNA stability, at least in part through O2·– and the p38 MAPK pathway (17 , 18 , 66 67 68 69) . Furthermore, iNOS binds COX-2, and iNOS-derived ·NO increases the catalytic activity of COX-2 through S-nitrosylation in a macrophage cell line (70) . In our study (Fig. 5) , ONOO– significantly increased COX-2 but not COX-1 expression in inflamed paw tissues. These findings confirm that nitrooxidative species, including O2·– and ONOO–, activate redox-sensitive transcription factors such as NF-{kappa}B and AP-1 as well as MAPK kinases such as p38 kinase (22 23 24 25) . Because the induction of COX-2 in inflammation is regulated in part through the NF-{kappa}B pathway (25) , it is likely that ONOO–-induced activation of NF-{kappa}B and subsequent induction of COX-2 contributes to hyperalgesia. In view of these considerations, we propose (Fig. 10 ) that ONOO–-mediated thermal hyperalgesia is driven at least in part by 1) activation of COX-1 and COX-2 in tandem with NF-{kappa}B induction of COX-2 in paw tissues, increasing in situ formation of PGE2; and 2) facilitation of PGE2-induced peripheral sensitization, as ·NO facilitates cAMP-dependent PGE2-induced hyperalgesia (71) . Whether ONOO– is also involved in the development of tactile hyperalgesia remains to be established, as its direct intraplantar injection failed to elicit tactile hyperalgesia despite the presence of significant inflammation (72) . The drugs used in our study were given systemically, and paw tissue expression of COX and PGE2 release from paw tissues were measured. Consequently, additional analyses are needed to unravel the relative contributions of both peripheral and central sensitization processes in the development of ONOO–-mediated hyperalgesia. We are currently undertaking these studies. As a final note, once formed, ONOO– is converted to several other species, such as nitrosoperoxycarbonate or its conjugate acid, peroxynitrous acid (73) . At this stage it is not possible to address the in vivo contribution of such downstream species derived from ONOO– in the overall development of thermal hyperalgesia, because the appropriate molecular, biochemical, and pharmacological tools are not yet available. These exciting questions will be addressed as these tools become available.


Figure 10
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  		Figure 10. Cartoon summarizing the key findings of this study. ONOO– is a potent hyperalgesic nitrooxidative species acting, at least in part, via the activation of COX-1 and COX-2 and the induction of COX-2 through the NF-{kappa}B pathway. Subsequent formation of PGE2 is an essential component to the development of ONOO– hyperalgesia. Dual inhibition of ONOO– and COX activity may provide an alternative therapeutic approach to the management of pain: effective analgesia with reduced side -effects typically associated with the use of COX inhibitors.

An important finding of this study was that the degrees of inhibition of hyperalgesia and edema obtained by combining suboptimal doses of FeTM-4-PyP5+ and NS398 or FeTM-4-PyP5+ and indomethacin were synergistic. Especially notable was that maximal inhibition of hyperalgesia could be achieved with combinations of FeTM-4-PyP5+ and NS398 or FeTM-4-PyP5+ and indomethacin at doses at least 10-fold lower than those needed to elicit maximal inhibition with these agents given individually. Besides its role in acute inflammatory pain shown in this study, it has been recently reported that neutrophil-derived ONOO– also contributes to hyperalgesia associated with chronic inflammation arthritis (74) . Collectively, these findings have broad implications. Data from the Centers for Disease Control and Prevention indicate over 40 million annual cases of inflammatory pain in the United States alone, and NSAIDs are the most commonly used class of analgesics for moderate to severe inflammatory pain. Indeed, selective inhibitors of COX-2 were designed to prevent the adverse effects of nonselective COX inhibitors while retaining their potent anti-inflammatory and pain-relieving actions (75) . Although this goal was reached to some extent (76 , 77) , some COX-2 inhibitors increase the risk of thrombosis (77) . Our results suggest that ONOO– decomposition catalysts are useful as novel nonnarcotic analgesics for inflammatory pain, either as stand-alone therapeutic agents or as adjuncts to COX inhibitors. The latter combination paradigm may provide an alternative strategy for the safe and effective management of pain states currently limited by the side-effect profile of selective and nonselective COX inhibitors.


   ACKNOWLEDGMENTS
 
This work was supported by Saint Louis University Seed Fund (D.S.) and IRCCS Centro Neurolesi "Bonino-Pulejo" grants (S.C.).


   FOOTNOTES
 
1 Current address: VA Medical Center, 915 North Grand Blvd., St. Louis, MO 63106, USA.

Received for publication March 13, 2008. Accepted for publication April 24, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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