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Abstract

Tension-type headache is associated with noxious input from neck muscles. Due to the importance of purinergic mechanisms in muscle nociception, experimental studies typically inject α,β-methyleneadenosine 5′-triphosphate (α,β-meATP). In contrast to native adenosine 5′-triphosphate (ATP), α,β-meATP has a narrow receptor profile and remains stable in tissue. The present study administered α,β-meATP or ATP in semi-spinal neck muscles in anaesthetized mice (n = 65) in order to address different effects in neck muscle nociception. The jaw-opening reflex monitored the impact of neck muscle noxious input on brainstem processing. Injection of α,β-meATP induced reflex facilitation in a dose-dependent manner. In contrast, only the lowest ATP dosage evoked facilitation. Preceding P2Y₁ receptor blockade revealed facilitation even under high-dosage ATP. Ongoing facilitation after α,β-meATP injection neutralized under subsequent activation of P2Y₁ receptors. Results demonstrate opposing excitatory P2X and inhibitory P2Y effects of ATP in neck muscle nociception. These mechanisms may be involved in the pathophysiology of neck muscle pain in man.

Keywords

ATP brainstem tension-type headache orofacial trigeminal

Introduction

Neck muscle nociception and pain probably play a decisive role in the pathophysiology of tension-type headache (TTH) (1–5). Consequently, a recently developed experimental animal model addressed the impact of nociceptive afferent input from neck muscles on central nervous system pain processing (6–11). Intramuscular (i.m.) injection of an adenosine 5′-triphosphate (ATP) analogue induces a sustained increase of neuronal excitability in the brainstem. This long-term excitatory effect on neck muscle nociception is mediated by P2X receptors (9).

Involvement of ATP in nociception and pain was recognized first in 1966 (12). Meanwhile, ATP is known to be an important neurotransmitter that interacts with ionotropic P2X receptors and metabotropic P2Y receptors (13). Of all P2X receptors especially homomeric P2X₃ and heteromeric P2X_2/3 subtypes mediate nociception, as shown in genetically manipulated mice and by pharmacological studies (14–17). Whereas ionotropic P2X₃ and P2X_2/3 receptors transmit excitatory actions, metabotropic P2Y₁ receptor mediates inhibitory effects (18–20). Under in vitro conditions P2Y₁ receptors inhibit P2X₃ receptor channels in dorsal root ganglion neurons, indicating an antagonist interaction at cellular level (19).

ATP seems to be particularly suitable for experimental induction of noxious input from muscles, for various reasons. Interstitial ATP concentration in muscles increases with muscle contraction (21–24). ATP excites nociceptive group III and IV afferents in skeletal muscles (25, 26). Ionotropic P2X₃ and P2X_2/3 receptors are localized in pericranial muscles (27). Finally, ATP injection into human trapezius muscle induces local tenderness and strong pain (28).

Due to several decisive pharmacological differences, experimental studies usually favour administration of the ATP analogue α,β-methyleneadenosine 5′-triphosphate (α,β-meATP) over native ATP. ATP and α,β-meATP differ mainly in enzymatic degradation and receptor interaction. Degradation studies have demonstrated dephosphorylation of ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) with half-lives of about 15–20 min (29, 30). These studies have also shown that α,β-meATP is very slowly degraded to α,β-meADP, with approximately 80% remaining in tissue after 1 h. Whereas native ATP interacts with most P2X and P2Y receptors, α,β-meATP exclusively addresses P2X receptors with high sensitivity to P2X₁, P2X₃ and P2X_2/3 (13, 31, 32). Biochemical stability and narrow receptor profile are the main reasons for selecting the P2X receptor agonist α,β-meATP in most experimental studies.

A single local injection of α,β-meATP into murine neck muscles increases neuronal excitability in the brainstem for many hours. This sustained effect is obviously mediated by ongoing excitation of P2X receptors on peripheral nociceptors in neck muscles (9). Due to the stability of α,β-meATP in tissue and its consequential long residence time in muscle, the experimental model with a single injection mimics a more prolonged application. The short half-life of native ATP and a broad receptor profile suggest different effects of ATP in the mouse model compared with α,β-meATP. In vitro studies, in particular, predict inhibitory actions of ATP. Thus, the present study addressed different effects of α,β-meATP and ATP in neck muscle nociception.

Methods

Electrophysiological experiments were performed in 65 adult male C57BL/6 mice (approximately 12 weeks old; 20–28 g; Charles River Laboratories, http://www.criver.com). All procedures received institutional approval from the local ethics committee (ref. no. 50.203.2-AC 15, 16/03). The principles of laboratory animal care and use of laboratory animals (European Council Directive of November 24, 1986(86/609/EEC)) were followed. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data.

A detailed description of anaesthesia, surgery and electrophysiological recording has been published (33). Mice were anaesthetized by an initial intraperitoneal (i.p.) injection of a 0.5% pentobarbital sodium salt solution (Sigma-Aldrich, http://www.sigmaaldrich.com) with a dose of 70 mg/kg. The depth of anaesthesia was checked by ensuring that noxious pinch stimulation (blunt forceps) of hindpaw, forepaw and ear did not evoke any sensorimotor reflexes. When the animal was sufficiently deeply anaesthetized, the skin of the throat was carefully shaved and lidocaine hydrochloride gel (Xylocaine® 2%; AstraZeneca, http://www.astrazeneca.com) was applied to induce local anaesthesia. Dexpanthenol eye ointment (Bepanthen®; Roche, http://www.roche.com) was applied to cornea and conjunctiva of both eyes to protect them from drying. The right external jugular vein was catheterized for continuous administration of a 2% methohexital sodium salt solution (Brevimytal®; Hikma, http://www.hikma.de) with a dose of 60 mg kg⁻¹ h⁻¹ corresponding to a flow rate of about 0.07 ml/h for a 23-g mouse. A pair of Teflon-coated stainless steel wires (140 µm diameter) was inserted into the right anterior digastric muscle (Dig) to record electromyographic activity (EMG) and the jaw-opening reflex (JOR) via a differential amplifier. After tracheotomy, animals were placed in a stereotaxic frame and were artificially respired with a stroke volume of about 150 µl and about 200 strokes per minute during the complete experiment (MiniVent Model 845; Harvard Apparatus, http://www.harvardapparatus.com). Body core temperature was maintained at 37.5°C with a heating blanket and a fine rectal thermal probe (FMI, http://www.fmigmbh.de). One platinum needle electrode each (300 µm diameter) was subcutaneously inserted into the left forepaw and right hindpaw to record the electrocardiogram (ECG) via a differential amplifier. Two stainless steel needle electrodes (150 µm diameter) were longitudinally inserted into the tongue musculature (parallel, 2 mm distance) in order to apply electrical stimuli and to evoke JOR. The oral cavity was filled up with white vaseline (Riemser, http://www.riemser.de) to protect oral mucous membrane from drying. Semispinal neck muscles on both sides were carefully exposed. One injection canula each (0.4 mm diameter) was inserted into muscle belly of both semispinal neck muscles. Each canula was connected via thin and short tubing to a liquid switch (CMA/110, http://www.microdialysis.se). Glass microsyringes (1 ml) were connected to the liquid switch by thin tubing and were fixed in a microdialysis pump (CMA 102, http://www.microdialysis.se). Use of the liquid switch enabled repeated injections at the same i.m. site without changing canula and without interruption of flow during the in vivo experiment. This procedure allowed bilateral induction of noxious input from neck muscles in order to mimic bilateral neck muscle pain in TTH patients. The following substances were intramuscularly administered with a volume of 20 µl per muscle during a time period of 1 min (corresponding to flow rate of 20 µl/min): α,β-meATP (100 nmol/l, 1 µmol/l; Sigma-Aldrich, http://www.sigmaaldrich.com), ATP (100 nmol/l, 1 µmol/l, 7.6 mmol/l; Sigma-Aldrich, http://www.sigmaaldrich.com), the competitive P2Y₁ receptor antagonist 2′-deoxy-N⁶-methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS2179, 1 µmol/l; Tocris, http://www.tocris.com), the P2Y₁ receptor agonist 2-(methylthio)adenosine 5′-diphosphate trisodium salt hydrate (2-MeSADP, 1 µmol/l; Sigma-Aldrich, http://www.sigmaaldrich.com) and isotonic saline (0.9%, Delta-Select GmbH, http://www.deltaselect.de). A maximum of two injections were performed in one muscle during one experiment.

After surgery and placement of all electrodes, the anaesthetized animal was rested for at least 1 h. During this time period the level of anaesthesia and heart rate were routinely checked and documented, and the depth of anaesthesia maintained. All electrical signals (EMG, ECG) were recorded by bioamplifiers and led into a data collection system (CED Micro1401, http://www.ced.co.uk) and a personal computer using Signal® and Spike2® software programs (CED, http://www.ced.co.uk).

The JOR was elicited by rectangular electrical pulses of 500 µs duration with a stimulation frequency of 0.1 Hz. The electrical threshold of the JOR was determined by applying increasing and decreasing stimulus intensities from 0 to 2 mA in steps of 100 µA. The lowest stimulus intensity that just evoked a reflex response was defined as the JOR threshold (I _JOR). Test stimulus intensity was adjusted to about 140% of the I _JOR. The JOR was evoked in series of eight stimuli each. These series were repeated every 5 min. After three stable baseline JOR series, substances were administered intramuscularly and the effect was monitored in three different time schemes (Fig. 1).

Figure 1

Stimulation protocol. The jaw-opening reflex (JOR) was evoked in series (vertical bars) of eight stimuli each (top right-hand corner). The reflex series were repeated every 5 min (grey bars). In three different experimental groups, substances were injected into both semspinal neck muscles (as marked by arrows). (a) After three baseline series either α,β-meATP (100 nmol/l, n = 10; 1 µmol/l, n = 10) or native ATP (100 nmol/l, n = 10; 1 µmol/l, n = 10; 7.6 mmol/l, n = 10) was intramuscularly (i.m.) administered and the effect on JOR was monitored for at least 2 h. (b) After three baseline series MRS2179 (1 µmol/l, n = 10) was injected intramuscularly and the reflex was recorded for three series. Subsequently, native ATP (1 µmol/l) was administered intramuscularly and effects on JOR were monitored for 2 h. (c) After three baseline series α,β-meATP (1 µmol/l, n = 10) was injected intramuscularly and the effect was monitored for 2 h. 2-MeSADP (1 µmol/l, n = 5) or saline (n = 5) was then administered intramuscularly and reflex effects were monitored for another 2 h.

In the first scheme (Fig. 1a), after three baseline series either α,β-meATP (100 nmol/l, n = 10; 1 µmol/l, n = 10) or native ATP (100 nmol/l, n = 10; 1 µmol/l, n = 10; 7.6 mmol/l, n = 10) was administered intramuscularly and the effect on JOR was monitored for at least 2 h. In the second scheme (Fig. 1b), after three baseline series MRS2179 (1 µmol/l, n = 10) was injected intramuscularly and the reflex was recorded for three series. Subsequently, native ATP (1 µmol/l) was administered intramuscularly and effects on JOR were monitored for 2 h. In the third scheme (Fig. 1c), after three baseline series α,β-meATP (1 µmol/l, n = 10) was injected intramuscularly and the effect was monitored for 2 h. 2-MeSADP (1 µmol/l, n = 5) or saline (n = 5) was then administered intramuscularly and reflex effects were monitored for another 2 h.

Onset latency, duration, and integral of JOR were analysed in each single sweep. In recent studies reflex integral turned out to be the key parameter of reflex alteration in the present animal model (6, 7, 9, 10). Reflex integral, indeed, partly depends on duration of reflex. For the sake of clarity and readability, the present study focuses on statistical analysis of reflex integral. Arithmetic mean and standard error were calculated (mean ± s.e.m.). One-way repeated measures analysis of variance (anova) or Friedman repeated measures anova on ranks (Friedman anova) were applied in order to analyse differences within groups. Multiple comparison procedures within groups were performed by Student–Newman–Keuls (SNK) post hoc test. Differences between groups were analysed by one-way anova (normality) or Kruskal–Wallis one-way anova on ranks (Kruskal–Wallis anova). Multiple comparison procedures between groups were performed by SNK post hoc test. Furthermore, mean integrals before and after i.m. injection were analysed by paired t-test or Mann–Whitney rank sum test.

Results

In 65 mice electrical tongue stimulation elicited the JOR with a threshold intensity of I _JOR = 694 ± 23 µA (mean ± s.e.m.). Test stimulus intensity was adjusted to 955 ± 30 µA corresponding to 139 ± 2% of I _JOR.

Sole administration of α,β-meATP

In 20 mice α,β-meATP solution (100 nmol/l, 1 µmol/l, n = 10 each) was injected into semispinal neck muscles (Figs 1a and 2). Reflex integrals significantly increased within 2 h after injections (Fig. 2). A higher concentration of α,β-meATP caused stronger facilitation of the reflex (100 nmol/l: median +97.5%; 1 µmol/l: median +171.2%; Mann–Whitney rank sum test: T = 77, P < 0.05).

Figure 2

Reflex facilitation after sole intramuscular (i.m.) injection of α,β-meATP. (a) Integral of jaw-opening reflex (JOR) increased significantly within 2 h after i.m. administration of 100 nmol/l α,β-meATP (black triangle; Friedman anova) and 1 µmol/l α,β-meATP (white triangle; anova) in 10 mice each. Data are presented as mean ± s.e.m. (b) After 100 nmol/l α,β-meATP, the reflex integral significantly increased within the first hour (Friedmann anova χ² = 104.4) and second hour (anova F = 13.5). The reflex integral significantly increased within the first hour (Friedmann anova χ² = 93.6) and second hour (Friedmann anova χ² = 73.3) after i.m. injection of 1 µmol/l α,β-meATP. With both concentrations the reflex integral showed a significant increment from the first to second hour (paired t-tests: 100 nmol/l, t = −6.1; 1 µmol/l, t = −3.8). Data are presented as box plots (dotted line: arithmetic mean). Asterisks mark significant differences: **P < 0.01; ***P < 0.001.

Sole administration of ATP

In 30 mice native ATP was injected in semispinal neck muscles (Figs 1a and 3). Within 2 h after administration of 100 nmol/l ATP, reflex integral significantly increased (Fig. 3). After 1 µmol/l, ATP reflex remained unchanged. With 7.6 mmol/l ATP reflex integral decreased in the first hour without any effects in the second hour.

Figure 3

Reflex facilitation after sole intramuscular (i.m.) injection of native ATP. (a) Integral of jaw-opening reflex (JOR) significantly increased within 2 h after i.m. administration of 100 nmol/l ATP (white circle; Friedman anova) and with successive injections of 1 µmol/l MRS2179 (MRS) and 1 µmol/l ATP (white triangle; Friedman anova) in 10 mice each. After 1 µmol/l and 7.6 mmol/l ATP administration, the reflex integral remained statistically unchanged for a complete 2-h time slot. Data are presented as mean ± s.e.m. (b) After 100 nmol/l ATP, the reflex integral significantly increased within the first hour (Friedmann anova χ² = 32) and second hour (Friedmann anova χ² = 27.8). Reflex did not change under 1 µmol/l ATP. Administration of 7.6 mmol/l ATP induced a decrease of reflex integral in the first hour (Friedman anova χ² = 32.2). After successive injections of 1 µmol/l MRS2179 (MRS) and subsequent 1 µmol/l ATP, the reflex integral increased in the first (Friedman anova χ² = 78.6) and second hour (Friedman anova χ² = 61.5). Within the first and second hour after ATP injection, mean reflex integrals differed significantly between groups (first hour: one-way anova, second hour: Kruskal–Wallis anova). Data are presented as box plots (dotted line: arithmetic mean). Asterisks mark significant differences: *P < 0.05; **P < 0.01; ***P < 0.001.

Combined administration of MRS and subsequent ATP

I.m. injection of native ATP in semispinal neck muscles induced reflex facilitation only with a low concentration of 100 nmol/l but not with 1 µmol/l and 7.6 mmol/l (Fig. 3). This might be due to inhibitory effects of native ATP and its metabolites on nociceptors via metabotropic P2Y₁ receptors. In order to test masking of a suggested excitatory effect of ATP by a competitive inhibitory effect of ATP and its metabolites (e.g. ADP), the potent P2Y₁ receptor antagonist MRS2179 (MRS) was applied intramuscularly 20 min before homotopic injection of native ATP (1 µmol/l) in 10 mice (Figs 1b and 3). MRS itself did not induce any changes of baseline reflex. Within 2 h after ATP administration, the reflex integral increased significantly (Fig. 3). Within the first and second hours after ATP injection, mean reflex integrals significantly differed between various ATP concentrations and combined MRS and ATP injection (Fig. 3b). Facilitatory effects on JOR within the first (SNK: q = 5.5, P < 0.05) and second hour (SNK: q = 6, P < 0.05) were significantly greater after combined administration of MRS and ATP than after sole injection of 1 µmol/l ATP (Fig. 3b).

Combined administration of α,β-meATP and 2-MeSADP

Preceding blockade of P2Y₁ receptors revealed a facilitatory effect of i.m. ATP injection (see above, Fig. 3). The result seemed to support competitive antagonism between purinergic inhibitory and excitatory effects on myofascial nociception mediated by P2Y₁ and P2X₃ receptors, respectively. This concept predicted inhibition of α,β-meATP-evoked facilitation by subsequent activation of P2Y₁ receptors. In order to test the concept, the P2Y₁ receptor agonist 2-MeSADP (1 µmol/l) was intramuscularly administered during established reflex facilitation 2 h after injection of 1 µmol/l α,β-meATP in semispinal neck muscles (n = 5) (Fig. 1c). The results were compared with experiments where a first injection of 1 µmol/l α,β-meATP was followed by a saline injection in order to exclude any influence of dilution by second injection (n = 5) (Fig. 1c). Within 2 h after i.m. administration of 1 µmol/l α,β-meATP, the reflex integral similarly increased in both groups (saline group: anova F = 32, P < 0.001; 2-MeSADP group: anova F = 6.8, P < 0.001) (Fig. 4). After a second injection, statistical comparison between groups showed a significant reduction after 1 µmol/l 2-MeSADP within the first hour (t-test: t = 3.2, P < 0.05) and second hour (t-test: t = 7.2, P < 0.001) (Fig. 4b). Facilitated reflex reversed completely back to baseline within 1 h after 2-MeSADP injection.

Figure 4

Subsequent administration of 2-MeSADP reverses reflex facilitation. (a) After three baseline jaw-opening reflex (JOR) series, 1 µmol/l α,β-meATP was intramuscularly (i.m.) injected in both semispinal neck muscles (n = 10). In five experiments each, either saline (black triangles) or the P2Y₁ receptor agonist 2-MeSADP (white triangles) were administered intramuscularly 2 h after injection of α,β-meATP. Reflex integrals are given as mean ± s.e.m. (b) Reflex integral changes are presented as box plots (dotted line: arithmetic mean) per hour after injection of α,β-meATP (first injection) and subsequent injection of saline (grey bars) or 2-MeSADP (white bars) (second injection). Reflex integral after 2-MeSADP significantly differed from the saline group during the first hour (t-test: t = 3.2) and second hour (t-test: t = 7.2).

Discussion

Local administration of α,β-meATP in murine semispinal neck muscles induces sustained facilitation of brainstem nociception in a dose-dependent manner. In contrast, native ATP injection evokes facilitation only with low dosage. Preceding i.m. blockade of P2Y₁ receptors reveals reflex facilitation even under a high dosage of native ATP. Ongoing facilitation after injection of α,β-meATP is abolished by subsequent activation of P2Y₁ receptors. Results indicate significant interactions between excitatory P2X and inhibitory P2Y₁ receptors in myofascial nociceptive processing from neck muscles in mice.

Experimental studies on myofascial nociception usually apply the ATP analogue α,β-meATP due to its prolonged chemical stability, with 80% remaining active in the tissue after 1 h. One major reason for prolonged stability in tissue is the ability of α,β-meATP to inhibit endogenous ecto-ATPases located on the cell surface and thus limiting emergence of metabolization products (34). α,β-meATP has a narrow receptor profile with high affinity to homomeric P2X₁, P2X₃, and heteromeric P2X_2/3 receptors (13, 31, 32). Several animal studies have documented the algogenic character of α,β-meATP (15, 35–38). In a previous study applying the same mouse model of neck muscle nociception, direct evidence for involvement of P2X₃ receptor as well as indirect evidence for an involvement of P2X_2/3 receptor in reflex facilitation after α,β-meATP injection were demonstrated (9, 39). Due to lack of homomeric P2X₁ receptors in skeletal muscles and rapid desensitization of P2X₃ receptors after ligand interaction, α,β-meATP effects seemed to be mainly mediated by heteromeric P2X_2/3 receptors (13, 16, 27, 40, 41).

On the other hand, native ATP is under in vivo conditions immediately degraded into its main metabolites (ADP, AMP, adenosine) within 15–20 min (29, 30, 42) and couples to a broad range of various ionotropic and metabotropic purinergic receptors (32, 43, 44).

In general, purinergic receptors are subdivided into ATP-gated ion channels (P2X) and G-protein-coupled P2Y receptors. Today, seven P2X and eight P2Y receptors have been identified (45, 46). Purinergic P2X receptors in particular play an important role in nociceptive excitatory processing (16, 40, 47, 48), with ATP being the most potent native agonist (40). Homomeric P2X₃ and heteromeric P2X_2/3 receptors have previously been identified to mediate the primary sensory effects of ATP (40, 49). P2X receptors are localized in a wide variety of tissues, including skeletal muscle as well as the central and peripheral neuronal system (32, 44). In muscle, the nociceptive properties of ATP were shown to be conducted via P2X receptors, located on group III and IV muscle afferents. Single nerve fibre recordings in decerebrate cats have demonstrated excitation of group III and IV muscle afferents from the triceps surae muscle under application of α,β-meATP into the poplietal artery (25). Studies in P2X₂ and P2X₃ double-knockout animals have shown a loss of α,β-meATP-induced currents in dissociated dorsal root ganglion neurons (17). In rat, P2X receptors are located in the spinal dorsal horn and especially in the substantia gelatinosa (50). With focus on trigeminal sensory neurons, P2X₂ and P2X₃ receptors were identified to be predominantly located on small to medium size nociceptive neurons and attributed a critical role in the modulation of craniofacial pain (51). Afferents from neck muscles in mice synapse in superficial layers of the upper cervical cord (8, 52). Immuno-histochemical techniques indicate that 22% of masseter muscle afferent neurons were positive for P2X₃ receptor, and 77% of these neurons were classified as heteromeric P2X_2/3 neurons (27).

Regarding the source of ATP involved in purinergic receptor activation, it is accepted that ATP is not only released during cell damage, but is actively released from skeletal muscle and neuronal cells (53–55). Recent studies have shown that in skeletal muscle the interstitial ATP concentration increases with muscle contraction (21–24) and compression (56). In various types of resting cells, extracellular ATP metabolism is balanced by constitutive release (57).

Metabotropic P2Y receptors were previously identified to interact with ionotropic P2X receptors. In nociceptive processing, the P2Y₁ receptor, in particular, seems to play an important role. Several in vitro studies have proposed an inhibitory effect of the metabotropic P2Y₁ receptor onto the excitatory effects being mediated through P2X₃ receptor (18–20).

In the present study, the blockade of the P2Y₁ receptor revealed the excitatory effects of higher concentrations of native ATP. Confirming the inhibitory role of the P2Y₁ receptor, injection of an agonist (2-MeSADP) led to a complete reversal of P2X-mediated facilitation.

Various studies using immunostaining techniques have shown that P2Y₁ receptor antibodies stained > 80% of dorsal root and trigeminal ganglia cells (58, 59) and that P2Y₁ and P2X₃ receptors are frequently colocalized on the same neuron (58, 60). P2Y₁ and P2Y₂ purinergic receptors were shown to be typically expressed in small, putatively nociceptive cells corresponding to known distribution of P2X₃ receptors (58, 61). In rat, distribution in skeletal muscle and neuronal cells has been identified (58, 62).

The physiologically most potent agonist at the P2Y₁ receptor is ADP as well as ATP at high concentrations, whereas MRS2179, which was also used in this study, is a highly selective antagonist (43, 63).

Due to rapid degradation of ATP, effects of application in tissue are probably induced by ATP and its metabolites. In order to minimize possible side-effects of ATP metabolites, an inhibitor of the endogenous ecto-ATPases (ARL67156) was applied in the present study (data not presented). Because of endogenous agonistic properties of ARL67516 (dirty drug) at both the excitatory P2X₃ and the inhibitory P2Y₁ receptor, no coherent results could be obtained (64). Since no other substances with similar effects on enzymes are available, experiments were stopped.

Different molecular mechanisms are involved in interaction of P2X₃ and P2Y₁ receptors (18–20). P2X₃ receptors are ion channels and are selectively permeable to cations (32, 65–68). Metabotropic P2Y₁ receptors are known to exert their effects through a G-protein-coupled cascade (69, 70). P2Y receptors modulate a number of voltage- and ligand-gated membrane channels such as M-type K⁺ channels, voltage-activated Ca²⁺ channels, N-methyl-D-aspartate receptors, as well as vanilloid-, pH- and heat-sensitive TRPV1 receptors (18). Recent studies have demonstrated that activation of metabotropic P2Y₁ receptors inhibits the conductance of P2X₃ receptor channels through facilitation of their desensitization and suppression of their recovery from the desensitized state (18–20).

ATP binds with high affinity to the P2X receptors (in higher concentrations also to P2Y receptors), whereas its main metabolite ADP shows greater affinity to the P2Y₁ receptor (43, 71). ADP has been shown to exhibit an EC₅₀ value at purified P2Y₁ receptors of approximately 1 µmol/l (72). This matches with our results, where lower concentrations of ADP (merging from the breakdown of ATP, 100 nmol/l) did not show significant effects. At that concentration, excitatory effects of native ATP at P2X receptors appear to predominate. For higher concentrations of native ATP and resulting higher concentrations of the metabolite ADP, the amount of ADP binding to the inhibitory P2Y₁ receptor is probably sufficient to counterbalance the ATP effect at the excitatory P2X receptor.

The present study has applied a recently developed model of neck muscle nociception in mice (6–9, 11). The suggested link between TTH on one hand and the animal model on the other may be the importance of neck muscle pain processing in patients. TTH patients typically suffer from pericranial tenderness and hardness of head and neck muscles (1, 73–75). Shoulder and neck pain is induced by static exercise in TTH patients more frequently than in healthy controls (76). Tonic muscle activation with subsequently induced ATP release may therefore be connected to the pathophysiology of TTH. The present study has demonstrated that even low intramuscular levels of ATP may facilitate neck muscle nociception.

By using native ATP as a pharmacological tool in the present study, interactions between P2X and P2Y₁ purinergic receptors in muscle nociception could for the first time be shown under in vivo conditions in mice. Divergent interactions of purinergic P2X and P2Y₁ receptors might be useful for a future pharmacological strategy in the treatment of TTH.

Acknowledgements

This research project was supported by grants of the German Headache Consortium (Federal Ministry of Education and Research, 01EM0516, project A3). This paper is part of the doctoral thesis at the Medical Faculty of RWTH Aachen University by cand. med. M.R.

References

Ashina

Bendtsen

Jensen

Sakai

Olesen

. Muscle hardness in patients with chronic tension-type headache: relation to actual headache state. Pain 1999; 79:201–5.

Bendtsen

Jensen

Olesen

. Decreased pain detection and tolerance thresholds in chronic tension-type headache. Arch Neurol 1996; 53:373–6.

Jensen

Bendtsen

Tension-type headache: why does this condition have to fight for its recognition?

Curr Pain Headache Rep 2006; 10:454–8.

Bendtsen

Jensen

. Tension-type headache: the most common, but also the most neglected, headache disorder. Curr Opin Neurol 2006; 19:305–9.

Kim

Chung

Kim

Lee

. Pain-pressure threshold in the head and neck region of episodic tension-type headache patients. J Orofac Pain 1995; 9:357–64.

Makowska

Panfil

Ellrich

. Long-term potentiation of orofacial sensorimotor processing by noxious input from the semi-spinal neck muscle in mice. Cephalalgia 2005; 25:109–16.

Makowska

Panfil

Ellrich

. Nerve growth factor injection into semi-spinal neck muscle evokes sustained facilitation of the jaw-opening reflex in anesthetized mice—possible implications for tension-type headache. Exp Neurol 2005; 191:301–9.

Panfil

Makowska

Ellrich

. Brainstem and cervical spinal cord Fos immunoreactivity evoked by nerve growth factor injection into neck muscles in mice. Cephalalgia 2006; 26:128–35.

Makowska

Panfil

Ellrich

. ATP induces sustained facilitation of craniofacial nociception through P2X receptors on neck muscle nociceptors in mice. Cephalalgia 2006; 26:697–706.

10.

Ellrich

Makowska

. Nerve growth factor and ATP excite different neck muscle nociceptors in anaesthetized mice. Cephalalgia 2007; 27:1226–35.

11.

Ellrich

. Development of an experimental model for investigation of the pathophysiology of tension-type headache. Akt Neurol 2007; 34:448–51.

12.

Burnstock

. Historical review: ATP as a neurotransmitter. Trends Pharmacol Sci 2006; 27:166–76.

13.

Burnstock

. Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 2007; 87:659–797.

14.

Cockayne

Hamilton

Zhu

Dunn

Zhong

Novakovic

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature 2000; 407:1011–15.

15.

Souslova

Cesare

Ding

Akopian

Stanfa

Suzuki

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors. Nature 2000; 407:1015–17.

16.

North

. P2X3 receptors and peripheral pain mechanisms. J Physiol 2004; 554:301–8.

17.

Cockayne

Dunn

Zhong

Rong

Hamilton

Knight

GE.

P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP. J Physiol 2005; 567:621–39.

18.

Gerevich

Zadori

Muller

Wirkner

Schroder

Rubini

Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their desensitization. Br J Pharmacol 2007; 151:226–36.

19.

Gerevich

Muller

Illes

. Metabotropic P2Y1 receptors inhibit P2X3 receptor-channels in rat dorsal root ganglion neurons. Eur J Pharmacol 2005; 521:34–8.

20.

Gerevich

Borvendeg

Schroder

Franke

Wirkner

Norenberg

Inhibition of N-type voltage-activated calcium channels in rat dorsal root ganglion neurons by P2Y receptors is a possible mechanism of ADP-induced analgesia. J Neurosci 2004; 24:797–807.

21.

Hellsten

Maclean

Radegran

Saltin

Bangsbo

. Adenosine concentrations in the interstitium of resting and contracting human skeletal muscle. Circulation 1998; 98:6–8.

22.

Ashina

Stallknecht

Bendtsen

Pedersen

Schifter

Galbo

Tender points are not sites of ongoing inflammation—in vivo evidence in patients with chronic tension-type headache. Cephalalgia 2003; 23:109–16.

23.

King

Sinoway

. Interstitial ATP and norepinephrine concentrations in active muscle. Circulation 2005; 111:2748–51.

24.

King

Sinoway

. ATP concentrations and muscle tension increase linearly with muscle contraction. J Appl Physiol 2003; 95:577–83.

25.

Hanna

Kaufman

. Activation of thin-fiber muscle afferents by a P2X agonist in cats. J Appl Physiol 2004; 96:1166–9.

26.

Reinöhl

Hoheisel

Unger

Mense

. Adenosine triphosphate as a stimulant for nociceptive and non-nociceptive muscle group IV receptors in the rat. Neurosci Lett 2003; 338:25–8.

27.

Ambalavanar

Moritani

Dessem

. Trigeminal P2X3 receptor expression differs from dorsal root ganglion and is modulated by deep tissue inflammation. Pain 2005; 117:280–91.

28.

Mork

Ashina

Bendtsen

Olesen

Jensen

. Experimental muscle pain and tenderness following infusion of endogenous substances in humans. Eur J Pain 2003; 7:145–53.

29.

Welford

Cusack

Hourani

. The structure-activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency. Eur J Pharmacol 1987; 141:123–30.

30.

Welford

Cusack

Hourani

. ATP analogues and the guinea-pig taenia coli: a comparison of the structure-activity relationships of ectonucleotidases with those of the P2-purinoceptor. Eur J Pharmacol 1986; 129:217–24.

31.

Khakh

. Molecular physiology of P2X receptors and ATP signalling at synapses. Nat Rev Neurosci 2001; 2:165–74.

32.

Gever

Cockayne

Dillon

Burnstock

Ford

. Pharmacology of P2X channels. Pflugers Arch 2006; 452:513–37.

33.

Ellrich

Wesselak

. Electrophysiology of sensory and sensorimotor processing in mice under general anesthesia. Brain Res Brain Res Protoc 2003; 11:178–88.

34.

Chen

Lin

. Inhibition of ecto-ATPase by the P2 purinoceptor agonists, ATPgammaS, alpha,beta-methylene-ATP, and AMP-PNP, in endothelial cells. Biochem Biophys Res Commun 1997; 233:442–6.

35.

Tsuda

Ueno

Inoue

. Evidence for the involvement of spinal endogenous ATP and P2X receptors in nociceptive responses caused by formalin and capsaicin in mice. Br J Pharmacol 1999; 128:1497–504.

36.

Hamilton

Wade

McMahon

. The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat. Br J Pharmacol 1999; 126:326–32.

37.

Tsuda

Koizumi

Kita

Shigemoto

Ueno

Inoue

. Mechanical allodynia caused by intraplantar injection of P2X receptor agonist in rats: involvement of heteromeric P2X2/3 receptor signaling in capsaicin-insensitive primary afferent neurons. J Neurosci 2000; 20:RC90.

38.

Sinoway

. ATP stimulates chemically sensitive and sensitizes mechanically sensitive afferents. Am J Physiol Heart Circ Physiol 2002; 283:H2636–43.

39.

Makowska

Hausmann

Panfil

Schmalzing

Ellrich

. ATP injection into neck muscles induces sustained nociceptive facilitation via P2X2/3 receptors in mice. Acta Physiol 2006; 186:122.

40.

North

. Molecular physiology of P2X receptors. Physiol Rev 2002; 82:1013–67.

41.

Liu

Salter

. Purines and pain mechanisms: recent developments. Curr Opin Investig Drugs 2005; 6:65–75.

42.

Zimmermann

. Ectonucleotidases in the nervous system. Novartis Found Symp 2006; 276:113–28.

43.

von Kugelgen

Wetter

. Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol 2000; 362:310–23.

44.

Abbracchio

Burnstock

Purinoceptors: are there families of P2X and P2Y purinoceptors?

Pharmacol Ther 1994; 64:445–75.

45.

Ralevic

Burnstock

. Receptors for purines and pyrimidines. Pharmacol Rev 1998; 50:413–92.

46.

Hollopeter

Jantzen

Vincent

England

Ramakrishnan

Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001; 409:202–7.

47.

Liu

Salter

. Purines and pain mechanisms: recent developments. Curr Opin Investig Drugs 2005; 6:65–75.

48.

Kennedy

Assis

Currie

Rowan

. Crossing the pain barrier: P2 receptors as targets for novel analgesics. J Physiol 2003; 553:683–94.

49.

Burnstock

. Purine-mediated signalling in pain and visceral perception. Trends Pharmacol Sci 2001; 22:182–8.

50.

Kidd

Grahames

Simon

Michel

Barnard

Humphrey

. Localization of P2X purinoceptor transcripts in the rat nervous system. Mol Pharmacol 1995; 48:569–73.

51.

Staikopoulos

Sessle

Furness

Jennings

. Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons. Neuroscience 2007; 144:208–16.

52.

Sessle

. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive transmission and neuroplasticity, and their clinical correlates. Crit Rev Oral Biol Med 2000; 11:57–91.

53.

Boyd

Forrester

The release of adenosine triphosphate from frog skeletal muscle in vitro . J Physiol 1968; 199:115–35.

54.

Forrester

Lind

. Identification of adenosine triphosphate in human plasma and the concentration in the venous effluent of forearm muscles before, during and after sustained contractions. J Physiol 1969; 204:347–64.

55.

Evans

Derkach

Surprenant

. ATP mediates fast synaptic transmission in mammalian neurons. Nature 1992; 357:503–5.

56.

Taguchi

Kozaki

Katanosaka

Mizumura

. Compression-induced ATP release from rat skeletal muscle with and without lengthening contraction. Neurosci Lett 2008; 434:277–81.

57.

Lazarowski

Boucher

Harden

. Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol Pharmacol 2003; 64:785–95.

58.

Ruan

Burnstock

. Localisation of P2Y1 and P2Y4 receptors in dorsal root, nodose and trigeminal ganglia of the rat. Histochem Cell Biol 2003; 120:415–26.

59.

Xiao

Huang

Zhang

Bao

Guo

Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain. Proc Natl Acad Sci USA 2002; 99:8360–5.

60.

Borvendeg

Gerevich

Gillen

Illes

. P2Y receptor-mediated inhibition of voltage-dependent Ca2+ channels in rat dorsal root ganglion neurons. Synapse 2003; 47:159–61.

61.

Gerevich

Illes

. P2Y receptors and pain transmission. Purinergic Signalling 2004; 1:3–10.

62.

Tokuyama

Hara

Jones

Fan

Bell

. Cloning of rat and mouse P2Y purinoceptors. Biochem Biophys Res Commun 1995; 211:211–18.

63.

Moro

Guo

Camaioni

Boyer

Harden

Jacobson

. Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites. J Med Chem 1998; 41:1456–66.

64.

Joseph

Pifer

Przybylski

Dubyak

. Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation. Br J Pharmacol 2004; 142:1002–14.

65.

Buell

Lewis

Collo

North

Surprenant

. An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J 1996; 15:55–62.

66.

Evans

Lewis

Virginio

Lundstrom

Buell

Surprenant

Ionic permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X receptors) expressed in mammalian cells. J Physiol 1996; 497:413–22.

67.

Lewis

Neidhart

Holy

North

Buell

Surprenant

. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature 1995; 377:432–5.

68.

Valera

Hussy

Evans

Adami

North

Surprenant

A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature 1994; 371:516–19.

69.

Communi

Gonzalez

Detheux

Brezillon

Lannoy

Parmentier

Identification of a novel human ADP receptor coupled to G(i). J Biol Chem 2001; 276:41479–85.

70.

Harden

Boyer

Nicholas

. P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol 1995; 35:541–79.

71.

Lazarowski

Boucher

Harden

. Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol Pharmacol 2003; 64:785–95.

72.

Waldo

Harden

. Agonist binding and Gq-stimulating activities of the purified human P2Y1 receptor. Mol Pharmacol 2004; 65:426–36.

73.

Bendtsen

. Central sensitization in tension-type headache—possible pathophysiological mechanisms. Cephalalgia 2000; 20:486–508.

74.

Jensen

Rasmussen

Pedersen

Olesen

. Muscle tenderness and pressure pain thresholds in headache. A population study. Pain 1993; 52:193–9.

75.

Langemark

Olesen

. Pericranial tenderness in tension headache. A blind, controlled study. Cephalalgia 1987; 7:249–55.

76.

Christensen

Bendtsen

Ashina

Jensen

. Experimental induction of muscle tenderness and headache in tension-type headache patients. Cephalalgia 2005; 25:1061–7.

Excitatory and Inhibitory Purinergic Control of Neck Muscle Nociception in Anaesthetized Mice

Abstract

Keywords

Introduction

Methods

Results

Sole administration of α,β-meATP

Sole administration of ATP

Combined administration of MRS and subsequent ATP

Combined administration of α,β-meATP and 2-MeSADP

Discussion

Acknowledgements

References