**4. Involvement of the serotonergic system**

was due to metabolization of *p*-aminophenol into AM404 because *p*-aminophenol-induced

To accurately establish the location of the involvement of TRPV1 in paracetamol action, systemic administration of paracetamol was challenged with the selective blockade of TRPV1 in the brain. Injection of capsazepine into the lateral ventricle of mice abolished the antinociceptive effects of paracetamol [7]. Similarly, the antinociceptive activity of *p*-aminophenol was also lost in mice intracerebroventricularly preinjected with capsazepine [6]. Collectively,

Arachidonic-related compounds such as anandamide and 2-arachidonylglycerol also interact with T-type calcium channels, especially the Cav3.2 subtype, an effect which mediates their analgesic property [32]. Silencing of Cav3.2 using oligonucleotide antisense [33], knockout mice [34], or pharmacological tools [35] resulted in impairment of pain in several pain tests, thereby confirming the strong role of this calcium channel in nociception. Because AM404 is the arachidonicrelated metabolite of paracetamol, the role of Cav3.2 in paracetamol action was investigated [30].

Mice with deletion of the Cav3.2−/− gene did not show any analgesic effect after paracetamol administration. In addition, the intracerebroventricular injection of AM404 did not induce an

To determine whether Cav3.2 in the brain is involved in the antinociceptive effect of paracetamol, we injected TTA-A2, a Cav3.2 blocker, intracerebroventricularly before administration of paracetamol. This treatment prevented the effect of paracetamol. Spinal involvement of Cav3.2 receptors was also studied by coadministering paracetamol with an intrathecal injection of TTA-A2. In contrast to the previous results, spinal blockade of Cav3.2 did not alter the analgesic effect of paracetamol, indicating that the antinociceptive effect of paracetamol is

AM404 seems to have an indirect action because it only weakly inhibited Cav3.2 currents (IC50 = 13.7 μM) recorded in DRG neurons by a whole-cell patch clamp method [30]. By comparison, in the same assay, TTA-A2 had an IC50 of 9.0 nM. As expected, neither paracetamol

We thus addressed the putative role of TRPV1, another calcium channel, in the mobilization of Cav3.2 in the analgesic action of paracetamol. To determine whether Cav3.2 was involved upstream or downstream of the action of TRPV1, we assessed the analgesic effect of intracerebroventricular injection of either TRPV1 agonist (capsaicin) or Cav3.2 antagonist (TTA-A2) in Cav3.2−/− and TRPV1−/− mice, respectively. Unlike the action of TTA-A2, which is maintained in TRPV1−/− mice, the analgesic effect of capsaicin is lost in Cav3.2−/− mice. These results show that brain TRPV1 activation needs Cav3.2 to mediate its action and suggest that the first target

To analyze more fully the relationship between TRPV1 and Cav3.2 channels, we performed electrophysiological recordings to study the Cav3.2 current in HEK cells stably expressing the

calcium mobilization was lost in cells pretreated with an FAAH inhibitor.

these findings identify brain TRPV1 as an important effector of paracetamol.

**3.4. Cav3.2 calcium channel**

212 Pain Relief - From Analgesics to Alternative Therapies

analgesic effect in these knockout mice.

dependent on Cav3.2 located in the brain.

nor *p*-aminophenol inhibited Cav3.2 currents.

of AM404 is TRPV1.

The involvement of the serotonergic system in the action of paracetamol was first described by Tjolsen et al. [37] in 1991 and by Pini et al. [38] in 1996. They demonstrated that the analgesic effect of paracetamol was reduced after lesion of the serotonergic bulbospinal pathway by 5,6-dihydroxytryptamine or total depletion of the central serotonin (5-HT) synthesis by *p*-chlorophenylalanine. These results were confirmed by another team using 5,7-dihydroxytryptamine [39]. Studies showing that paracetamol did not bind serotonin receptors [38, 40] prompted investigation of the mobilization of the serotonin neurotransmitter. The results showed that paracetamol increased in a dose-dependent manner the tissue concentrations of 5-HT in the cortex, hypothalamus, striatum, hippocampus and brainstem [38, 41].

Later studies showed that the spinal role of 5-HT1A, 5-HT2A, 5-HT2C and 5-HT<sup>7</sup> receptor subtypes of serotonin receptors was involved in the action of paracetamol [39, 42–48]. However, investigations of the involvement of 5-HT3 receptors yielded conflicting results in both animals [36, 42, 43, 47, 49, 50] and humans [51–54]. Interestingly, some of these studies showed that tropisetron, a nonspecific 5-HT<sup>3</sup> receptor antagonist, blocked the analgesic effect of paracetamol. Libert et al. [36] reported that the inhibitory effect of tropisetron on the action of paracetamol was not mediated by 5-HT<sup>3</sup> receptor because (1) other 5-HT<sup>3</sup> antagonists (granisetron and ondansetron) or (2) antisense oligodeoxynucleotides directed against 5-HT<sup>3</sup> receptors did not reverse the paracetamol-induced antinociceptive effect, which suggests the involvement of a spinal tropisetron-sensitive receptor that is not the 5-HT3 receptor. More work is needed to identify this spinal receptor.

These results should be treated with caution. Serotonin receptor subtypes are differently involved in paracetamol action, depending on the nature of the stimulus. For example, spinal 5-HT1A is involved in the analgesic action of paracetamol assessed in the formalin test (chemical stimulus) [44] but not in the paw pressure test (mechanical stimulus) [47]. This discrepancy could be explained by the differential efficacy and power of serotonin itself relative to the noxious tests [55]. In addition, the analgesic action of spinal-administered serotonin, like that of paracetamol, is suppressed in the formalin test [44, 45] and conserved in the paw pressure test [45, 56] following the inhibition of spinal 5-HT1A receptors.

Like paracetamol, *p*-aminophenol elicited antinociception through the serotonergic bulbospinal pathway because its effect was reversed after lesion of the pathway by 5,7-dihydroxytryptamine [6]. In addition, spinal pretreatment of rats with WAY-100,635, a 5-HT1A receptor antagonist and tropisetron, a nonspecific 5-HT3/4 receptor antagonist, reduced the analgesic effect of *p*-aminophenol in the formalin test and the paw pressure test, respectively [6].

In light of evidence showing that paracetamol and *p*-aminophenol involved CB1 receptors [5], we investigated the serotonergic descending bulbospinal pathways and spinal 5-HT receptors in the antinociceptive effect of arachidonyl-2′-chloroethylamide (ACEA), a CB1 receptor agonist. Our results showed that ACEA needed intact descending bulbospinal serotonergic pathways. Elsewhere, it was shown that the antinociceptive action of ACEA was suppressed by intrathecal injection of WAY-100,635 and tropisetron in the formalin test and the paw pressure test, respectively [5]. The similar serotonergic profiles of ACEA and paracetamol suggest that CB1 receptor is an important link between paracetamol and serotonin in the production of antinociception.
