TRPV1 in brain is involved in acetaminophen-induced antinociception

Abstract

Background: Acetaminophen, the major active metabolite of acetanilide in man, has become one of the most popular over-the-counter analgesic and antipyretic agents, consumed by millions of people daily. However, its mechanism of action is still a matter of debate. We have previously shown that acetaminophen is further metabolized to N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z -eicosatetraenamide (AM404) by fatty acid amide hydrolase (FAAH) in the rat and mouse brain and that this metabolite is a potent activator of transient receptor potential vanilloid 1 (TRPV(1)) in vitro. Pharmacological activation of TRPV(1) in the midbrain periaqueductal gray elicits antinociception in rats. It is therefore possible that activation of TRPV(1) in the brain contributes to the analgesic effect of acetaminophen.

Methodology/principal findings: Here we show that the antinociceptive effect of acetaminophen at an oral dose lacking hypolocomotor activity is absent in FAAH and TRPV(1) knockout mice in the formalin, tail immersion and von Frey tests. This dose of acetaminophen did not affect the global brain contents of prostaglandin E(2) (PGE(2)) and endocannabinoids. Intracerebroventricular injection of AM404 produced a TRPV(1)-mediated antinociceptive effect in the mouse formalin test. Pharmacological inhibition of TRPV(1) in the brain by intracerebroventricular capsazepine injection abolished the antinociceptive effect of oral acetaminophen in the same test.

Conclusions: This study shows that TRPV(1) in brain is involved in the antinociceptive action of acetaminophen and provides a strategy for developing central nervous system active oral analgesics based on the coexpression of FAAH and TRPV(1) in the brain.

Figure 1. Dose-effect relationship of acetaminophen on locomotor activity and nociception in mice.

Left and right panels of the figure show the effects of acetaminophen after oral (p.o.) and intraperitoneal (i.p.) administration, respectively. (A) Animals were placed in actimetry boxes (Actisystem, Apelex) and their movements were assessed by determining the number of crossings of light beams during 15 min. Intraperitoneal doses induced a higher reduction in spontaneous activity than oral doses. The test was performed 20 min after acetaminophen administration. Data are given as mean ± SEM (n = 6). *P<0.05, **P<0.01 compared to vehicle. (B–D) Mice were submitted to chemical (B), thermal (heat; C) and mechanical (D) stimuli before and 20 min after oral or intraperitoneal administration of acetaminophen at the highest dose that was without effect on locomotor activity (200 mg/kg p.o. and 100 mg/kg i.p.). Data are given as mean ± SEM (n = 6). *P<0.05, **P<0.01 compared to vehicle (A, B) or before treatment (C, D).

Figure 2. The antinociceptive effect of acetaminophen is dependent on FAAH.

The effect of acetaminophen (AcAP; 200 mg/kg p.o.) was assessed in (A) the formalin test (B) the tail immersion test and (C) the von Frey test. In FAAH^−/− mice, acetaminophen failed to produce antinociception, while it was effective in FAAH^+/+ mice in the same tests. All tests were performed 20 min after acetaminophen administration. In the tail immersion and von Frey test tests, results are expressed as MPE (%): [(post-treatment score – pre-treatment score)/(cut-off value – pre-treatment score) ×100]. Basal pre-treatment threshold responses were 7.73±0.32 and 8.05±0.29 s in the tail immersion test and 0.55±0.03 and 0.62±0.06 g in the von Frey test for FAAH^+/+ and FAAH^−/− mice, respectively. Data are presented as mean ± SEM (n = 6–8 per group). *P<0.05, **P<0.01 compared to vehicle.

Figure 3. Comparison of the effects of acetaminophen and ibuprofen in various pain tests.

(A) The antinociceptive effects of oral administration of acetaminophen (AcAP; 200 mg/kg in mice and 300 mg/kg in rats) and intraperitoneal injection of ibuprofen (Ibu; 100 mg/kg) were assessed in (A,B) the formalin test, (C) the tail immersion test and (D) the von Frey test. In the first phase of the formalin test as well as the von Frey and the tail immersion tests, acetaminophen produced an antinociceptive effect, whereas ibuprofen was ineffective. Acetaminophen and ibuprofen inhibited the nociceptive behavior during the second phase of the formalin test. In the tail immersion and von Frey test tests, results are expressed as MPE (5): [(post-treatment score – pre-treatment score)/(cut-off value – pre-treatment score) ×100]. Basal pre-treatment threshold responses were 7.29±0.43 s in the tail immersion test and 0.67±0.08 g in the von Frey test, respectively. All tests were performed 20 min after acetaminophen or ibuprofen administration. Data are presented as mean ± SEM (n = 6–8 per group). *P<0.05, **P<0.01 compared to vehicle.

Figure 4. The acetaminophen metabolite AM404 produces antinociception by activation of TRPV₁ in the brain.

(A) Intracerebroventricular (i.c.v.) injection of AM404 (10 nmol) 5 min before injection of formalin into the mouse hind paw decreased biting and licking during the first, but not the second phase of the formalin test (n = 5–6). When AM404 (10 nmol) was administered 10 min after formalin, it also decreased the second phase (15 to 30 min post-formalin) (n = 10–12). (B) The role of TRPV₁ in the action of AM404 was further investigated on the first phase of the formalin test. The effect of AM404 (10 nmol) was inhibited by coinjection of AM404 with capsazepine (Cz; 100 nmol) and absent in TRPV₁ ^−/− mice (n = 5–8). (C) Injection of AM404 into the mouse hind paw induced a pronounced nocifencive behavior, as recorded during 5 min after the injections (n = 5–6). The nociceptive response to intraplantar injection AM404 (60 nmol) was absent in TRPV₁ ^−/− mice (n = 6). Data are presented as mean ± SEM. *P<0.05, ***P<0.001 compared to vehicle in wild-type mice.

Figure 5. The antinociceptive effect of acetaminophen is dependent on TRPV₁ in brain.

The effect of acetaminophen (AcAP; 200 mg/kg p.o.) was assessed in (A) the formalin test, (B) the tail immersion test and (C) the von Frey test. In TRPV₁ ^−/− mice, acetaminophen failed to produce antinociception, while it was effective in TRPV₁ ^+/+ mice in the same tests. All tests were performed 20 min after acetaminophen administration. In the tail immersion and von Frey test tests, results are expressed as MPE (%): [(post-treatment score – pre-treatment score)/(cut-off value – pre-treatment score) ×100]. Basal pre-treatment threshold responses were 7.59±0.79 and 8.63±0.88 s in the tail immersion test and 0.67±0.08 and 0.68±0.10 g in the von Frey test for TRPV₁ ^+/+ and TRPV₁ ^−/− mice, respectively. (D) The effect of acetaminophen (300 mg/kg p.o.) in the formalin test was also examined in rats pre-treated with capsazepine (Cz; 10 mg/kg i.p.) or vehicle 45 min before injection of formalin. Pre-treatment with capsazepine suppressed the antinociceptive effect of acetaminophen. (E) The antinociceptive effect of acetaminophen (AcAP; 200 mg/kg) on both phases of the formalin test was inhibited by intracerebroventricular injection of capsazepine (100 nmol) 5 min before oral administration of acetaminophen. Data are presented as mean ± SEM (n = 5–8 per group). *P<0.05, **P<0.01, ***P<0.001 compared to vehicle.

Figure 6. Mechanism behind the TRPV₁-mediated antinociceptive effect of acetaminophen.

Acetaminophen is metabolized to p-aminophenol (p-AP) mainly in the liver. p-Aminophenol is subsequently conjugated with arachidonic acid (AA) in FAAH-containing cells in the nervous system, including neurons expressing TRPV₁, leading to the formation of the TRPV₁ activator AM404. As suggested for capsaicin, AM404 may activate TRPV₁ from inside of the cell , . In contrast to AM404, acetaminophen and p-aminophenol do not directly interact with TRPV₁ . Activation of TRPV₁ could produce antinociception by stimulation of bulbospinal descending inhibitory pathways in the periaqueductal gray .