CB1 receptor-dependent desensitisation of TRPV1 channels contributes to the analgesic effect of dipyrone in sensitised primary sensory neurons

Abstract

Background and purpose: While dipyrone is a widely used analgesic, its mechanism of action is not completely understood. Recently, we have reported that the dipyrone metabolite 4-aminoantipyrine (4-AA) reduces PGE₂ -induced pain-related behaviour through cannabinoid CB₁ receptors. Here, we ascertained, in naive and PGE₂ -induced "inflamed" conditions, both in vivo and in vitro, the molecular mechanisms involved in the 4-AA-induced analgesic effects.

Experimental approach: The effect of local administration of 4-AA (160 μg per paw) on capsaicin (0.12 μg per paw) injection-induced pain-related behaviour and 4-AA's effect on 500-nM capsaicin-induced changes in intracellular calcium concentration ([Ca²⁺ ]_i ) in cultured primary sensory neurons were assessed in vivo and in vitro, respectively.

Key results: 4-AA reduced capsaicin-induced nociceptive behaviour in naive and inflamed conditions through CB₁ receptors. 4-AA (100 μM) reduced capsaicin-induced increase in [Ca²⁺ ]_i in a CB₁ receptor-dependent manner, when PGE₂ was not present. Following PGE₂ application, 4-AA (1-50 μM) increased the [Ca²⁺ ]_i . Although 4-AA activated both TRPV1 and TRPA1 channels, increased [Ca²⁺ ]_i was mediated through TRPV1 channels. Activation of TRPV1 channels resulted in their desensitisation. Blocking CB₁ receptors reduced both the excitatory and desensitising effects of 4-AA.

Conclusion and implications: CB₁ receptor-mediated inhibition of TRPV1 channels and TRPV1-mediated Ca²⁺ -influx- and CB₁ receptor-dependent desensitisation of TRPV1 channels contribute to the anti-nociceptive effect of 4-AA in naive and inflamed conditions respectively. Agonists active at both CB₁ receptors and TRPV1 channels might be useful as analgesics, particularly in inflammatory conditions.

Keywords: CB1; TRPA1; TRPV1; calcium imaging; dipyrone; dorsal root ganglion.

FIGURE 1

4‐aminoantipyrine (4‐AA) reduces the excitatory effect of capsaicin through CB₁ receptor activation in naive (non‐inflamed) conditions. (a) Local administration of capsaicin (CAP; 0.12 μg per paw) induces flinches. The capsaicin‐induced nociceptive behaviour is reduced by the administration of 4‐AA (160 μg per paw). AM251 (50 μg per paw) reversed the anti‐nociceptive effect of 4‐AA. *P< .05, significantly different from 0.9% NaCl, vehicle and (capsaicin+4‐AA). # P< .05, significantly different from 0.9% NaCl, vehicle, capsaicin and (capsaicin+4‐AA+AM251); one‐way ANOVA, with Tukey's test. (b–d) Representative recordings of calcium imaging from cultured primary sensory neurons. Capsaicin (500 nM) induces a rise in the intracellular calcium concentration ([Ca²⁺]_i; (b)) that is reversed by 4‐AA (100 μM; (c)). AM251 (10 μM) reversed the inhibitory effect of 4‐AA on the capsaicin‐induced calcium‐influx (ANOVA, Tukey's test, P < .05; (d)). (e) Average normalised amplitudes of capsaicin‐evoked changes in the [Ca²⁺]_i in various conditions. (f) Proportions of neurons responding the capsaicin at various conditions; 4‐AA reduces the proportion of neurons that respond to capsaicin and AM251 reduces the inhibitory effect of 4‐AA (Fisher's exact test, P < .05). In (e–f), *P< .05, significantly different from capsaicin and (capsaicin+4‐AA+AM251); one‐way ANOVA, with Tukey's test

FIGURE 2

PGE₂ increases capsaicin‐induced pain‐related behaviour and capsaicin‐induced increase in the [Ca²⁺]_i in primary sensory neurons. (a) and (b) Local administration of capsaicin (0.12 μg per paw) into subcutaneous tissues of the hind paw induces pain‐related behaviour. Pretreatment with PGE₂ (100 nM, 150 min) increases the capsaicin‐induced responses. *P< .05, significantly different from capsaicin alone; one‐way ANOVA with Tukey's test. (c, d) Typical recordings on changes in the [Ca²⁺]_i in cultured primary sensory neurons. The pretreatment with PGE₂ (1 μM, for 5 min) increases capsaicin‐induced increase in the [Ca²⁺]_i (d) when compared to responses without pretreatment (c). (e) Average normalised amplitudes of capsaicin‐evoked responses in cultured primary sensory neurons without and with pretreatment with PGE₂. *P< .05, significantly different from capsaicin alone; one‐way ANOVA with Tukey's test. (f) PGE₂ pretreatment does not affect the number of capsaicin‐responsive neurons (Fisher's exact test, P < .05)

FIGURE 3

4‐AA reduces capsaicin‐induced pain‐related behaviour following PGE₂ pretreatment. (a, b) 4‐AA reduces capsaicin‐induced pain‐related behavioural responses following pretreatment with PGE₂. *P< .05, significantly different from (PGE₂+capsaicin), #P< .05, significantly different from (PGE₂+capsaicin+4‐AA); one‐way ANOVA with Tukey's test

FIGURE 4

4‐AA increases [Ca²⁺]_i through activation of TRPV1 channels in cultured primary sensory neurons following pretreatment of the cells with PGE₂. (a) 4‐AA (50, 25, 10, and 1 μM) increases [Ca²⁺]_i in primary sensory neurons, sensitised by PGE₂ in a concentration‐dependent manner. (b) The proportion of 4‐AA‐responding cells is also increased following PGE₂ pretreatment. *P< .05, significant effect of 4‐AA. (c–f) Representative recordings of changes in the [Ca²⁺]_i by various conditions. Following PGE₂ pretreatment, 4‐AA (50 μM) induces an increase in the [Ca²⁺]_i in the presence of PGE₂ (c). While the 4‐AA‐induced increases in the [Ca²⁺]_i were not affected by the TRPA1 channel antagonist HC‐030031 (10 μM) in the presence of PGE₂ (1 μM), at the start of washing PGE₂, HC‐030031 and 4‐AA out from the recording chamber, a “buffer‐evoked” calcium transient emerged (d). Importantly, no such “buffer‐evoked” calcium transient was observed when 4‐AA was missing from the superfusate (e). The TRPV1 channel antagonist capsazepine (CAPZ; 10 μM), but not the TRPA1 channel antagonist, blocked the 4‐AA‐evoked calcium transient in the presence of PGE₂ (f). (g) Average normalised amplitudes of responses of cultured primary sensory neurons in various conditions. Data shown are means SEM from a number of neurons, as follows: 4‐AA, n = 156; PGE₂ +4‐AA, n = 156; PGE₂+4‐AA+CAPZ, n = 142; PGE₂+4‐AA+HC, n = 100; PGE₂ +CAPZ, n = 115; PGE₂+HC, n = 289: PGE₂, n = 156. * P< .05, significant differences; one‐way ANOVA with Tukey's test

FIGURE 5

4‐AA desensitises TRPV1 channels, involving CB₁ receptors. (a) Average normalised amplitudes of primary sensory neurons to 4‐AA (1–50 μM) or capsaicin (CAPS; 500 nM) with and without PGE₂ (1 μM) pretreatment. Note that 4‐AA applied before capsaicin, at and above 10 μM, significantly reduces the amplitude of capsaicin‐evoked responses. *P< .05, significantly different from capsaicin alone; one‐way ANOVA with Tukey's test. (b) Representative recording of changes in the [Ca²⁺]_i from a cultured primary sensory neuron by the application of 4‐AA (10 μM), capsaicin (CAP; 500 nM) in the presence of PGE₂ (1 μM). Note that capsaicin induces only a small response. (c) Average normalised amplitudes of primary sensory neurons to 4‐AA (10 μM) and capsaicin (500 nM) in the presence of PGE₂ (1 μM) and without or with AM251 (10 μM) pretreatment. Note the opposing changes in the 4‐AA‐ and 4‐AA + capsaicin‐evoked increase in the [Ca2+]_i by AM251; while the 4‐AA‐induced responses are decreased, the 4‐AA + capsaicin‐evoked responses are increased. *P< .05, significantly different from capsaicin, #P< .05, significantly different from capsaicin, †P< .05, significantly different from 4‐AA, §P< .05, significantly different from (capsaicin+4‐AA); one‐way ANOVA with Tukey's test. (d) A typical recording of changes in the [Ca²⁺]_i by 4‐AA (10 μM) and capsaicin (500 nM) in the presence of PGE₂ (1 μM) and AM251 (10 μM) from a cultured primary sensory neuron. (e) AM251 (10 μM) attenuates the effect of 4‐AA on the number of capsaicin‐responding cells whereas it has no effect on the number of cells responsive to 4‐AA (Fisher's exact test, P < .05). *P< .05, significantly different from capsaicin, #P< .05, significantly different from capsaicin; one‐way ANOVA with Tukey's test