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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 anti-migraine triptans, which act at 5-HT1B/D Gi-GPCRs have been implicated in pain chronification. We determined if 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 (PKCε), but not a PKA 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 pre-treated with pertussis toxin or IB4-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 co-injection of antagonists for the 5-HT1B and 5-HT1D, but not 5-HT7, serotonin receptors, and completely prevented by co-administration 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 via Gi-GPCRs, induces type I hyperalgesic priming, unlike agonists at other Gi-GPCRs, which induce type II priming.
While the mechanism underlying the transition from acute to chronic pain remains poorly understood, it has been suggested that some analgesics can produce pain chronification. Thus, opioids, which are used clinically to treat moderate-to-severe pain, especially agonists at the mu-opioid receptor (MOR), may come to exacerbate or actually produce pain during their chronic administration, a phenomenon referred to as opioid-induced hyperalgesia (OIH) [6; 60; 63]. Similarly, the triptan class of drugs, used to treat migraine [25; 35; 76], also facilitates pain chronification and/or contributes to migraine chronification, a condition referred to as medication overuse headache (MOH; [9; 11; 26; 28; 38; 41; 45; 77]). The target for the therapeutic effect of triptans is thought to be 5-HT1B and 5-HT1D receptors that, like MOR are inhibitory G-protein coupled receptors (Gi-GPCRs) [37; 42; 64; 86; 95; 96]. Therefore, in the present study we explored the possibility that, like MOR and A1-adenosine receptor agonists [7; 8], triptans would also induce type II hyperalgesic priming. In addition, we explored the 5-HT receptor subtypes at which triptans act (5-HT1B, 5-HT1D and 5-HT7) to induce priming. We report that while sumatriptan, a prototypical 5-HT1B/D receptor agonist induces hyperalgesic priming, this priming meets the criteria for type I rather than type II priming.
Experiments were performed on 230–280 g male and female Sprague–Dawley rats (Charles River Laboratories, Hollister, CA, USA). Rats were housed in a controlled environment in the animal care facility at the University of California, San Francisco, under a 12-h light/dark cycle. Food and water were available ad libitum. The experimental protocols were approved by the Institutional Animal Care and Use Committee at UCSF and adhered to the guidelines of the American Association of Laboratory Animal Care, the National Institutes of Health (NIH), and the Committee for Research and Ethical Issues of the International Association for the Study of Pain (IASP), for the use of animals in research. In the design of experiments, all efforts were made to minimize the number of animals used and their suffering.
Mechanical nociceptive threshold was quantified using an Ugo Basile Analgesymeter® (Randall-Selitto paw-withdrawal test; Stoelting, Chicago, IL), which applies a linearly increasing mechanical force to the dorsum of the rat's hind paw, as previously described [32; 78; 80]. Nociceptive threshold was defined as the force in grams at which the animal withdrew its paw; baseline paw-pressure nociceptive mechanical threshold was defined as the mean of the 3 readings taken just before a test agent was injected. Each paw was treated as an independent measure and each experiment performed on a separate group of rats. Data are presented as mean change from baseline mechanical nociceptive threshold.
The chemicals used in this study were: cordycepin 5′-triphosphate sodium salt (protein translation inhibitor; [7; 8; 34]), prostaglandin E2 (PGE2; a hyperalgesic agent that acts at receptors on nociceptors to sensitizes them), sumatriptan succinate (a 5-HT1B and 5-HT1D receptor agonist), CP-93129 dihydrochloride hydrate (a 5-HT1B receptor agonist), pertussis toxin (Gi-protein inhibitor) and SB-269970 (a 5-HT7 receptor antagonist), all from Sigma-Aldrich (St. Louis, MO, USA); PKCεV1–2 (PKCε-I, a PKCε-specific translocation inhibitor peptide; [7; 8; 48; 58]), from Calbiochem (La Jolla, CA, USA); H-89 dihydrochloride (inhibitor of protein kinase A [PKA]; [7; 8]) from Santa Cruz Biotechnology (Dallas, TX, USA); L-694,247 (a 5-HT1D receptor agonist), BRL 15572 (a 5-HT1D receptor antagonist) and NAS-181 (a 5-HT1B receptor antagonist), all from Tocris Bioscience (Avonmouth, Bristol, UK).
The selection of doses was based on previous studies that showed their effectiveness when injected intradermally on the dorsum of the hind paw [3; 7; 8; 32; 52]. The stock solution of PGE2 (1 μg/μL) was prepared in 10% ethanol and additional dilutions made with physiological saline (0.9% NaCl), yielding a final ethanol concentration of less than 1%. Cordycepin, sumatriptan, CP-93129, pertussis toxin, PKCεV1–2 and SB-269970 were dissolved in saline. All other drugs were dissolved in 100% DMSO (Sigma-Aldrich) and further diluted in saline containing 2% Tween 80 (Sigma-Aldrich). The final concentration of DMSO and Tween 80 was 2%. All drugs were injected intradermally on the dorsum of the hind paw, in a volume of 5 μL, using a 30-gauge hypodermic needle adapted to a 50 μl Hamilton syringe (Reno, NV, USA). The injection of cordycepin, H-89, PKCεV1–2 and pertussis toxin, was preceded by a hypotonic shock (2 μL of distilled water, separated by a bubble of air to avoid mixing in the same syringe), to facilitate the entry of compounds into the nerve terminal [14; 16].
Isolectin B4 (IB4)-saporin, an IB4-positive nociceptor neurotoxin (Advanced Targeting Systems, San Diego, CA), was diluted in saline, and a dose of 3.2 μg, in a volume of 20 μL was administered intrathecally, 14 days prior to the priming experiments. The dose of IB4-saporin and time protocol were chosen based on previous reports from us and other groups [7; 8; 51; 52; 66; 89].
The neurotoxin [Sar9,Met(O2)11]-substance P-saporin (SSP-Saporin, Advanced Targeting Systems, San Diego, CA) was diluted in saline, and a dose of 100 ng, in a volume of 20 μL was administered intrathecally, 14 days prior to the priming experiments. The addition of [Sar9Met(O2)11] to substance P conjugated to saporin makes the agent more stable and potent than when substance P is bound to saporin. The dose and pre-treatment interval that we used were based on previous reports [22; 93], that demonstrated the effectiveness of this agent to deplete substance P-containing fibers with no loss of lumbar dorsal horn neurons expressing the neurokinin 1 (NK1) receptor in deeper laminae and prominent loss of NK1 receptor in laminae I [56; 59; 87; 92].
Rats were briefly anesthetized with 2.5% isoflurane (Phoenix Pharmaceuticals, St. Joseph, MO, USA) in 97.5% O2. Then, a 30-gauge hypodermic needle was inserted, on the midline, into the subarachnoid space, between the L4 and L5 vertebrae. The control treatment consisted of intrathecal injection of saline (vehicle; 20 μL). Animals regained consciousness approximately 1 min after removal from the anesthetic chamber. There was no effect of IB4-saporin or SSP-saporin on the mechanical nociceptive threshold per se.
Our group has previously shown that a single intradermal injection of a selective 5-HT1 receptor agonist produced mechanical hyperalgesia (Fig. 1A; [2; 79; 81]). To investigate the changes in nociceptor function produced by the injection of sumatriptan (a 5-HT1B/1D receptor agonist, 10 ng), 5-HT1B receptor agonist (CP-93129, 10 or 100 ng) or 5-HT1D receptor agonist (L-694,247, 10 or 100 ng) — measured as prolonged response to a hyperalgesic mediator, at a point in time when the mechanical nociceptive threshold was not different from pre-sumatriptan baseline-PGE2 (100 ng) was injected at the same site, and the change in nociceptive threshold evaluated 30 min and 4 h later. Presence of hyperalgesia at the 4th h is characteristic of priming [4; 7; 8; 31]. To evaluate the intracellular signaling pathways that play a role in priming induced by sumatriptan, and to investigate the mechanisms that play a role in the induction of the changes in nociceptor function produced by the activation of the 5-HT1B/D receptor, pharmacological agents were injected before sumatriptan (prevention protocol). To investigate the second messengers involved in the expression of the neuroplasticity, inhibitors were administered before the injection of PGE2 in the already primed paw (inhibition protocol), at a time when mechanical nociceptive threshold was not different from pre-sumatriptan levels.
In all experiments, the dependent variable was mechanical paw-withdrawal threshold, expressed as percentage change from baseline. The average paw withdrawal thresholds before the injection of sumatriptan and before the tests with PGE2 (3 or more days, depending on the experiment) were 123.0 ± 1.05 g and 123.3 ± 1.12 g, respectively; paired Student's t-test showed no significant difference between these values (t125 = 0.4285, p = 0.6753). The total number of paws used in this study was 126. To compare the mechanical hyperalgesia induced by injection of the neuroplasticity-inducing agents (i.e., sumatriptan, 5-HT1B [CP-93129] or 5-HT1D [L-694,247] receptor agonists) and to compare the effect of PGE2 in different groups, in the presence or absence of inhibitors of signaling pathways, two-way repeated-measures ANOVA, followed by Bonferroni post hoc test, was performed. Graph Pad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA) was used to plot graphs and to perform statistical analyses; a p-value less than 0.05 was considered statistically significant. Data are presented as mean ± SEM.
One of the defining features of both type I and II priming is the prolongation of hyperalgesia induced by pronociceptive mediators, prototypically prostaglandin E2 (PGE2) [4; 12; 33; 57; 69; 73]. To determine if sumatriptan induces hyperalgesic priming we injected PGE2 intradermally, at the site of nociceptive testing. We report that a single low dose of sumatriptan (10 ng) induces mechanical hyperalgesia (Fig. 1A). This differs from the pronociceptive effects of MOR and A1-adenosine agonists that took repeated administration before they induced hyperalgesia [7; 8]. Therefore, in the following experiments, we tested for priming after a single administration of sumatriptan. Unlike type II priming, in which the prolongation of PGE2 hyperalgesia is observed immediately after the repeated injections of mu-opioid or A1-adenosine receptor agonists, no prolongation of PGE2 hyperalgesia was observed when PGE2 was administered 24 hours post sumatriptan (Fig. 1B). However, when it was injected 72 hours (Fig. 1C) or 30 days (Fig. 1D) after sumatriptan, we observed prolonged hyperalgesia. This demonstrates the time course for the onset of priming to be similar to that observed for type I hyperalgesic priming [4; 12].
Since triptans are agonists at two Gi-GPCRs, 5-HT1B and 5-HT1D [5; 24; 35; 39; 43; 82; 84] we evaluated the ability of antagonists at these two receptors to prevent sumatriptan-induced hyperalgesic priming. We found that, while alone the 5-HT1B and 5-HT1D antagonists (NAS-181 and BRL 15572, respectively) each partially attenuate the magnitude of the prolongation of PGE2 hyperalgesia (Fig. 2, gray and white bars), the co-administration of the two antagonists completely prevented sumatriptan-induced prolongation of PGE2 hyperalgesia (Fig. 2, dotted bars). Since, it has been suggested that triptans also produce some of their effects by action at the 5-HT7 receptor [71; 75; 88; 90; 91], we also determined if a 5-HT7 receptor antagonist (SB-269970) would attenuate sumatriptan-induced priming. We found that the SB-269970 did not attenuate sumatriptan-induced priming (Fig. 2, horizontally striped bars).
We found that the prolongation of PGE2-induced hyperalgesia by injection of sumatriptan (10 ng) is dependent of activation of both 5-HT1B and 5-HT1D receptors (Fig. 2). Therefore, we next tested the effect of selective agonists for 5-HT1B (CP-93129; Fig. 3, gray bars) and 5-HT1D (L-694,247; Fig. 3, white bars). We observed that the dose of 100 ng, but not 10 ng, of both agonists, induced priming as manifest by prolongation of PGE2-induced hyperalgesia (Fig. 3).
Another mechanism that distinguishes type I from type II priming is the second messengers mediating the prolongation of PGE2 hyperalgesia, PKCε for type I priming [4; 70] and PKA for type II [7; 8]. Compatible with the above findings, which support the suggestion that sumatriptan induces type I priming, we observed that the prolongation of PGE2 hyperalgesia induced by sumatriptan was attenuated by a PKCε (Fig. 4A), but not by a PKA (Fig. 4B) inhibitor.
A third distinction between type I and type II priming is the dependence of type I  but not type II [7; 8] priming, on protein translation in the peripheral terminal of the primary afferent nociceptor. We report that intradermal injection of cordycepin, an inhibitor of protein translation, permanently reversed sumatriptan-induced priming (Fig. 5), further suggesting that sumatriptan induces type I priming.
Since the 5-HT1B and 5-HT1D receptors are Gi-GPCRs [37; 42; 64; 86; 95; 96], to determine the signaling pathway downstream of the 5-HT1B/D receptor that contributes to the prolongation of PGE2-induced hyperalgesia, we tested the effect of the treatment with the G-protein αi subunit inhibitor, pertussis toxin (PTX), injected before sumatriptan. We found that the prolongation of PGE2-induced hyperalgesia was inhibited by PTX (Fig. 6). Of note, the ability of PTX to inhibit the induction of sumatriptan-induced priming (Fig. 6), the expression of type II priming induced by A1-adenosine receptor agonist CPA  and also type I hyperalgesic priming [29; 33; 57] contrasts with mu-opioid receptor agonist DAMGO-induced type II priming where PTX failed to inhibit the prolongation of PGE2-induced hyperalgesia induced by DAMGO .
We have previously demonstrated that the induction of type I priming is prevented by spinal intrathecal treatment with IB4-saporin, a neurotoxin that eliminates IB4-positive nociceptors [7; 8; 51; 52; 66; 89], whereas type II priming induction by the MOR agonist DAMGO is prevented by intrathecal administration of a stabilized form of substance P, conjugated to saporin (SSP-saporin; unpublished results), a specific neurotoxin that destroys neurons with processes containing NK1 receptors in the superficial but not the deeper laminae of the dorsal horn [22; 59; 62; 93], by eliminating substance P (SP) containing sensory neurons. In the present experiments we found that, in rats pretreated with IB4-saporin, but not SSP-saporin, the injection of sumatriptan did not induce prolongation of PGE2 hyperalgesia, evaluated 4 days later (Fig. 7).
We have previously shown that the administration of agonists for cell surface receptors (such as TNF-α, MCP-1 and IL-6), induce type I priming only in male rats . On the other hand, induction of type II priming by Gi-GPCR agonists is observed in females as well as males [7; 8]. In the present study we observed that sumatriptan did not induce priming in female rats (Fig. 8), another characteristic feature of type I hyperalgesic priming .
The triptans, agonists at serotonin 5-HT1B and 5-HT1D receptors, are an important class of drugs used in the treatment of migraine [1; 35; 36; 40; 47; 55; 84]. One limitation in their clinical use is that they can contribute to migraine chronification and more frequent episodes of headache, a condition referred to as medication overuse headache (MOH), recently characterized as a global epidemic [9; 11; 27; 28; 38; 41; 45; 77]. The triptan receptors are both Gi-protein coupled, which they share with other classes of analgesics, including those that act at mu-opioid or CB cannabinoid receptors [67; 74]. Importantly, opioids can also produce exacerbation of the pain for which they are clinically administered [21; 23; 44; 60; 65], a phenomenon referred to as opioid-induced hyperalgesia (OIH). We have studied opioid-induced hyperalgesia, using a model in the primary afferent nociceptor, hyperalgesic priming (type II) [7; 8]. Therefore, in the present experiments we determined if sumatriptan, a clinically used triptan [35; 36; 49; 83] would also induce type II priming.
In the present experiments we observed that intradermal injection of sumatriptan produces mechanical hyperalgesia at the injection site, as has been reported in patients being treated for migraine [17; 18; 68; 85]. Also, the frequent use of triptans can lead to transition to MOH or chronic migraine [9; 10; 27; 28; 38; 41; 68; 77; 85]. We observed that sumatriptan produces a neuroplastic change in nociceptor function such that PGE2-induced mechanical hyperalgesia is markedly prolonged, a characteristic feature of both type I [4; 29; 33; 50; 57; 69; 73] and type II [7; 8] hyperalgesic priming. However, the mechanism underlying priming induced by sumatriptan resembled type I rather than type II priming. Thus, unlike type II priming, which develops within a matter of a few hours [7; 8], that induced by sumatriptan required 3 days to develop, characteristic of type I priming [4; 12]. A second difference was that, unlike type II priming, in which the prolongation of PGE2 hyperalgesia is PKA dependent, that induced by sumatriptan was PKCε dependent, another characteristic feature of type I priming [4; 70]. Furthermore, characteristic of type I , but not type II priming [7; 8], that induced by sumatriptan was reversed by administration of a protein translation inhibitor to the peripheral terminal of the primed nociceptor. Moreover, the ability of pertussis toxin to inhibit the induction of sumatriptan-induce priming is also characteristic of type I priming [33; 52; 57] and type II priming induced by CPA , but not type II priming induced by DAMGO . In addition, sumatriptan is unable to produce priming in rats in which IB4-positive (non-peptidergic), but not IB4-negative (peptidergic), nociceptors have been destroyed, a further feature of type I  but not type II priming [7; 8]. Finally, unlike the type II priming induced by mu-opioid or A1-adenosine receptor agonists [7; 8], which occurs in female as well as male rats, sumatriptan induced priming only in males. Thus, we conclude that while the triptans act at Gi-protein coupled receptors, which are involved in pain chronification as well as analgesia, when tested in a preclinical model, unlike other Gi-protein coupled receptor agonists, sumatriptan induces type I priming. However, the downstream signaling pathway through which an agonist at a Gi-protein coupled receptor induces type I priming remains to be established. In Fig. 9 the signaling pathways that participate in sumatriptan-induced type I hyperalgesic priming are illustrated.
Since the triptans act at two serotonin receptors, 5-HT1B and 5-HT1D, both of which are GPCRs, we evaluated the role that each subtype of receptor plays in the induction of hyperalgesic priming. We observed that while alone the antagonists of 5-HT1B and 5-HT1D partially attenuate the prolongation of PGE2 hyperalgesia, their co-administration completely prevented sumatriptan-induced prolongation of PGE2 hyperalgesia. Furthermore, when agonists for each of these two serotonin receptors were injected, alone, each induced prolongation of PGE2-induced hyperalgesia.
Why agonists at 5-HT1B and 5-HT1D induce type I priming, while agonists at other Gi-protein coupled receptors, namely mu-opioid and A1-adenosine, induce type II priming remains to be explained. One possible way to address the underlying differences would be to determine if repeated administration of agonists at mu- or A1-receptors induce a state whereby triptan agonists will now produce prolonged hyperalgesia, or vice versa. In preliminary experiments we observed that while the repeated administration of the A1-adenosine receptor agonist CPA induces a state whereby the mu-agonist DAMGO induces hyperalgesia, the repeated administration of the mu-opioid receptor agonist DAMGO did not induce a state whereby the CPA induced hyperalgesia (unpublished results). Given this initial complexity, even for receptors whose agonists both induce type II priming, these experiments are beyond the scope of the present analysis.
In conclusion, in a model of pain chronification, induced by agonists at the triptan receptors 5-HT1B and 5-HT1D, key to the treatment of migraine, we have demonstrated that a clinically used member of this class of agonists, sumatriptan, induces both mechanical hyperalgesia at the site of injection and type I hyperalgesic priming, in nociceptors innervating the cutaneous injection site. While our studies were executed outside of the trigeminal system, the site of migraine, the basic neurovascular unit at both spinal and trigeminal levels is innervated by nociceptors that contain 5-HT1B and 5-HT1D [13; 15; 46; 61; 71; 72; 94]. Moreover, support for shared mechanisms in spinal and trigeminal distributions comes from both the presence of a migraine variant, abdominal migraine , which has been reported to be treatable with triptans in some patients , as well as the observation that migraineurs do have lowered threshold in extra-cranial sites .
This study was funded by a grant from the National Institutes of Health (NIH), NS084545.
Conflict of Interest: The authors declare no competing financial interests.
Dioneia Araldi, Departments of Medicine and Oral Surgery, and Division of Neuroscience, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.
Luiz F. Ferrari, Departments of Medicine and Oral Surgery, and Division of Neuroscience, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.
Jon D. Levine, Departments of Medicine and Oral Surgery, and Division of Neuroscience, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.