Severe pain syndromes reduce quality of life in patients with inflammatory and neoplastic diseases, often because chronic opiate therapy results in reduced analgesic effectiveness, or tolerance, leading to escalating doses and distressing side effects. The mechanisms leading to tolerance are poorly understood. Our studies revealed that development of antinociceptive tolerance to repeated doses of morphine in mice was consistently associated with the appearance of several tyrosine-nitrated proteins in the dorsal horn of the spinal cord, including the mitochondrial isoform of superoxide (O 2 -) dismutase, the glutamate transporter GLT-1, and the enzyme glutamine synthase. Furthermore, antinociceptive tolerance was associated with increased formation of several proinflammatory cytokines, oxidative DNA damage, and activation of the nuclear factor poly(ADPribose) polymerase. Inhibition of NO synthesis or removal of O 2 -blocked these biochemical changes and inhibited the development of tolerance, pointing to peroxynitrite (ONOO -), the product of the interaction between O 2 -and NO, as a signaling mediator in this setting. Indeed, coadministration of morphine with the ONOO -decomposition catalyst, Fe(III) 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, blocked protein nitration, attenuated the observed biochemical changes, and prevented the development of tolerance in a dose-dependent manner. Collectively, these data suggest a causal role for ONOO -in pathways culminating in antinociceptive tolerance to opiates. Peroxynitrite (ONOO -) decomposition catalysts may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

Peroxynitrite (ONOO(-)), the reaction product of the interaction between superoxide (O(2)(*-)) 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 O(2)(*-) (1 microM) led to the development of thermal hyperalgesia associated with a profound localized inflammatory response. Both events were blocked by L-NAME (N(G)-nitro-L-arginine methyl ester, 3-30 mg/kg), a nitric oxide synthase inhibitor, or by FeTM-4-PyP(5+) [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 O(2)(*-) 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 microM) similarly led to inflammatory hyperalgesia. ONOO(-) generated by the interaction between exogenous administration of O(2)(*-) and endogenous *NO, or provided by direct injection of ONOO(-), activated the transcription factor NF-kappaB 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) E(2) antibody (200 microg). In another established model of inflammation-related hyperalgesia by intraplantar injection of carrageenan in rats, inhibition of ONOO(-) with FeTM-4-PyP(5+) (3-30 mg/kg) inhibited the development of hyperalgesia and the release of PGE(2) in paw tissue exudates. Furthermore, FeTM-4-PyP(5+) 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-PGE(2) 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.

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The effective treatment of pain is typically limited by a decrease in the pain-relieving action of morphine that follows its chronic administration (tolerance). Therefore, restoring opioid efficacy is of great clinical importance. In a murine model of opioid antinociceptive tolerance, repeated administration of morphine significantly stimulated the enzymatic activities of spinal cord serine palmitoyltransferase, ceramide synthase, and acid sphingomyelinase (enzymes involved in the de novo and sphingomyelinase pathways of ceramide biosynthesis, respectively) and led to peroxynitrite-derive nitroxidative stress and neuroimmune activation [activation of spinal glial cells and increase formation of tumor necrosis factor-␣, interleukin (IL)-1␤, and IL-6]. Inhibition of ceramide biosynthesis with various pharmacological inhibitors significantly attenuated the increase in spinal ceramide production, nitroxidative stress, and neuroimmune activation. These events culminated in a significant inhibition of the development of morphine antinociceptive tolerance at doses devoid of behavioral side effects. Our findings implicate ceramide as a key upstream signaling molecule in the development of morphine antinociceptive tolerance and provide the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.

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