Endocannabinoids mediate anxiolytic-like effect of acetaminophen via CB1 receptors

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Abstract

Acetaminophen (Paracetamol), a most commonly used antipyretic/analgesic agent, is metabolized to AM404 (N-arachidonoylphenolamine) that inhibits uptake and degradation of anandamide which is reported to mediate the analgesic action of acetaminophen via CB1 receptor. AM404 and anandamide are also reported to produce anxiolytic-like behavior. In view of the implication of endocannabinoids in the effect of acetaminophen, we contemplated that acetaminophen may have anxiolytic-like effect. Therefore, this possibility was tested by observing the effects of various doses of acetaminophen in mice on anxiety-related indices of Vogel conflict test and social interaction test. The results from both the tests indicated that acetaminophen (50, 100, or 200 mg/kg, i.p.) or anandamide (10 or 20 µg/mouse, i.c.v.) dose dependently elicited anxiolytic-like effect, that was comparable to diazepam (2 mg/kg, i.p.). Moreover, co-administration of sub-effective dose of acetaminophen (25 mg/kg, i.p.) and anandamide (5 µg/mouse, i.c.v) produced similar anxiolytic effect. Further, pre-treatment with AM251 (a CB1 receptor antagonist; 1 mg/kg, i.p.) antagonized the effects of acetaminophen and anandamide with no per se effect at 1 mg/kg dose, while anxiogenic effect was evident at a higher dose (5 mg/kg, i.p.). None of the treatment/s was found to induce any antinociceptive or locomotor impairment effects. In conclusion, the findings suggested that acetaminophen (50, 100, or 200 mg/kg, i.p.) exhibited dose dependent anxiolytic effect in mice and probably involved endocannabinoid-mediated mechanism in its effect.

Introduction

Acetaminophen (Paracetamol) is a household analgesic/antipyretic agent with weak anti-inflammatory activity and low incidence of adverse effects on gastrointestinal tract as compared to other non-steroidal anti-inflammatory drugs. This effect has been attributed to the inhibition of cyclo-oxygenase activity (Muth-Selbach et al., 1999, Ouellet and Percival, 2001), decrease in nitric oxide synthesis (Bjorkman et al., 1994) and facilitation of serotonergic system (Pini et al., 1996, Bonnefont et al., 2003). However, none of the effects are sufficient enough to explain the therapeutic effect of acetaminophen and hence its mechanism of action is still debatable.

Recently it was shown that pre-treatment of rats with CB1 receptor antagonist eliminated the analgesic effect of acetaminophen, thus implicating the role of endocannabinoids in its action (Ottani et al., 2006, Dani et al., 2007, Mallet et al., 2008). The proposed involvement of endocannabinoid is not surprising particularly because acetaminophen is known to metabolize to N-arachidonoylphenolamine (AM404) (Högestätt et al., 2005), which is reported to inhibit the cellular uptake (Beltramo et al., 1997, Giuffrida et al., 2001) and degradation (Glaser et al., 2003) of anandamide — an endocannabinoid. The agents that inhibit anandamide transport, or prevent its degradation by inhibiting FAAH are shown to enhance the CB1 receptor-mediated behavioral effects of anandamide (Giuffrida et al., 2000, Kathuria et al., 2003). Moreover, anandamide and AM404 both are reported to exhibit antinociceptive and hypothermic effect in rodents (Sulcova et al., 1998, Costa et al., 1999, Rawls et al., 2006, Mitchell et al., 2007).

Another well demonstrated effect of anandamide includes its influence on anxiety-related behavior. It produces anxiolytic-like behavior in experimental animals via CB1 receptors (Moreira et al., 2007, Lisboa et al., 2008). In addition, stress-subjected animals that exhibited anxiogenic-like behavior were found to have low levels of endocannabinoids (Patel et al., 2004). Anandamide might also induce anxiogenic-like responses in rodents when administered at high doses by activating TRPV1 receptors (Rubino et al., 2008), which has been shown to contribute to anxiogenic responses in mice (Marsch et al., 2007, Di Marzo et al., 2008, Micale et al., 2009). However, a recent study has shown that anandamide induced anxiogenic-like effect in open field was not blocked by capasazapine — a TRPV1 blocker (Panlilio et al., 2009). In addition, AM404 is also shown to produce anxiolytic-like, and that was attenuated by CB1 receptor antagonist (Bortolato et al., 2006). Similar to anandamide, whether AM404 has any anxiogenic effect at any higher dose is not known.

In view of the fact that acetaminophen favours the accumulation of anandamide via AM404 (Högestätt et al., 2005); and anandamide can produce anxiolytic-like (Moreira et al., 2007, Lisboa et al., 2008), we speculated that acetaminophen may also influence anxiety-related behavior by possibly involving endocannabinoids. Feel good effect of acetaminophen in neuropathy patients (Murray, 1973) and rewarding effect in rats (Abott and Helleman, 2000) indicate such possibility.

We tested this possibility by studying the influence of acetaminophen on anxiety-related behavior in mice by employing Vogel conflict test and social interaction test. The involvement of endocannabinoids was further tested by using CB1 blockers.

Section snippets

Animals

The experiments were carried out in strict accordance with the guidelines approved by the Institutional Animal Ethical Committee constituted for the purpose of control and supervision of experimental animals under Ministry of Environment and Forests, Government of India, New Delhi, India. The investigations were carried out in young healthy male Swiss mice (22–25 g), born and reared in the animal house of Department of Pharmaceutical Sciences; RTM Nagpur University, Nagpur, from a stock

Influence of acetaminophen, diazepam, anandamide and AM251 on anxiety and locomotor activity

In Vogel conflict test, statistical analysis with one-way ANOVA revealed that the treatments had a significant effect {acetaminophen: [F(5, 36) = 27.44, P < 0.0001]; anandamide [F(6, 42) = 44.27, P < 0.0001]; AM251 [F(5, 36) = 39.59, P < 0.0001]} (Fig. 1). Post hoc test revealed that acetaminophen (50, 100 and 200 mg/kg, i.p.), anandamide (10 and 20 µg/mouse, i.c.v.) and diazepam (2 mg/kg, i.p.) caused significant increase in the number of shocks per 3 min when compared to the respective vehicle group,

Discussion

The present investigations revealed that acetaminophen induced anxiolytic-like effect in mice, as indicated by dose dependent increase in the number of shocks per 3 min in Vogel conflict test and increase in interaction time in social interaction test. In a similar set of experiment, i.c.v. administration of anandamide showed anxiolytic-like effect in lower doses, while produced anxiogenic effect at higher dose. This biphasic response of anandamide is well in line with the earlier reports (

Conclusion

Taken together, the present investigation revealed for the first time that acetaminophen produces anxiolytic-like effect that could be consequent to the activation of CB1 receptors via endocannabinoid system. However, actual estimation of anandamide in anxiety-related brain regions would further support the involvement of endocannabinoid in acetaminophen-induced anxiolytic-like effect.

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