Marked sexual dimorphism in 5-HT1 receptors mediating pronociceptive effects of sumatriptan

doi:10.1016/j.neuroscience.2016.12.031

. 2017 Mar 6:344:394-405.

doi: 10.1016/j.neuroscience.2016.12.031. Epub 2016 Dec 29.

Marked sexual dimorphism in 5-HT₁ receptors mediating pronociceptive effects of sumatriptan

Dioneia Araldi¹, Luiz F Ferrari¹, Paul Green², Jon D Levine³

Affiliations

¹ Department of Oral & Maxillofacial Surgery, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Division of Neuroscience, University of California at San Francisco, San Francisco, CA 94143-0440, United States.
² Department of Oral & Maxillofacial Surgery, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Department of Preventative & Restorative, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Division of Neuroscience, University of California at San Francisco, San Francisco, CA 94143-0440, United States.
³ Department of Oral & Maxillofacial Surgery, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Division of Neuroscience, University of California at San Francisco, San Francisco, CA 94143-0440, United States. Electronic address: [email protected].

PMID: 28040566
PMCID: PMC5321636
DOI: 10.1016/j.neuroscience.2016.12.031

Marked sexual dimorphism in 5-HT₁ receptors mediating pronociceptive effects of sumatriptan

Dioneia Araldi et al. Neuroscience. 2017.

. 2017 Mar 6:344:394-405.

doi: 10.1016/j.neuroscience.2016.12.031. Epub 2016 Dec 29.

Authors

Dioneia Araldi¹, Luiz F Ferrari¹, Paul Green², Jon D Levine³

Affiliations

¹ Department of Oral & Maxillofacial Surgery, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Division of Neuroscience, University of California at San Francisco, San Francisco, CA 94143-0440, United States.
² Department of Oral & Maxillofacial Surgery, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Department of Preventative & Restorative, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Division of Neuroscience, University of California at San Francisco, San Francisco, CA 94143-0440, United States.
³ Department of Oral & Maxillofacial Surgery, University of California at San Francisco, San Francisco, CA 94143-0440, United States; Division of Neuroscience, University of California at San Francisco, San Francisco, CA 94143-0440, United States. Electronic address: [email protected].

PMID: 28040566
PMCID: PMC5321636
DOI: 10.1016/j.neuroscience.2016.12.031

Abstract

Amongst the side effects of triptans, a substantial percentage of patients experience injection site pain and tenderness, the underlying mechanism of which is unknown. We found that the dose range from 10fg to 1000ng (intradermal) of sumatriptan induced a complex dose-dependent mechanical hyperalgesia in male rats, with distinct peaks, at 1pg and 10ng, but no hyperalgesia at 1ng. In contrast, in females, there was 1 broad peak. The highest dose (1000ng) did not produce hyperalgesia in either sex. We evaluated the receptors mediating sumatriptan hyperalgesia (1pg, 1 and 10ng). In males, the injection of an antagonist for the serotonin (5-HT) receptor subtype 1B (5-HT_1B), but not 5-HT_1D, markedly inhibited sumatriptan (1pg)-induced hyperalgesia, at 10ng a 5-HT_1D receptor antagonist completely eliminated hyperalgesia. In contrast, in females, the 5-HT_1D, but not 5-HT_1B, receptor antagonist completely blocked sumatriptan (1pg and 10ng) hyperalgesia and both 5-HT_1B and 5-HT_1D receptor antagonists attenuated hyperalgesia (1ng) in females, which is GPR30 estrogen receptor dependent. While selective 5-HT_1D or 5-HT_1B, agonists produce robust hyperalgesia in female and male rats, respectively, when co-injected the hyperalgesia induced in both sexes was attenuated. Mechanical hyperalgesia induced by sumatriptan (1pg and 10ng) is dependent on the G-protein α_i subunit and protein kinase A (PKA), in IB4-positive and negative nociceptors. Understanding the mechanisms responsible for the complex dose dependence for triptan hyperalgesia may provide useful information for the design of anti-migraine drugs with improved therapeutic profiles.

Keywords: 5-HT(1B) receptor; 5-HT(1D) receptor; hyperalgesia; migraine; triptans.

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Figures

**Figure 1. Sex differences in dose dependence for sumatriptan-induced mechanical hyperalgesia**
Female (A) and male (B) rats received a single intradermal injection of vehicle (V; saline, 5 μL) or sumatriptan (10 fg at 1000 ng) on the dorsum of the hindpaw and 30 min later, the mechanical nociceptive threshold was evaluated using the Randall-Sellitto paw withdrawal test. A. Significant hyperalgesia was observed in female rats treated with sumatriptan 100 fg (** p = 0.0068), 1 pg (*** p < 0.0001), 10 pg (** p = 0.0013), 100 pg (*** p < 0.0001), 1 ng (*** p = 0.0007), 10 ng (*** p < 0.0001) and 100 ng (* p = 0.0272) when were compared to vehicle (*dark gray bar*; unpaired Student’s t-test). However, non-significant (NS) changes in the mechanical nociceptive threshold were observed in females that received the doses of 10 fg (NS; p = 0.5165) and 1000 ng (NS; p = 0.2457) when were compared to vehicle (*dark gray bar*; unpaired Student’s t-test). (N = 6 paws per dose). B. In male rats, we observed a significant hyperalgesia in the groups treated with the doses of 1 and 10 pg (*** p < 0.0001), 100 pg (*** p = 0.0002) and 10 ng (*** p < 0.0001) of sumatriptan when were compared to vehicle group (*dotted bar*; unpaired Student’s t-test). However, in males that received 10 fg (NS; p = 0.7816), 100 fg (NS; p = 0.8959), 1 ng (NS; p = 0.7689), 100 ng (NS; p = 0.7501) and 1000 ng (NS; p = 0.2471) non-significant (NS) changes in the mechanical nociceptive threshold were observed when compared to vehicle group (*dotted bar*; unpaired Student’s t-test). (N = 6 paws per dose). C. The mechanical nociceptive threshold was evaluated 1, 3, 5, 10, 15, 20 and 30 minutes after a single injection of sumatriptan (10 ng) on the dorsum of the hindpaw of male rats. Significant hyperalgesia was observed 5 min after injection of sumatriptan (F_7,63 = 51.92, ***p < 0.0001; one-way repeated-measures ANOVA followed by Bonferroni *post hoc* test). BL: baseline. (N = 10 paws)

**Figure 2. Sumatriptan (1 ng) induces mechanical hyperalgesia in male with the implant of estrogen and not in ovariectomized female rats**
Two weeks after male rats had received a subcutaneous implant of 17β-estradiol (males (17β-estradiol); *dotted bar*), sumatriptan (1 ng) was injected on the dorsum of the hind paw and the mechanical threshold was evaluated 30 min after its injection. Only in male rats implanted with 17β-estradiol (*gray bar*) was sumatriptan (1 ng) able to induce hyperalgesia (t = 18.24, *** p < 0.0001; unpaired Student’s t-test). Female rats were ovariectomized (females OVX; *dotted bar*), and 5 weeks later, sumatriptan (1 ng) was injected showing that OVX females did not develop mechanical hyperalgesia (1 ng; *dotted bar*). However, in females that were not OVX, sumatriptan (1 ng) was able to induce hyperalgesia (*white bar*; t = 5.689, *** p = 0.0002; when females and females OVX were compared; unpaired Student’s t-test) indicating that the hyperalgesia induced by sumatriptan (1 ng) is dependent of estrogen. (N = 6 paws per group).

**Figure 3. Sumatriptan (1 ng)-induced hyperalgesia in female rats is GPR30 dependent**
A. Female rats were treated with daily spinal intrathecal injections of ODN mismatch sequence (*black bars*) or ODN antisense (*gray bars*) for ER-α, ER-β or GPR30, for 3 consecutive days. On the fourth day, intradermal injection of sumatriptan (1 ng) on the dorsum of the hind paw was performed and, the mechanical nociceptive threshold was evaluated 30 minutes later. Treatment with ODN-antisense for ER-α and ER-β did not affect sumatriptan (1 ng)-induced hyperalgesia. However, in females treated with ODN antisense for GPR30, compared to ODN mismatch for GPR30, 30 minutes after injection of sumatriptan (1 ng) the mechanical hyperalgesia was significantly attenuated (t = 8.336, *** p < 0.0001; when ODN mismatch and ODN antisense for GPR30-treated groups were compared; unpaired Student’s t-test), indicating that the GPR30 plays a role in the sumatriptan (1 ng)-induced hyperalgesia in female rats. (N = 6 paws per group). B. A separate group of female rats received an injection on the dorsum of the hind paw of vehicle (5 μL; *dark gray bar*) or G-36 (1 μg; a GRP30 receptor antagonist; *dotted bar*) followed by the injection of sumatriptan (1 ng) at the same site. The mechanical nociceptive threshold was evaluated 30 minutes after sumatriptan injection. Treatment with G-36 (*dotted bar*), compared to vehicle (*dark gray bar*), significantly inhibited sumatriptan (1 ng)-induced hyperalgesia measured 30 min after its injection (t = 9.002, *** p < 0.0001; when vehicle and G-36-treated groups were compared; unpaired Student’s t-test), supporting a role for GPR30 in sumatriptan (1 ng)-induced hyperalgesia. (N = 6 paws per group)

**Figure 4. Sexual dimorphism in the effect of 5-HT_1B and 5-HT_1D receptor antagonists**
***Upper panel:*** Female rats received vehicle (5 μL; *black bar*), NAS-181 (1 μg; 5-HT_1B receptor antagonist; *gray bar*) or BRL 15572 (1 μg; 5-HT_1D receptor antagonist; *white bar*) co-injected with sumatriptan [1 pg (A), 1 ng (B) or 10 ng (C)] on the dorsum of the hind paw. The mechanical nociceptive threshold was evaluated 30 min after sumatriptan injection. A. In the group co-injected with BRL 15572, but not with NAS-181, mechanical hyperalgesia induced by sumatriptan (1 pg) was completely prevented (*white bar*; F = 89.24, *** p < 0.0001; when vehicle, NAS-181, and BRL 15572 groups were compared; one-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test*). B. A dose of 1 ng of sumatriptan, injected with vehicle (*black bar*), was able to induce mechanical hyperalgesia in female rats that was significantly attenuated by NAS-181 (*gray bar*) and BRL 15572 (*white bar*; F = 12.32, ** p = 0.0020, when vehicle, NAS-181, and BRL 15572 groups were compared; one-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test*). C. When the dose of 10 ng of sumatriptan was co-injected with vehicle (*black bar*) or NAS-181 (*gray bar*) a robust hyperalgesia was observed; however, when co-injected with BRL 15572 (*white bar*) the hyperalgesia was completely blocked (F = 91.70, *** p < 0.0001; when vehicle, NAS-181, and BRL 15572 groups were compared; one-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test*). (N = 6 paws per group) ***Lower panel:*** Male rats received vehicle (5 μL; *black bar*), NAS-181 (1 μg; 5-HT_1B receptor antagonist; *gray bar*) or BRL 15572 (1 μg; 5-HT_1D receptor antagonist; *white bar*) co-injected on the dorsum of the hind paw, with sumatriptan [1 pg (D), 1 ng (E) or 10 ng (F)]. The mechanical nociceptive threshold was evaluated 30 min after sumatriptan injection. D. Mechanical hyperalgesia induced by co-injection of vehicle and sumatriptan (1 pg; *black bar*) was prevented by co-injection of NAS-181 (*gray bar*; F = 46.67, *** p < 0.0001; when vehicle, NAS-181, and BRL 15572 groups were compared; one-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test*) but not by BRL 15572. E. When sumatriptan (1 ng; *black bar*) was co-injected with vehicle, we did not observe changes in mechanical threshold, neither when sumatriptan (1 ng) was co-injected with NAS-181 (gray bar) nor BRL 15572 (*white bar*). F. Vehicle co-injected with sumatriptan (10 ng; *black bar*) induced a robust hyperalgesia that was significantly attenuated by the co-injection of NAS-181 (*gray bar*) and was completely inhibited by BRL 15572 (*white bar*; F = 62.94, *** p < 0.0001; when vehicle, NAS-181 or BRL 15572 groups were compared; one-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test*). (N = 6 paws per group)

**Figure 5. Agonists for the 5-HT_1B, but not 5-HT_1D, receptor induce hyperalgesia**
Rats were treated on the dorsum of the hind paw with a single injection of an agonist for 5-HT_1B receptor (CP-93129; 10 or 100 ng; *black bars*), 5-HT_1D receptor (L-694,247; 10 or 100 ng; *gray bars*) or a combination of CP-93129 + L-694,247 (10 or 100 ng; *dotted bars*). Thirty minutes later the mechanical nociceptive threshold was evaluated. We found that the agonist for 5-HT_1B (CP-93129; *black bars*) at the dose of 10 and 100 ng induced a decrease in the mechanical nociceptive threshold (** p = 0.0031 and *** p < 0.0001, respectively; unpaired Student’s t-test) when compared to baseline (before the agonist injection). However, the agonist for 5-HT_1D (L-694,247; *gray bars*) did not induce changes in mechanical nociceptive threshold, at the doses of 10 (NS; p = 0.0963), 100 (NS; p = 0.0907) or 1000 ng (NS; p = 0.0999) when compared to baseline (unpaired Student’s t-test). When co-injected on the dorsum of the hind paw CP-93129 + L-694,247 (10 ng; *dotted bar*) we observed non-significant (NS) change in the mechanical nociceptive threshold (NS; p = 0.4980) when compared to baseline; however, when compared to CP-93129 (10 ng, *black bar*; ^### p = 0.0010) or L-694,247 (10 ng, *gray bar*; ^## p = 0.0030) we observed an increase in the mechanical nociceptive threshold (unpaired Student’s t-test). At the dose of 100 ng of a combination of CP-93129 + L-694,247 (*dotted bar*), we observed a decrease in mechanical nociceptive threshold when was compared to baseline and L-694,247 (*gray bar*; ^○○○ p < 0.0001; unpaired Student’s t-test); on the other hand, when the combination (100 ng; *dotted bar*) was compared to CP-93129 (100 ng; *black bar*) we observed an attenuation in the mechanical hyperalgesia induced by CP-93129 (^○○ p = 0.0048; unpaired Student’s t-test). (N = 6 paws per group)

**Figure 6. Mechanical hyperalgesia induced by sumatriptan (1 pg or 10 ng) depends on G-protein α_i subunit and PKA**
Male rats were treated, on the dorsum of the hind paw, with vehicle (5 μL; *black bar*), pertussis toxin (PTX; 1 μg; *gray bar*) or PKA inhibitor (H-89, 1 μg; *dotted bar*). Ten minutes after the treatment, sumatriptan [1 pg (A) or 10 ng (B)] was injected at the same site and the mechanical hyperalgesia evaluated 30 min after its injection. A. Treatment with pertussis toxin (PTX; *gray bar*) significantly attenuated the sumatriptan (1 pg)-induced mechanical hyperalgesia (** p = 0.0012, when vehicle and PTX groups were compared; unpaired Student’s t-test). A PKA inhibitor (H-89; *dotted bar*), completely blocked the sumatriptan (1 pg)-induced hyperalgesia (*** p < 0.0001; unpaired Student’s t-test; when vehicle and PKA inhibitor groups were compared). B. In a group of rats treated with PTX (*gray bar*) the mechanical hyperalgesia induced by sumatriptan (10 ng) was completely blocked 30 min after its injection (*** p = 0.0003; when vehicle and PTX groups were compared; unpaired Student’s t-test) and, in the group treated with H-89 (*dotted bar*), sumatriptan (10 ng)-induced hyperalgesia was attenuated (** p = 0.0085; when vehicle and H-89 groups were compared; unpaired Student’s t-test). (N = 6 paws per group)

**Figure 7. Mechanical hyperalgesia induced by sumatriptan depends on IB4-positive and IB4-negative neurons (1 pg) or only IB4-positive neurons (10 ng)**
Male rats were treated with vehicle (control, *black bars*), IB4-saporin (3.2 μg/20 μL; *white bars*) or SSP-saporin (100 ng/20 μL; *gray bars*) by intrathecal injection. Fifteen days later, sumatriptan [1 pg (A) or 10 ng (B)] was injected on the dorsum of the hind paw and the mechanical nociceptive threshold was evaluated 30 min later. A. One-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test* demonstrated a significant attenuation of sumatriptan (1 pg)-induced hyperalgesia in the group previously treated with IB4-saporin (F = 32.13, *** p < 0.0001) and with SSP-saporin (** p < 0.0001; when vehicle, IB4-saporin, and SSP-saporin groups were compared; one-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test*). B. A complete inhibition of sumatriptan (10 ng)-induced hyperalgesia was observed in the group previously treated with IB4-saporin (F = 78.92, *** p < 0.0001; when the vehicle, IB4-saporin, and SSP-saporin groups were compared; one-way repeated-measures ANOVA followed Dunnett’s multiple comparison *test*) but not with SSP-saporin. BL: baseline. (N = 6 paws per group)

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References

1. Al-Hasani R, Bruchas MR. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology. 2011;115:1363–1381. - PMC - PubMed
1. Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, Reichling DB, Levine JD. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron. 2003;39:497–511. - PubMed
1. Alvarez P, Bogen O, Levine JD. Role of nociceptor estrogen receptor GPR30 in a rat model of endometriosis pain. Pain. 2014;155:2680–2686. - PMC - PubMed
1. Araldi D, Ferrari LF, Levine JD. Repeated Mu-Opioid Exposure Induces a Novel Form of the Hyperalgesic Priming Model for Transition to Chronic Pain. J Neurosci. 2015;35:12502–12517. - PMC - PubMed
1. Araldi D, Ferrari LF, Levine JD. Adenosine-A1 receptor agonist induced hyperalgesic priming type II. Pain. 2016a;157:698–709. - PMC - PubMed

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[1] Al-Hasani R, Bruchas MR. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology. 2011;115:1363–1381. - PMC - PubMed

[2] Al-Hasani R, Bruchas MR. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology. 2011;115:1363–1381. - PMC - PubMed

[3] Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, Reichling DB, Levine JD. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron. 2003;39:497–511. - PubMed

[4] Alessandri-Haber N, Yeh JJ, Boyd AE, Parada CA, Chen X, Reichling DB, Levine JD. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron. 2003;39:497–511. - PubMed

[5] Alvarez P, Bogen O, Levine JD. Role of nociceptor estrogen receptor GPR30 in a rat model of endometriosis pain. Pain. 2014;155:2680–2686. - PMC - PubMed

[6] Alvarez P, Bogen O, Levine JD. Role of nociceptor estrogen receptor GPR30 in a rat model of endometriosis pain. Pain. 2014;155:2680–2686. - PMC - PubMed

[7] Araldi D, Ferrari LF, Levine JD. Repeated Mu-Opioid Exposure Induces a Novel Form of the Hyperalgesic Priming Model for Transition to Chronic Pain. J Neurosci. 2015;35:12502–12517. - PMC - PubMed

[8] Araldi D, Ferrari LF, Levine JD. Repeated Mu-Opioid Exposure Induces a Novel Form of the Hyperalgesic Priming Model for Transition to Chronic Pain. J Neurosci. 2015;35:12502–12517. - PMC - PubMed

[9] Araldi D, Ferrari LF, Levine JD. Adenosine-A1 receptor agonist induced hyperalgesic priming type II. Pain. 2016a;157:698–709. - PMC - PubMed

[10] Araldi D, Ferrari LF, Levine JD. Adenosine-A1 receptor agonist induced hyperalgesic priming type II. Pain. 2016a;157:698–709. - PMC - PubMed

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Marked sexual dimorphism in 5-HT₁ receptors mediating pronociceptive effects of sumatriptan

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Marked sexual dimorphism in 5-HT₁ receptors mediating pronociceptive effects of sumatriptan

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