Date Published: April 3, 2013
Publisher: Public Library of Science
Author(s): Katarzyna Starowicz, Wioletta Makuch, Michal Korostynski, Natalia Malek, Michal Slezak, Magdalena Zychowska, Stefania Petrosino, Luciano De Petrocellis, Luigia Cristino, Barbara Przewlocka, Vincenzo Di Marzo, Bernard Le Foll. http://doi.org/10.1371/journal.pone.0060040
Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.
The endocannabinoid system includes the cannabinoid CB1 and CB2 receptors (two G-protein-coupled receptors activated also by the main psychotropic component of Cannabis sativa, Δ9-tetrahydrocannabinol ), and the endogenous agonists at these receptors, i.e. the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG)]. AEA also activates the transient receptor potential vanilloid-1 (TRPV1) channel , which transduces the pronociceptive and heat-like effects of another plant natural product, the pungent component of hot peppers, capsaicin , , , . TRPV1 usually, but not always, plays in pain transmission a role opposite to cannabinoid receptors ,  and, therefore, the possible effects on pain of the activation of this channel by AEA have been thoroughly studied , , .
In the present study, the spinal administration of a high dose of the FAAH inhibitor, URB597, caused anti-allodynic and anti-hyperalgesic effects in neuropathic rats. The effects of URB597 were blocked by a selective TRPV1 receptor antagonist (I-RTX) and significantly attenuated by a 12/15-LOX inhibitor (baicalein). CCI was accompanied by increased levels of AEA and increased co-localization of TRPV1 with AEA catabolic enzymes: 15-LOX and FAAH in the lumbar spinal cord. The analgesic effects of URB597 (200 µg) were accompanied by increased spinal levels of PEA, OEA and 2-AG and, surprisingly, decreased levels of AEA as compared with matched vehicle-treated CCI controls. Our pharmacological and molecular data support the possibility that the full inactivation of FAAH, together with CCI-induced over-expression of TRPV1 and 15-LOX in the DH of the spinal cord, trigger: 1) an alternative pathway of AEA metabolism leading to15-LOX-derived metabolites that inhibit nociception via TRPV1 desensitization; and 2) elevation of the levels of AEA-related acylethanolamides with possibly similar TRPV1-mediated analgesic actions, i.e. PEA and OEA. Our findings, together with our previous investigation of the effects of lower intrathecal doses of URB597, or of varying doses of AEA , suggest that elevation of spinal AEA levels with increasing doses of a locally injected FAAH inhibitor, produce anti-hyperalgesic and anti-allodynic actions via mechanisms that progressively involve TRPV1 channel desensitization rather than CB1 receptor activation (CB2 receptors are not a likely target for cannabinoid-mediated antinociception in CCI model and AEA is only a partial agonist at CB2 receptors , , ). FAAH inhibition would result first in elevation of AEA levels to an extent that is high enough to activate/desensitize this channel , and then in the elevation of the levels of other TRPV1-active acylethanolamides and in the activation of AEA catabolic pathways leading to other TRPV1-active metabolites (present data). Thus, potent inhibition of FAAH may cause fully effective analgesic actions against neuropathic pain as a result of a complex readaptation of endocannabinoid metabolism and signaling, involving AEA-lipoxygenation, PEA and OEA.
Following full inhibition of its hydrolysis, AEA might be metabolized to 15(S)-hydroxy-AEA, which, together with OEA and PEA, may produce TRPV1-mediated anti-hyperalgesic and anti-allodynic effects in neuropathic rats.