Exploration of purinergic receptors as potential anti-migraine targets using established pre-clinical migraine models

Myograph studies in hMMA

Samples of dura mater were perioperatively obtained from 10 patients (two males and eight females; 48–69 years) undergoing neurosurgical procedures. Tissues were collected in a sterile organ-protecting solution and were immediately brought to the laboratory. Subsequently, hMMAs (internal diameter 0.5–1.5 mm) were dissected and used the same day or stored overnight in cold oxygenated Krebs solution of the following composition: 119 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃ and 11.1 mM glucose; pH 7.4. Study protocols were approved by the ethics committee at the Erasmus Medical Center, Rotterdam.

The hMMAs were cut into 1–2 mm long segments and mounted on stainless-steel wires (Ø 40 µm) in Mulvany myographs (Danish Myo Technology, Aarhus, Denmark) filled with carbogenated Krebs buffer solution at 37 ℃. The segments were stretched to their optimal lumen diameter (0.9 × L100), where L100 is an estimate of the diameter of the vessel under a passive transmural pressure of 100 mmHg (13.3 kPa). Data were obtained through a LabChart data acquisition system (AD Instruments Ltd, Oxford, UK). hMMAs were exposed to 30 mM KCl, followed by 100 mM KCl to determine the reference contractile response. Using the indicated agonists, a cumulative concentration response curve (using half log-steps unless otherwise indicated) was constructed. This cumulative approach might lead to an underestimation of the maximal contractions in case of desensitization, but in view of the scarcity of human tissue an alternative approach, using only one single concentration per vessel segment, is not feasible. The functional integrity of the endothelium was tested with substance P (10 nM) on arteries precontracted with the thromboxane A₂ analogue U46619 (100 nM). The same precontraction was used to construct vasodilation curves.

Intravital microscopy on a closed cranial window in rats

Twenty-seven normotensive male Sprague-Dawley rats (300–400 g), purchased from Harlan (Horst, The Netherlands), were maintained on a 12/12-h light-dark cycle (with light beginning at 7 a.m.) and housed at a constant temperature (22 ± 2 ℃) and humidity (50%), with food and water ad libitum. The animals were anaesthetized with sodium pentobarbital (60 mg/kg i.p., followed by 18 mg/kg i.v. per hour when necessary). The adequacy of anaesthesia was judged by the absence of ocular reflexes and a negative tail flick test. The Institutional Ethics Committee approved all experimental protocols (Erasmus MC; permission protocol number EMC 1931 [118-09-04]).

The protocol was identical to our previous studies (18). Briefly, the left femoral vein and artery were cannulated for intravenous (i.v.) administration of drugs and continuous monitoring of blood pressure. Rats were placed in a stereotaxic frame and the skull was carefully drilled thin, until the MMA was visible. An intravital microscope (Leica MZ 16; Leica Microsystem Ltd, Heerbrugg, Switzerland) was used to record the artery diameter with a cyan filter on a cold source of light. A zoom lens (80 × magnification) and camera (DCx V3.52, Thorlabs LTD, Ely, UK) were used to capture the image of the dural artery, which was displayed and measured on a computer using a dedicated software package (IDA-Intravital Dimension Analyser; http://www.beneryx.co.uk) integrated with an ADC/DAC board (DI-158, DATAQ instruments, ‘s-Hertogenbosch, The Netherlands). For the periarterial electrical stimulation, a bipolar stimulating electrode (NE 200X, Clark Electromedical, Edenbridge, Kent, U.K.) was placed within 200 µm from the MMA. The cranial window surface was stimulated at 5 Hz, 1 ms for 10 s (stimulator model S88, Grass Instruments, West Warwick, RI, USA). As we previously demonstrated, dural vasodilator responses are reproducible after repeated stimuli for at least four times (18,19). Animals were randomized, and we allowed 30 min between stimulations/treatments for recovery to the baseline diameter. MRS2211 (P2Y13 receptor antagonist) or ADPβS (ADP analogue, P2Y1/12/13 receptor agonist) was administered 15 min before periarterial electrical stimulation. The duration of each experiment was approximately 2.5 hours after stabilization. The detection limit for arterial diameter was about 10 µm; in case an artery was not visible due to pronounced constriction, its diameter was set to be 10 µm.

CGRP release

Twelve male Sprague-Dawley rats (300–400 g), purchased from Taconic (Ejby, Denmark) were maintained on a 12/12-h light-dark cycle (with dark beginning at 7 am) and housed at a constant temperature (22 ± 2 ℃) and humidity (55 ± 10%), with food and water ad libitum. Rats were generally housed in Eurostandard cages (Type VI with 123-Lid) 2–6 together and single housed (Type III with 123-Lid) directly before undergoing the procedure.

Rats were anaesthetized by CO₂ inhalation and decapitated. All procedures are approved by the Danish Animal Experimentation Inspectorate. The protocol is described in detail elsewhere (19). Shortly, the trigeminal nucleus caudalis (TNC) was excised from the brain stem, measuring approximately 6 mm in length. The skull was cut mid-sagittally and the brain halves were carefully removed while the cranial dura was left attached to the skull, and the trigeminal ganglion (TG) was dissected out. TGs and TNCs were immersed in 10  ml synthetic interstitial fluid (SIF, composition: 108 mM NaCl, 3.5 mM KCl, 3.5 mM MgSO₄ , 26 mM NaHCO₃, NaH₂PO₄, 1.5 mM CaCl₂, 9.6 mM NaGluconate, 5.6 mM glucose and 7.6 mM sucrose; pH 7.4.) at 37 ℃ for 30  min. Skull halves were transferred to a beaker containing SIF and washed two times (each wash, 15 minutes) in 250  ml SIF.

TGs and TNCs were randomized, placed in Eppendorf tubes in a heating block at 37 ℃. TGs and TNCs were again washed five times (each wash, 10 minutes), with 300 µl SIF. Both skull halves were randomized and placed in a humid chamber above a water bath to maintain temperature at 37 ℃ and washed five times (each wash 10 minutes) with 300 µl SIF. After 10 min incubation with 300 µl SIF, 200 µl samples for measuring the basal CGRP release were collected from all the tissues, mixed with 50 µl enzyme immunoassay buffer (containing protease inhibitors) and stored at −20 ℃ until analysis, within a week after the experiment was performed. Previous studies have shown that there is no significant difference between the basal CGRP release from the left and right side of the tissues; thus, one side served as a control for the other, which has been shown to reduce the experimental and biological variations (19). The release of CGRP from the skull halves, TG and TNC was induced by 60 mM potassium. To maintain equal osmolarity, a proportional amount of Na⁺ was removed from the buffer. Experiments by others have shown that 10 min incubation is sufficient for a significant and reproducible release of CGRP over basal levels (19). The tissues were pre-incubated for 20 minutes with different concentrations of inhibitors/vehicle and agonist/vehicle, and during the final KCl challenge the same concentrations were maintained.

The samples were processed using commercial EIA kits (SPIbio, Paris, France) to study CGRP release. From all tissues, 200 µl of sample was mixed with 50 µl of EIA buffer, which was also added to the CGRP standard. Determination of CGRP content was calculated based on this standard curve. Previous studies on the samples obtained from TNC showed that the amount of CGRP release was too high to fall in the linear range of the standard curve (19). Therefore, TNC samples were diluted six times. Antibody in the CGRP EIA kit is directed against human-CGRP α/β, but it has 100% cross-reactivity with rat and mouse CGRP (19,20). The protocol was performed following the manufacturer's instructions. Briefly, samples were incubated at 4 ℃ for 16–20 h, washed, and incubated with Ellman's reagent. Following 60 min of incubation, the optical density was measured at 410 nm using a micro-plate photometer (Tecan, Infinite M200, software SW Magellan v.6.3, Männedorf, Switzerland). Samples (a total of two dura and one TNC) that did not generate any CGRP release in response to 60 mM potassium, and one TG with baseline over 100 pg/ml CGRP, were not included in the analysis. Since data were paired, both the left and right side were excluded in the above cases.

Immunohistochemistry

Human TGs were collected at autopsy, within 48 h post-mortem, from four patients (two males and two females; 68–82 years). The patients were without disorders related to the central nervous system. The specimens were fixed after removal in 2% paraformaldehyde (PFA) and 0.2% picric acid in 0.1 mol/l phosphate buffer, pH 7.2, tyrode solution. The tissue samples were kept at −80 ℃ until embedding and cryo-sectioning. The study followed the guidelines of the European Communities Council (86/609/ECC) in accordance with the Szeged University Medical School guidelines for ethics in human tissue experiments and was approved by the local Hungarian Ethics Committee. The hMMA was obtained from the same patients that were used for the myograph studies. Study protocols were approved by the ethics committee at the Erasmus Medical Center, Rotterdam.

Human TGs and MMAs were cryosectioned. Details on immunohistochemistry are described elsewhere (21). In short, slides with 10 µm sections were washed in PBS containing 2.5% Triton (PBS-T) for 15 min. Next, the primary antibody, rabbit anti-P2Y13 receptor (Alomone, APR#017, 1:50), rabbit anti-P2X3 receptor (Alomone, APR#026, 1:200) or mouse anti-CGRP (Abcam Ab81887, 1:100) was added. Following incubation overnight in moisturized chambers (+8 ℃), cryosections were washed in PBS-T twice for 15 min and incubated for 1 hour with secondary antibodies (FITC anti-mouse, Jackson Immunoresearch 1:200, and Cy3 anti-rabbit, Jackson Immunoresearch, 1:300) in the dark. After three washes for 15 min, the slides were mounted with Vectashield mounting medium containing DAPI (4′,6-diamidino-2-phenylindole, Vector Laboratories, Burlingame, CA, USA). Negative controls were performed omitting the primary antibody. Immunofluorescence was observed using an epifluorescence microscope (Nikon 80i, Tokyo, Japan) combined with a Nikon DS-2MV camera. The images were then processed using Adobe Photoshop CS3 (v10.0 Adobe 3 Systems, Mountain View, CA, USA). Potential colocalization was determined by superimposing the images taken with different wavelength filters.

Data presentation and statistical evaluation

All quantitative data are presented as mean ± SEM. The n-number represents one rat or one human. The peak increases in dural meningeal artery diameter, as well as changes in mean arterial blood pressure (MAP, in vivo experiment) were expressed as percent change from baseline. The difference between two concentration response curves was determined by two-way analysis of variance (ANOVA) and the Bonferroni post-test. The difference between the variables within one group was compared by using a one-way repeated measures ANOVA followed by Dunnet's test. When only two variables were compared, a paired Student's t-test was used.

Compounds

The compounds used in this study were: Sodium pentobarbital (Nembutal; Ceva Sante Animale B.V., Maassluis, The Netherlands); MRS2211, αβmetATP, olcegepant (Tocris); ADPβS, UDPβS, UTPγS, ATPγS (Biolog.de) and CGRP (NeoMPS S.A., Strasbourg, France). All other chemicals were from Sigma Chemicals Co. (Steinheim, Germany). All compounds were dissolved in distilled water, with the exception of capsaicin and olcegepant, which were dissolved in DMSO. All solutions were further diluted in saline for in vivo studies or distilled water for in vitro studies.

Myograph studies on the hMMA

hMMAs were obtained from 10 patients (two males and eight females; 48–69 years) during neurological procedures requiring trepanation of the skull.

In vitro relaxation of hMMA

Since the hMMA has a much larger diameter than the rMMA it can therefore be mounted in myographs without damaging the endothelium, making it possible to investigate endothelium-dependent vasodilation. The presence of functional endothelium was checked with the addition of substance P (79 ± 8% relaxation from 100 nM U46619) in the vessels used below. We investigated the main known purinergic vasodilators, UTPγS (P2Y2 and P2Y4 receptor agonist), UDPβS (P2Y6 receptor agonist) and ADPβS (P2Y1, P2Y12, and P2Y13 receptor agonist). ADPβS (Figure 1(a), pEC₅₀ 6.82 ± 0.28, E_MAX 72 ± 10%) had similar potency as UTPγS (Figure 1(b), pEC₅₀ 6.27 ± 0.39, 84 ± 9%). UDPβS did not cause any vasodilation, but further contracted the artery by 45 ± 21% (Figure 1(c), pEC₅₀ ∼ 4). OPEN IN VIEWER

In vitro contraction of the hMMA

For the contractile studies, we performed the experiment in endothelium-free hMMA, which was confirmed by a complete lack of dilation in response to substance P. Since the P2X receptors desensitize very fast, we constructed a two-log step concentration response curve to αβmetATP, an agonist at P2X receptors. αβmetATP was the most potent agonist (Figure 2(a), pEC₅₀ 6.40 ± 0.20, E_MAX 117 ± 11%) on the hMMA, while ATPγS (Figure 2(b), pEC₅₀ 5.05 ± 0.09, E_MAX 173 ± 47%) was less potent than αβmetATP. For the pyrimidine receptors, the most pronounced contraction was observed after activation of the P2Y2 receptor and P2Y4 receptor (Figure 2(c), UTPγS, pEC₅₀ 4.73 ± 0.10, E_MAX 170 ± 44%). Since UTPγS and ATPγS had similar potency, this indicates that most of the effect is mediated by the P2Y2 receptors. A specific agonist for the P2Y6 receptor caused minor contraction (Figure 2(d), UDPβS, pEC₅₀ 5.48 ± 0.41, E_MAX 36 ± 14%). OPEN IN VIEWER

All of the above receptors have previously been investigated in human coronary and cerebral arteries where they induced contraction (22,23), similarly as for the triptans. Therefore, they are not preferable targets for anti-migraine agonists. In contrast, ADPβS, an agonist for the P2Y1 receptor, P2Y12 receptor and P2Y13 receptor, has been shown to be devoid of contractile properties in human coronary and cerebral arteries (22,23). In contrast to the arteries above, in the hMMA, ADPβS caused a minor contraction (Figure 2(e), pEC₅₀ 5.99 ± 0.20, E_MAX 19 ± 4%). Due to the relatively low EC₅₀ value, corresponding better to the P2Y12 receptor or P2Y13 receptor than the P2Y1 receptor, we applied a P2Y13 receptor antagonist (MRS2211, 10 µM) under two different conditions. First, in a pure contractile experiment, where MRS2211 prevented contraction from baseline (Figure 2(e)). Secondly, we added ADPβS with/without MRS2211 on arteries that were precontracted with 30 mM KCl and had subsequently been relaxed with CGRP (Figure 2)(f). The results show that ADPβS agonism can revert the CGRP-induced relaxation by binding to the P2Y13 receptor, similar to what has been observed with sumatriptan (24).

Effects on in vivo/in situ CGRP release in rat

Immunohistochemistry on hTG and hMMA: Localization of P2Y13 receptor and P2X3 receptor, co-localization with CGRP

We have here presented strong functional evidence for the involvement of the P2Y13 receptor and P2X receptor in in vivo and in situ rat models. Similar experiments are not possible in humans, but we had access to human tissue. Although the samples were obtained post mortem and could have been subject to potential changes in protein expression, we investigated whether similar receptor components are present in the human trigeminovascular system. Although sparsely expressed, we did detect neurons expressing both the P2Y13 receptor and CGRP in the human trigeminal ganglion (Figure 7(a)). In contrast, the P2X3 receptor is strongly expressed together with CGRP (Figure 7(b)). In the hMMA, CGRP and the P2Y13 receptor co-localized, in what appear to be C-fibre nerve endings (Figure 8(a), white arrows), whereas the P2X3 receptor is sparsely, if at all, present in the hMMA (Figure 8(b)). In one of the samples of hMMA, we observed a meningeal nerve (Figure 8(c)). Here, it is even more evident that the P2Y13 receptor co-localizes with CGRP in the C-fibres, but this is not the case for the P2X3 receptor (white arrows). OPEN IN VIEWER

Figure 7.

Immunohistochemical localization of P2Y13 receptor and P2X3 receptor in the human trigeminal ganglion. (a) Representative expression of P2Y13 receptor (green) and CGRP (red) in the human trigeminal ganglion co-localizes in some cells (merged). (b) Representative expression of P2X3 receptor (green) and CGRP (red) in the human trigeminal ganglion co-localizes in several of the cells (merged). DAPI was used to stain the nuclei for the merged picture. Scalebar is 100 µM.

OPEN IN VIEWER

Figure 8.

Immunohistochemical localization of P2Y13 receptor and P2X3 receptor in the hMMA. (a) Representative expression of P2Y13 receptor (green) and CGRP (red) in the hMMA, co-localizes in nerve endings, see arrows (merged). (b) Representative expression of P2X3 receptor (green) and CGRP (red) in the hMMA did not co-localize at the nerve endings, see arrows (merged). (c) Insert of a nerve following the hMMA further illustrates the co-localization of P2Y13 receptor (green) and CGRP (red), but not any co-localization of CGRP with the P2X3 receptor. DAPI was used to stain the nuclei for the merged picture. Scalebar is 100 µM.

Direct effects on the MMA

Until CGRP receptor antagonists were developed, all acutely acting specific anti-migraine drugs induced direct vasoconstriction. Although direct vasoconstriction of the MMA may not be an essential requirement in treating migraine (1), it still might play a relevant role (28). In addition, the MMA may serve as a proxy of the trigeminal system, where potential anti-migraine compounds can be tested. In the current study, we started with a characterization of purinergic receptors on the hMMA. The data show that purinergic receptors have important effects. Particularly, their activation on the endothelium may induce strong dilation (Figure 1), while their activation in smooth muscle cells may induce a strong contraction (Figure 2). Only the contractile responses can be compared to our previously obtained data in the rat, as there were no endothelial responses in the rMMA due to their limited diameter (29).

Vasoactive properties of purinergic ligands on the hMMA

The P2X receptor agonist αβmetATP caused the most potent contraction in the hMMA (pEC₅₀ 6.40 ± 0.20, Figure 2(a)). This is expected, as the P2X1 receptor is known to mediate potent contractions in smooth muscle cells (30). The main pyrimidine receptor in the hMMA is the P2Y2 receptor (Figure 1(b) and Figure 2(b)/(c)). When activated in the endothelium of the hMMA, it causes a strong vasodilation, with higher potency than in the human internal mammary artery (31). The P2Y2 receptor is also the receptor that leads to the strongest contraction of the MMA, similar to human coronary and omental arteries (22,23). Activation of the P2Y6 receptor caused a pure contractile effect (Figure 1(c) and Figure 2(d)). The E_MAX was not very high, which is similar to the human omental artery (22). In human coronary arteries, there was no contraction to UDPβS (23), which contrasts to the cerebral arteries, where UDPβS is a strong constrictor (22). ADPβS was a strong vasodilator (Figure 1(a)), which is typically attributed to the P2Y1 receptor (31). The contraction to ADPβS in the hMMA was potent, but with a relatively low E_MAX (Figure 2(e)). In conclusion, the functional purinergic profile of the hMMA is most similar to human omental arteries and shares strong similarities to the coronary arteries. The profile in hMMA contrasts with that in human cerebral arteries, where the main difference is strong contractility to UDPβS (P2Y6 receptor agonist). Contractions to ADPβS are not described in any of the other human arteries, making the hMMA unique.

Focus on the P2Y13 receptor in the hMMA

We were further particularly interested in ADP and its stable analogue ADPβS, since it is known that this does not cause contraction in the human coronary or cerebral vasculature (22,23). The side effect of coronary vasoconstriction is a current caveat of the current acutely acting antimigraine treatments such as the triptans (32). The contractile response to ADPβS in hMMA is small but significant. Furthermore, the response to ADPβS on the hMMA seems to be mediated purely by the P2Y13 receptor, as it is blocked by its antagonist MRS2211 (Figure 2(e)/(f)), at a concentration that should be devoid of effects on the P2Y1 and P2Y12 receptors (33). Although the contraction to ADPβS was minor, ADPβS and P2Y13 receptor activation could reverse the dilation caused by CGRP (Figure 2)(f). This has a strong link to anti-migraine potential, and the data are very similar to what has been observed for the triptans (24).

Comparison between hMMA and rMMA

In our previous study on rMMA, ADPβS caused a strong contraction compared to the current data on the hMMA. We did not explore the functional pharmacological identity of the ADPβS response in the rat study, but we detected a strong P2Y13 receptor signal in the rMMA by PCR, combined with some P2Y1 receptor expression, but no expression of the P2Y12 receptor (29). Regarding the difference in contraction between rMMAs and hMMAs, it appears that in both human and rat, fast acting and short-lasting effects are mediated via ATP on P2X receptors. In humans, long-term effects of purines are effectuated by ATP on the P2Y2 receptor, in contrast to rats where ADP is acting on the P2Y1 receptor (29). For pyrimidines, UTP through P2Y2 receptor activation is important in humans, while the breakdown product UDP acts on the P2Y6 receptor in rodents. It therefore seems that the human artery is more sensitive to ATP. In conclusion, the purinergic contractions in hMMA are caused by ATP/UTP, in contrast to the rMMA, which contracts to their breakdown-products UDP/ADP. Nevertheless, in vivo, the response is most likely similar, as the breakdown of UTP/ATP into UDP/ADP is nearly instantaneous (34), exemplified by studies on P2Y2 receptor and P2Y6 receptor knock-out mice (35) and functional data from rat meningeal arteries (36).

Trigeminovascular effects

In the light of the recent advances in migraine research, investigating the in vivo effects of potential antimigraine compounds on CGRP release is a highly relevant approach. We therefore used an established method to study the in vivo effects of ADPβS and αβmetATP, which activate P2Y13 and P2X receptors, respectively.

Inhibitory effects of P2Y13 receptor agonism

Our data show that ADPβS, through activation of the P2Y13 receptor, inhibits vasodilation caused by periarterial electrical stimulation (Figure 3(b)/(c)). Periarterial stimulation has been shown to cause in vivo release of CGRP (19,26). Therefore, our experiments are the first to show that P2Y receptors, coupled to G_i proteins, can have prejunctional inhibitory effects on CGRP release in vivo. Similar observations have been made on other prejunctional nerve endings, where P2Y13 receptor activation was demonstrated to inhibit noradrenaline release from sympathetic neurons (16) and cholinergic transmission on the neuromuscular junction (17). Importantly, we also show the direct inhibitory effect of ADPβS on CGRP release from both the dura and the TG in situ (Figure 4(a)). ADPβS is activating the P2Y13 receptor, as the specific P2Y13 receptor inhibitor MRS2211 was without effects per se but reversed the inhibitory effect of ADPβS (Figure 4(b)/(c)). This is similar to the experiments by Honey et al., where an adonsine A1 receptor antagonist (DPCPX) blocked the inhibitory effect of an A1 agonist (GR79236) on meningeal vasodilation but had no effects per se (37). Since inhibiting CGRP release (or CGRP signalling) is now believed to be the most important hallmark of all acute anti-migraine treatments (1), our data clearly demonstrate the therapeutic potential of a specific P2Y13 receptor agonist.

Trigeminal activation by P2X receptor activation

αβmetATP injections caused an activation of the trigeminovascular system, starting with a strong, short-lasting contraction of the rMMA, followed by a relaxation (Figure 5(a)). The relaxation is mediated by CGRP, as demonstrated by the blockade induced by the CGRP receptor antagonist olcegepant (Figure 5(b)). This is highly similar to the experiment by McCulloch and colleagues that was performed over 30 years ago, who showed that trigeminal nerve lesion prevented the relaxation following a constriction of the meningeal artery (38). Our data show that αβmetATP-induced activation of P2X receptors on the TG is the most likely mechanism of action, leading to CGRP release (Figure 6). Indeed, Yegutkin et al. showed that αβmetATP triggered trigeminal nerve fibre activity (36) and Eroli et al. have shown that αβmetATP evokes neuronal firing in trigeminal cultures (39). Therefore, stimulation of P2X3 receptors using αβmetATP could be a model of trigeminal activation, particularly for antidromic CGRP release occurring at the MMA. This is supported by: i) The relatively long time from stimulation until observable dilation (Figure 5(a)), as the signal to release CGRP might travel from the TG and ii) the ability of αβmetATP to trigger CGRP release only from the TG and not in the dura mater (Figure 6).

Expression in human TG and clinical relevance

We investigated whether the purinergic receptors found to be important in the rat trigeminovascular system are also expressed in the human TG. The P2X3 receptor was strongly expressed in CGRP-ergic neurons (Figure 7(a)), similar to what has been shown before in rats (9). There are also neurons that are positive for both the P2Y13 receptor and CGRP, although we only sporadically observed these (Figure 7(b)). In addition, we investigated the hMMA and its innervation. Here we found that, in contrast to the TG, the P2Y13 receptor is co-localized with CGRP (Figure 8(a)), in contrast to the P2X3 receptor (Figure 8(b)). Combined with the findings in the TG, these data match the functional data that we observed in the rat. Both the functional data and the immunohistochemistry indicate that P2X3 receptor activation and expression mainly occurs in the TG (Figures 5/6 and 7/8), and that P2Y13 receptor inhibition and expression can both exist in the TG (Figures 3/4 and 7) and on the nerve endings at the dura/MMA (Figures 3/4 and 8). Combined, the above findings suggest that there are similarities in the purinergic aspects of the neurovascular system between humans and rats.

The clinical applications and potential anti-migraine effects need to be further investigated; for this purpose, there is a particular need for the development of specific agonists. The main concern regarding the use of agonists based on ADP is their potential thrombic activity. ADP activating P2Y1 or P2Y12 receptors is considered a relatively weak platelet aggregation agonist by itself (we did not observe any acute thrombic events in the current experiments). Unfortunately, ADP still works as an amplifier of platelet aggregators such as thrombin (40), which is not compatible with treatment of patients. This potential side-effect should not be an issue for a specific P2Y13 receptor agonist.