**3.4. Cav3.2 calcium channel**

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 analgesic effect in these knockout mice.

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 dependent on Cav3.2 located in the brain.

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

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 of AM404 is TRPV1.

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 human Cav3.2 sequence. In these cells, the Cav3.2 current induced by depolarization was not affected by the bath application of capsaicin. However, when the cells were transfected with TRPV1, application of capsaicin suppressed the Cav3.2 current.

Altogether, these behavioral and electrophysiological findings show that Cav3.2 and TRPV1 act sequentially in concert to support the analgesic action of paracetamol [30].
