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, 150 (6), 766-81

Analgesic Actions of N-arachidonoyl-serotonin, a Fatty Acid Amide Hydrolase Inhibitor With Antagonistic Activity at Vanilloid TRPV1 Receptors

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Analgesic Actions of N-arachidonoyl-serotonin, a Fatty Acid Amide Hydrolase Inhibitor With Antagonistic Activity at Vanilloid TRPV1 Receptors

S Maione et al. Br J Pharmacol.

Abstract

Background and purpose: N-arachidonoyl-serotonin (AA-5-HT) is an inhibitor of fatty acid amide hydrolase (FAAH)-catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA-5-HT antagonizes the transient receptor potential vanilloid-1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia.

Experimental approach: AA-5-HT was tested in vitro, on HEK-293 cells overexpressing the human or the rat recombinant TRPV1 receptor, and in vivo, in rats and mice treated with formalin and in rats with chronic constriction injury of the sciatic nerve. The levels of the endocannabinoids, AEA and 2-arachidonoylglycerol, in supraspinal (periaqueductal grey, rostral ventromedial medulla), spinal or peripheral (skin) tissues were measured.

Key results: AA-5-HT behaved as an antagonist at both rat and human TRPV1 receptors (IC(50)=37-40 nM against 100 nM capsaicin). It exerted strong analgesic activity in all pain models used here. This activity was partly due to FAAH inhibition, elevation of AEA tissue levels and indirect activation of cannabinoid CB(1) receptors, as it was reversed by AM251, a CB(1) antagonist. AA-5-HT also appeared to act either via activation/desensitization of TRPV1, following elevation of AEA, or as a direct TRPV1 antagonist, as suggested by the fact that its effects were either reversed by capsazepine and 5'-iodo-resiniferatoxin, two TRPV1 antagonists, or mimicked by these compounds administered alone.

Conclusions and implications: Possibly due to its dual activity as a FAAH inhibitor and TRPV1 antagonist, AA-5-HT was highly effective against both acute and chronic peripheral pain.

Figures

Figure 1
Figure 1
AA-5-HT is a potent antagonist of TRPV1 receptors. (a) Effect of increasing concentrations of AA-5-HT on the effect of capsaicin (100 nM, concentration eliciting ∼90% of the maximal observable effect, determined with ionomycin (4 μM)) on intracellular calcium in HEK-293 cells overexpressing the human recombinant TRPV1. (b) Effect of increasing concentrations of AA-5-HT on the effect of anandamide (1 μM, concentration eliciting ∼90% of the maximal observable effect, determined with ionomycin (4 μM)) on intracellular calcium in HEK-293 cells overexpressing the human recombinant TRPV1. (c) Effect of increasing concentrations of AA-5-HT on the dose–response curve of capsaicin on intracellular calcium in HEK-293 cells overexpressing the human recombinant TRPV1. Here we have expressed the effect of the agonist (in this case capsaicin) as percent of the effect of 10−4M capsaicin in the absence of the antagonist, and not as percent of the maximum observable effect with ionomycin 4 μM. (d) Schild' plot calculated from the data in (c) and showing a slope of 0.73. (e) Effect of increasing concentrations of AA-5-HT on the effect of capsaicin (100 nM) on intracellular calcium in HEK-293 cells overexpressing the human recombinant TRPV1 at pH=7.4 or 6.0. (f) Effect of increasing concentrations of AA-5-HT on the effect of capsaicin (100 nM, concentration eliciting ∼90% of the maximal observable effect, determined with ionomycin (4 μM)) on intracellular calcium in HEK-293 cells overexpressing the rat recombinant TRPV1. All data are means of n=3 separate experiments. The s.e. bars are not shown for clarity and were never higher than 10% of the means. In (a, b, e and f) the effect of AA-5-HT is expressed as percent of the effect on [Ca2+]i exerted by the agonist alone. The net effect exerted by the agonists (100 nM capsaicin or 1 μM anandamide) are described in the Results section.
Figure 2
Figure 2
Effect of s.c. formalin (1.2%, 25 μl) injections into the hindpaw of mice on the time course of the nociceptive behaviour. Formalin was injected 15 min after the systemic administration of vehicle (10% DMSO in 0.9% NaCl, i.p.) (ac), AA-5-HT (5 mg kg−1, i.p.), alone or in combination with AM251 (3 mg kg−1, i.p.), AM630 (3 mg kg−1, i.p.) (a) or capsazepine (CPZ, 2.5 and 10 mg kg−1, i.p.) (b). AM251 and CPZ were administered 5 min before AA-5-HT. The effects of AM251 (3 mg kg−1 i.p.) and capsazepine (CPZ, 2.5 and 10 mg kg−1, i.p.) alone on the nociceptive behaviour induced by formalin are shown in (c). The data represent the total time of the nociceptive responses (mean±s.e.m.) of 10 mice per group, measured every 5 min. Significant differences from the corresponding controls are indicated by filled symbols. P<0.05 was considered statistically significant.
Figure 3
Figure 3
Effect of s.c. formalin (1.2%, 25 μl) injections into the hindpaw of mice on the time course of the nociceptive behaviour. Formalin was injected 15 min after the systemic administration of vehicle (10% DMSO in 0.9% NaCl, i.p.) (a and b), AA-5-HT (5 mg kg−1, i.p.), alone or in combination with 5′-iodoresiniferatoxin (I-RTX, 0.1 and 0.2 mg kg−1, i.p.) (a) or 5′-iodoresiniferatoxin alone (I-RTX, 0.1 and 0.2 mg kg−1, i.p.) (b). I-RTX was administered 5 min before AA-5-HT. The data represent the total time of the nociceptive responses (mean±s.e.m.) of 10 mice per group, measured every 5 min. Significant differences from the corresponding controls are indicated by filled symbols. P<0.05 was considered statistically significant.
Figure 4
Figure 4
Effect of s.c. formalin (5%, 50 μl) injections into the hindpaw of rat on the time course of the nociceptive behaviour. Formalin was injected 5 min after vehicle (10% DMSO in 0.9% NaCl, s.c. intra paw), AA-5-HT (1 mg per rat, s.c. intra paw) alone or in combination with AM251 (0.35 mg per rat, s.c. intra paw) or capsazepine (CPZ, 50 μg per rat, s.c. intra paw) into the dorsal surface of the hindpaw (a). AM251 and CPZ were co-injected with AA-5-HT. (b) The effect or lack thereof of AM251 (0.35 mg per rat, s.c. intra paw) or capsazepine (CPZ, 50 μg per rat, s.c. intra paw) on the time course of the nociceptive behaviour induced by formalin. In (c) AA-5-HT (1 mg rat−1, s.c.) was administered into the hindpaw 20 min before formalin. The data represent the total time of the nociceptive responses (mean±s.e.m.) of 10 rats per group, measured every 5 min. Significant differences from the corresponding controls are indicated by filled symbols. P<0.05 was considered statistically significant.
Figure 5
Figure 5
Effect of s.c. formalin (5%, 50 μl) injections into the hindpaw of rats on the time course of the nociceptive behaviour. Formalin was injected 15 min after the systemic administration of vehicle (10% DMSO in 0.9% NaCl, i.p.) (a and b), AA-5-HT (5 mg kg−1, i.p.), alone or in combination with AM251 (3 mg kg−1, i.p.) or capsazepine (CPZ, 2.5 mg kg−1, i.p.). AM251 and CPZ were administered 5 min before AA-5-HT. The effect, or lack thereof, of AM251 (3 mg kg−1 i.p.) and capsazepine (CPZ, 2.5 mg kg−1, i.p.) alone on the nociceptive behaviour induced by formalin are shown in (b). The data represent the total time of the nociceptive responses (mean±s.e.m.) of 10 rats per group, measured every 5 min. Significant differences from the corresponding controls are indicated by filled symbols. P<0.05 was considered statistically significant.
Figure 6
Figure 6
Effect of s.c. formalin (5%, 50 μl) injections into the hindpaw of rats on the time course of the nociceptive behaviour. Formalin was injected 15 min after the systemic administration of vehicle (10% DMSO in 0.9% NaCl, i.p.) (a and b), AA-5-HT (5 mg kg−1, i.p.), alone or in combination with I-RTX (0.1 and 0.2 mg kg−1, i.p.) (a) or I-RTX (0.1 and 0.2 mg kg−1, i.p.) (b). I-RTX was administered 5 min before AA-5-HT. The data represent the total time of the nociceptive responses (mean±s.e.m.) of 10 rats per group, measured every 5 min. Significant differences from the corresponding controls are indicated by filled symbols. P<0.05 was considered statistically significant.
Figure 7
Figure 7
Effects of 7-day repeated treatment with vehicle (veh, 10% DMSO in 0.9% NaCl, s.c.), URB597 (3 mg kg−1 s.c.), OL-135 (3 mg kg−1 s.c.) or AA-5-HT (5 mg kg−1 s.c.) on mechanical withdrawal threshold (a) and thermal withdrawal latency (b) in sham and CCI rats. (c and d) The effects in sham and CCI rats of 3-day repeated treatment with vehicle (veh, 10% DMSO in 0.9% NaCl, s.c.), URB597 (3 mg kg−1 s.c.), OL135 (3 mg kg−1 s.c.) or AA-5-HT (5 mg kg−1 s.c.) on mechanical withdrawal threshold and thermal withdrawal latency, respectively. Each point represents the mean±s.e.m. of 10 animals per group. (*) Indicates significant differences vs sham/veh, (°) significant differences vs CCI/veh. P-values <0.05 were considered statistically significant.
Figure 8
Figure 8
Effects of 7-day treatment with vehicle (veh, 10% DMSO in 0.9% NaCl, s.c.), capsazepine (CPZ, 2.5 and 10 mg kg−1, s.c.), AM251 (1 mg kg−1, s.c.), AA-5-HT (5 mg kg−1 s.c.) alone or in combination with AM251 (1 mg kg−1, s.c.), AM630 (1 mg kg−1, s.c.) or capsazepine (CPZ, 2.5 and 10 mg kg−1, s.c.) on mechanical withdrawal threshold (a) and on thermal withdrawal latency (b) in CCI rats. Each point represents the mean±s.e.m. of 10 animals per group. (*) Indicates significant differences vs veh, (°) significant differences vs AA-5-HT (5 mg kg−1, s.c.). P-values <0.05 were considered statistically significant.

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