Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming

Abstract

We have recently described a novel form of hyperalgesic priming (type II) induced by agonists at two clinically important Gi-protein-coupled receptors (Gi-GPCRs), mu-opioid and A1-adenosine. Like mu-opioids, the antimigraine triptans, which act at 5-HT1B/D Gi-GPCRs, have been implicated in pain chronification. We determined whether sumatriptan, a prototypical 5-HT1B/D agonist, produces type II priming. Characteristic of hyperalgesic priming, intradermal injection of sumatriptan (10 ng) induced a change in nociceptor function such that a subsequent injection of prostaglandin-E2 (PGE2) induces prolonged mechanical hyperalgesia. However, onset to priming was delayed 3 days, characteristic of type I priming. Also characteristic of type I priming, a protein kinase Cε, but not a protein kinase A inhibitor attenuated the prolongation phase of PGE2 hyperalgesia. The prolongation of PGE2 hyperalgesia was also permanently reversed by intradermal injection of cordycepin, a protein translation inhibitor. Also, hyperalgesic priming did not occur in animals pretreated with pertussis toxin or isolectin B4-positive nociceptor toxin, IB4-saporin. Finally, as observed for other agonists that induce type I priming, sumatriptan did not induce priming in female rats. The prolongation of PGE2 hyperalgesia induced by sumatriptan was partially prevented by coinjection of antagonists for the 5-HT1B and 5-HT1D, but not 5-HT7, serotonin receptors and completely prevented by coadministration of a combination of the 5-HT1B and 5-HT1D antagonists. Moreover, the injection of selective agonists, for 5-HT1B and 5-HT1D receptors, also induced hyperalgesic priming. Our results suggest that sumatriptan, which signals through Gi-GPCRs, induces type I hyperalgesic priming, unlike agonists at other Gi-GPCRs, which induce type II priming.

Figure 1. Sumatriptan induces mechanical hyperalgesia and hyperalgesic priming in male rats

A. Rats were treated with a single intradermal injection of vehicle (5 μL; black bar) or sumatriptan (10 ng in 5 μL; white bar) and 30 min later, mechanical nociceptive threshold was evaluated by the Randall-Sellitto paw withdrawal test. The group treated with sumatriptan showed significant mechanical hyperalgesia, when compared with the group treated with vehicle (***p < 0.0001; Unpaired Student's t-test), indicating that sumatriptan produces a pronociceptive effect. Average mechanical nociceptive threshold before the injection was 126.2 ± 2.7 g, for vehicle, and 118.5 ± 1.5 g, for sumatriptan group. B. Twenty-four hours later, when the mechanical nociceptive threshold was no longer different from pre-injection baseline (t₅ = 1.667; p = 0.1942, for the vehicle group; t₅ = 0.2116; p = 0.8460, for the sumatriptan group; paired Student's t-test), PGE₂ (100 ng) was injected intradermally at the same site on the dorsum of the hind paw, and mechanical nociceptive threshold evaluated 30 min and 4 h later. In both groups PGE₂ induced significant hyperalgesia at 30 min, which was no longer present at the 4^th h, in the both groups (NS, p > 0.05, when both groups are compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that hyperalgesic priming was not present 24 hours after the injection of vehicle or sumatriptan. However, 72 hours later (C) and 30 days later (D) when PGE₂ (100 ng) was again injected, at the same site, hyperalgesia induced by PGE₂ was present 30 min after injection in the group previously treated with sumatriptan, and was still present at the 4^th h (F_1,20 = 72.97, ***p < 0.0001, when both groups are compared at the 4^th h; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These data demonstrate that a single injection of sumatriptan, 72 hours or 30 days prior, produced long-term changes in nociceptor function characteristic of hyperalgesic priming. (N = 6 paws per group)

Figure 2. Role of 5-HT_1B and 5-HT_1D, but not 5-HT₇, receptors in sumatriptan-induced prolongation of PGE₂-induced hyperalgesia

Male rats received vehicle (5 μL; black bars), NAS-181 (1 μg; 5-HT_1B antagonist; gray bars), BRL 15572 (1 μg; 5-HT_1D antagonist; white bars), the combination of NAS-181 (1 μg)/BRL 15572 (1 μg; dotted bars) or SB-269970 (1 μg; 5-HT₇ antagonist; horizontally striped bars) on the dorsum of the hind paw. Ten minutes later, sumatriptan (10 ng) was injected at the same site. Four days later, when the mechanical nociceptive thresholds were not different from pre sumatriptan-injection control baseline (t₅ = 0.8165; p = 0.4740, for the vehicle group; t₅ = 1.260; p = 0.2967, for the NAS-181 group; t₅ = 0.3015; p = 0.7827, for the BRL 15572 group; t₅ = 1.000; p = 0.3910, for the NAS-181/BRL 15572 group; t₅ = 1.667; p = 0.1942, for the SB-269970 group; paired Student's t-test), PGE₂ (100 ng) was injected at the same site, and the mechanical nociceptive threshold evaluated. In all groups of rats evaluated 30 min after its injection PGE₂ induced significant hyperalgesia. However, in the groups previously treated with NAS-181 (gray bars) or BRL 15572 (white bars), the prolongation of PGE₂-induced hyperalgesia was significantly attenuated, and completely eliminated in the group previously treated with the combination of NAS-181/BRL 15572 (dotted bars; F_2,30 = 507.75; ***p < 0.0001, when NAS-181, BRL 15572 and NAS-181/BRL 15572 groups are compared with the vehicle-treated group; two-way repeated measures ANOVA followed by Bonferroni post hoc test), indicating the participation of both 5-HT_1B and 5-HT_1D, but not 5-HT₇, receptors in the induction of hyperalgesic priming by sumatriptan. (N = 6 paws per group)

Figure 3. Agonists for 5-HT_1B and 5-HT_1D receptors induced hyperalgesic priming

Male rats were injected intradermally with agonists for 5-HT_1B (CP-93129; 10 or 100 ng; gray bars) or 5-HT_1D (L-694,247; 10 or 100 ng; white bars) receptors. Four days later, PGE₂ (100 ng) was injected at the same site and the mechanical nociceptive threshold was evaluated 30 min and 4 h after its injection. In all groups of rats PGE₂ induced significant hyperalgesia, evaluated 30 min after injection. Also, we observed prolongation of PGE₂-induced hyperalgesia in the groups previously treated with 100 ng of agonist for 5-HT_1B (F_1,20 = 94.06, ***p < 0.0001) and 5-HT_1D (F_1,20 = 127.07, ***p < 0.0001; two-way repeated-measure ANOVA followed by Bonferroni post hoc test showed) receptor, when the 4^th h of the groups were compared. BL: baseline. (N = 6 paws per group)

Figure 4. PKCε but not PKA plays a role in the expression of hyperalgesic priming induced by sumatriptan

A. Male rats received a single injection of sumatriptan (10 ng) on the dorsum of the hind paw. One week later, when the mechanical nociceptive thresholds were not different from pre sumatriptan-injection baseline (t₅ = 0.3203; p = 0.7697, for black bars; t₅ = 0.2089; p = 0.8479, for white bars; paired Student's t-test), PGE₂ (100 ng) was injected at the same site, in the presence of vehicle (control, black bars) or PKCε inhibitor (1 μg, white bars). The mechanical nociceptive threshold was then evaluated 30 min and 4 h later, by Randall-Sellitto paw withdrawal test. In both groups PGE₂ induced significant hyperalgesia, evaluated 30 min after injection. However, we observed significant attenuation of PGE₂-induced prolongation of hyperalgesia in the group previously treated with PKCε inhibitor (F_1,15 = 132.58, ***p < 0.0001, when the vehicle and PKCε inhibitor group were compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B. A different group of male rats were primed with an intradermal injection of sumatriptan (10 ng) and, one week later, received at the same site, vehicle (5 μL) or a PKA inhibitor (H-89; 1 μg). Ten min later, PGE₂ (100 ng) was injected on the dorsum of the hind paw and the mechanical nociceptive thresholds were evaluated 30 min and 4 h later. While PGE₂-induced hyperalgesia was still present after 30 min, in the group that received vehicle, in the group treated with H-89 it was significantly attenuated. However, it was present at the 4^th h (F_2,12 = 132.95, ***p < 0.0001, when vehicle and H-89 groups are compared at 30 min; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the expression of priming induced by previous injection of sumatriptan is not dependent on PKA. BL: baseline. (N = 6 paws per group)

Figure 5. Hyperalgesic priming induced by sumatriptan is dependent on local protein translation

Male rats that were treated with intradermal injection of sumatriptan (10 ng) on the dorsum of the hind paw received, three days later, PGE₂ (100 ng) injected at the same site, in the presence of vehicle (5 μL, black bars) or the inhibitor of protein translation, cordycepin (1 μg, white bars), administered 15 min before. Mechanical nociceptive threshold was evaluated 30 min and 4 h after injection of PGE₂. While the hyperalgesia induced by PGE₂ was present 30 min after injection in the group previously treated with cordycepin, it was significantly inhibited at the 4^th hour (F_1,15 = 104.94, ***p < 0.0001, when both groups are compared at the 4^th h; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). BL: baseline. (N = 6 paws per group)

Figure 6. Role of inhibitory G-protein α_i subunit in sumatriptan-induced priming

Male rats were treated with vehicle (5 μL, black bars) or pertussis toxin (PTX; 1 μg, white bars) by intradermal injection and, 30 min later, sumatriptan (10 ng) was injected at the same side. Five days later, PGE₂ (100 ng) was injected intradermally at the same site on the dorsum of the hind paw, and the mechanical hyperalgesia was evaluated 30 min and 4 h later, by Randall-Sellitto paw withdrawal test. In both groups PGE₂ induced significant hyperalgesia, evaluated 30 min after injection. However, we observed significant attenuation of PGE₂-induced prolongation of hyperalgesia in the group previously treated with PTX (F_2,12 = 169.04, ***p < 0.0001, when the vehicle and PTX group were compared; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the α_i subunit plays a role in the induction of sumatriptan-induce priming. BL: baseline. (N = 6 paws per group)

Figure 7. Type I priming induced by sumatriptan is dependent on IB4-positive neurons

Male rats were treated with vehicle (5 μL, black bars), IB4-saporin (3.2 μg/20 μL; white bars) or SSP-saporin (100 ng/20 μL; gray bars) by intrathecal injection. Fourteen days later, sumatriptan (10 ng) was injected on the dorsum of the hind paw. Four days later, when mechanical thresholds were not different from pre-sumatriptan baseline, (t₅ = 1.663; p = 0.2010, for the vehicle group; t₅ = 1.667; p = 0.1942, for the IB4-saporin group; t₅ = 0.6547; p = 0.5799, for the SSP-saporin group; paired Student's t-test), PGE₂ (100 ng) was injected intradermally at the same site on the dorsum of the hind paw, and the mechanical hyperalgesia evaluated 30 min and 4 h later. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test showed PGE₂-induced hyperalgesia at 30 min in all groups, that was still present at the 4^th h, in vehicle (black bars) and SSP-saporin (gray bars)-treated groups, but not in IB4-saporin (white bars) treated-group, which at the 4^th hour, the PGE₂-induced hyperalgesia was significant blocked (F_1,15 = 26.95, ***p = 0.0006, when vehicle-treated groups are compared at the 4^th h with IB4-saporin-treated group; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that the prolonged hyperalgesia induced by PGE₂ observed in the priming induced by sumatriptan occurs in IB4-positive neurons. BL: baseline. (N = 6 paws per group)

Figure 8. Sumatriptan did not induce prolongation of PGE₂ hyperalgesia in female rats

Female rats received intradermal injection of sumatriptan (10 ng, white bars) or vehicle (black bars) on the dorsum of the hind paw. Three days later, when the mechanical thresholds were not different from pre-vehicle or pre-sumatriptan baseline levels (t₅ = 0.7559; p = 0.4838, for the vehicle group; t₅ = 2.150; p = 0.0842, for the sumatriptan group, paired Student's t-test), PGE₂ (100 ng) was injected at the same site, and the mechanical hyperalgesia was evaluated 30 min and 4 h later. Two way ANOVA followed by Bonferroni post hoc test showed that PGE₂ induced significant hyperalgesia at 30 min in both groups, that was not present at the 4^th h after its injection (F_1,15 = 0.83; p = 0.3779, when compared both groups). BL: baseline. (N = 6 paws per group)

Figure 9. Schematic representation of the signaling pathways involved in sumatriptan-induced type I hyperalgesic priming

As shown in “A”, the administration of sumatriptan (a 5-HT_1B/D receptor agonist), applied at the terminal of the IB4-positive nociceptor, triggers the events that will lead to mechanical hyperalgesia and the development of type I hyperalgesic priming. Activation of 5-HT_1B and 5-HT_1D receptors by administration of sumatriptan and the following activation of G-protein α_i subunit (Gα_i), ultimately producing neuroplastic changes that are expressed as prolongation of the PGE₂-induced hyperalgesia. B. PGE₂-induced hyperalgesia, which is dependent only on PKA in the normal state [34], in the primed state is prolonged due to activation of an additional pathway involving PKCε and protein translation. Abbreviations: 1B, 5-HT_1B receptor subtype; 1D, 5-HT_1D receptor subtype; PKCε, protein kinase C epsilon; PGE₂, prostaglandin-E₂.