European Journal of Pharmacology 647 (2010) 55–61

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

Inhibition of nNOS prevents and inhibition of iNOS reverses α,β-meATP-inducedfacilitation of neck muscle nociception in mice

Dejan Ristic a, Peter Spangenberg b, Jens Ellrich a,⁎a Medical Physiology and Experimental Pharmacology Group, Department of Health Science and Technology, Medical Faculty, Aalborg University, Aalborg, Denmarkb Department of Neurosurgery, Ruhr University Bochum, Knappschaftskrankenhaus Bochum, Germany

⁎ Corresponding author. Medical Physiology and ExpeCenter for Sensory-Motor Interaction SMI, DepartmTechnology, Medical Faculty, Aalborg University, FredAalborg, Denmark. Tel.: +45 21307277.

E-mail address: jellrich@hst.aau.dk (J. Ellrich).

0014-2999/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ejphar.2010.08.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 March 2010Received in revised form 23 June 2010Accepted 10 August 2010Available online 4 September 2010

Keywords:BrainstemHeadacheJaw-opening reflexNitric oxide synthasePain

Infusion of α,β-methylene ATP (α,β-meATP) into murine neck muscle facilitates brainstem nociception.Unspecific nitric oxide synthase (NOS) inhibition prevents and reverses this sensitization. It is unclearwhether neuronal (nNOS), inducible (iNOS) or endothelial NOS isoenzymes are involved in this α,β-meATPeffect. Hypothesized involvement of nNOS isoenzyme was addressed by preceding (0.5, 1, and 2 mg/kg) andsubsequent (2 mg/kg) intraperitoneal injection of the nNOS-inhibitor NPLA. iNOS involvement wasaddressed by subsequent, intraperitoneal administration of the iNOS-inhibitor 1400 W (2 mg/kg). Brainstemnociception was monitored by the jaw-opening reflex elicited via electrical tongue stimulation in 45anesthetized mice. Preceding NPLA dose-dependently prevented α,β-meATP-induced reflex facilitation.Whereas subsequent inhibition of nNOS showed no effect, iNOS inhibition by 1400 W significantly reversedreflex facilitation. Data provide evidence that nNOS plays a major role in induction and iNOS in maintenanceof facilitation in neck muscle nociception. Divergent roles of NOS isoenzymes may promote research ontarget specific treatment for headache and neck muscle pain.

rimental Pharmacology Group,ent of Health Science andrik Bajers Vej 7D2, DK-9220

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Tension-type headache is themost frequent primary headachewithimportant socioeconomic impact (Stovner et al., 2007). Increasedpericranial muscle tenderness is a hallmark of tension-type headache(Ashina et al., 1999b; Buchgreitz et al., 2007). The involvement of nitricoxide (NO) in primary headache has been demonstrated (Ashina et al.,1999a; Ashina et al., 1999c; Ashina et al., 2000). Unspecific inhibition ofnitric oxide synthase (NOS) isoenzymes by L-NMMA relieves tension-type headache (Ashina et al., 1999a; Ashina et al., 1999c). Administra-tion of NO-donor glyceryl trinitrate evokes delayed headache attacksthat share characteristics with tension-type headache (Ashina et al.,2000). Therefore, an important role of NO in tension-type headachepathophysiology is assumed (Ashina, 2002; Ashina, 2007).

NO synthesis depends on oxidization of arginine toNO and citrullinecatalyzed by three different NOS isoenzymes (Alderton et al., 2001;Garthwaite, 2008;Olesen, 2008). NeuronalNOS(nNOS) is constitutivelyexpressed in nervous system and skeletal muscles. Inducible iNOS canbe expressed in a variety of cell types and has a pivotal role in thecytotoxic function of microphages. Endothelial eNOS was first found invascular endothelial cells but can also be found in neuronal tissue and

astrocytes. NO is involved in nociceptive transmission of central andperipheral nervous system (Garthwaite and Boulton, 1995; Luo andCizkova, 2000;Moncada et al., 1991). NOproductionmay trigger centralsensitization (Meller and Gebhart, 1993; Wu et al., 1998; Zhang et al.,2005). As NOS inhibition reduces central sensitization in animalexperiments (Hao and Xu, 1996; Mao et al., 1997; Meller et al., 1994),nociceptive responses can be augmented with NO donors in thesemodels (Coderre and Yashpal, 1994; Kitto et al., 1992).

A recently developed translational mouse model on putativepathophysiological mechanisms of tension-type headache addressesthe impact of nociceptive afferent input from neck muscles on centralnervous system nociceptive processing monitored by the jaw-openingreflex (Ellrich, 2007; Ellrich et al., 2010; Ellrich and Makowska, 2007;Makowska et al., 2005a;Makowska et al., 2006;Makowska et al., 2005b;Panfil et al., 2006; Reitz et al., 2009). This reflex is a commonly acceptedmodel to investigate alterations of excitability in sensory brainstemneurons with convergent afferent input from different craniofacialtissues such as neck muscles. Sustained reflex potentiation by noxiousinput fromneckmuscles evokedby intramuscular infusion (i.m.) ofα,β-methylene adenosine 5´-triphosphate (α,β-meATP) indicates hetero-synaptic facilitation due to access of nociceptive afferents from neckmuscle to the reflex neuronal network in the brainstem.

Similar to the above-mentioned therapeutic effect of L-NMMA inheadache patients (Ashina et al., 1999a; Ashina et al., 1999c), L-NMMAprevents and reverses α,β-meATP-induced facilitation of neck musclenociception inmice (Ellrich et al., 2010). L-NMMAequally inhibits nNOS,iNOS, and eNOS. It remains unclear which NOS isoenzymes mainly

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contribute to analgesic and antinociceptive effects. This manuscriptaddresses the involvement of NOS isoenzymes with particular focus onnNOS in α,β-meATP-mediated facilitation of neck muscle nociception.Preliminary results of the study were presented at the InternationalHeadache Congress 2009 (Ristic and Ellrich, 2009).

Fig. 1. Stimulation protocol. (A) The jaw-opening reflex was evoked by electricalstimulation of afferent nerve fibers in the tongue via two needle electrodes. Reflexresponses were recorded in the anterior digastric muscle by electromyography (EMG)(a.u.: arbitrary units). The graph shows a typical reflex response elicited by a singlestimulus. Dotted lines mark onset and end latency which determine the reflex duration.In this time window, the reflex integral (area under the curve in grey) was analyzed asthemost prominent reflex parameter. (B) The jaw-opening reflexwas evoked in a seriesof eight stimuli each (black bars). Reflex serieswere repeated every 5 min (gray bars). Intwo different experimental groups, drugs were applied intraperitoneally (i.p.) orintramuscularly (i.m.) into both semispinal neckmuscles asmarked by arrows. (C) Afterthree baseline series, isotonic saline or the nNOS inhibitor NPLA (0.5, 1, 2 mg/kg)was i.p.injected. A putative baseline effect of NPLA or saline (vehicle) administration wasobserved for the following 30 min. Thereafter, α,β-meATP (20 μl, 1 μmol/l) was i.m.administered and the effect on the jaw-opening reflex was monitored for 90 min.(D) After three baseline series,α,β-meATPwas i.m. injected and the reflexwas recordedfor 90 min. Subsequently, isotonic saline, NPLA (2 mg/kg) or the iNOS inhibitor 1400 W(2 mg/kg) was i.p. administered. Saline served as vehicle for NPLA and 1400 Winjections. The effects on the reflex were monitored for one further hour.

2. Methods

Electrophysiological experimentswere performed in 45 adultmaleC57BL/6 mice (approximately 12 weeks old; 22 to 34 g; Taconic,www.taconic.com). All procedures received institutional approvalfrom the local ethics committee. The principles of laboratory animalcare and use of laboratory animals (European Council Directive ofNovember 24, 1986(86/609/EEC)) were followed. All efforts weremade to minimize animal suffering and to use only the number ofanimals necessary to produce reliable scientific data.

Detailed description of anesthesia, surgery and electrophysiologicalrecording has been published (Ellrich andWesselak, 2003). Briefly,micewere anesthetized by an initial intraperitoneal (i.p.) injection of a 0.5%pentobarbital sodium salt solution (Amgros I/S, Copenhagen, Denmark,www.amgros.dk) with a dose of 70 mg/kg. Depth of anesthesia waschecked by ensuring that noxious pinch stimulation (blunt forceps) ofhindpaw, forepaw, and ear did not evoke any sensorimotor reflexes.When the animal was sufficiently deeply anesthetized, the skin ofthe throat and neck were carefully shaved and lidocaine hydrochloridegel (Xylocaine® 2%, AstraZeneca A/S, Albertslund, Denmark, www.astrazeneca.com) was applied to the skin of the throat to induce localanesthesia. Dexpanthenol eye ointment (Bepanthen®, Roche, Grenzach-Wyhlen, Germany, www.roche.com) was applied to cornea andconjunctiva of both eyes to protect them from drying. The right externaljugular vein was catheterized for continuous administration of a 2%methohexital sodium salt solution (Brevimytal® Hikma, Hikma Phar-maceuticals PLC, London, United Kingdom, www.hikma.de) with a doseof 60 mg/kg per hour corresponding to a flow rate of about 0.07 ml/h fora 23 g mouse. A pair of Teflon-coated stainless steel wires (140 μmdiameter) was inserted into the right anterior digastric muscle (Dig) torecord electromyographic activity (EMG) and the jaw-opening reflex viaa differential amplifier. After tracheotomy, animals were placed in astereotactic frame and were artificially respired with a stroke volume ofabout 150 μl and about 200 strokes per min (MiniVent Model 845,Harvard Apparatus, Hugo Sachs Elektronik, March-Hugstetten,Germany, www.harvardapparatus.com). Body core temperature wasmaintained at 37.3 °C with a heating blanket and a fine rectal thermalprobe (FMI GmbH, Seeheim-Ober-Beerbach, Germany, www.fmigmbh.de). One platinum needle electrode each (300 μm diameter) wassubcutaneously inserted into right forepaw and left hind paw to recordthe electrocardiogram (ECG) via a differential amplifier. Two stainlesssteel needle electrodes (150 μm diameter) were longitudinally insertedinto the tongue musculature (parallel, 2 mm distance) in order to applyelectrical stimuli and to evoke the jaw-opening reflex. The oral cavitywasfilled upwithwhite vaseline (Riemser Arzneimittel AG, Greifswald -Insel Riems, Germany, www.riemser.de) to protect oral mucousmembrane from drying. Neck skin was locally anesthetized viaXylocaine®. Semispinal neck muscles on both sides were carefullyexposed. One injection cannula each (0.4 mm diameter) was insertedinto themuscle belly of both semispinal neckmuscles. Each cannulawasconnected via thin and short tubing to a liquid switch (CMA/110, CMAMicrodialysis AB, Solna, Sweden, www.microdialysis.se). Glass micro-syringes (1 ml) were connected to the liquid switch by thin tubing andwere fixed in a microdialysis pump (CMA 102, www.microdialysis.se).This procedure allowed bilateral induction of noxious input from neckmuscles in order to mimic bilateral neck muscle pain in tension-typeheadache patients. 1 μM α,β-meATP (Sigma-Aldrich Chemie Gmbh,Munich, Germany, www.sigmaaldrich.com) was i.m. administered witha volume of 20 μl per muscle during a time period of one min.

After preparation, the anesthetized animal was rested for at least1 h. During this time period level of anesthesia and heart rate wereroutinely checked and documented, and depth of anesthesia wasmaintained. All electrical signals (EMG, ECG) were recorded bybioamplifiers. EMG signals led into a data collection system (CEDMicro1401, CED, Cambridge Electronic Design Limited, Cambridge,United Kingdom, www.ced.co.uk) and a personal computer usingSignal® software program (CED, Cambridge Electronic DesignLimited, Cambridge, United Kingdom, www.ced.co.uk).

The jaw-opening reflex was elicited by electrical stimulation ofafferent nerve fibers in the tongue musculature via two needleelectrodes with rectangular electrical pulses of 500 μs duration and astimulation frequency of 0.1 Hz (Fig. 1). Reflex responses wererecorded in the anterior digastric muscle by electromyography(EMG). A typical reflex response elicited by a single stimulus isquantified by its onset latency, duration and integral. The durationcovers the time window between onset latency and end of the reflexresponse in the digastric EMG. In this time window, the reflex integral(area under the curve) was calculated. The electrical threshold of thejaw-opening reflex was determined by applying one series ofincreasing and decreasing stimulus intensities from 0 to 2 mA insteps of 100 μA. The lowest stimulus intensity that just evoked a reflexresponse was defined as the reflex threshold. Test stimulus intensitywas adjusted to approximately 125% of the reflex threshold. The jaw-opening reflex was evoked in series of eight stimuli (Fig. 1B). Theseseries were repeated every 5 min. After three stable baseline series,drugs were applied and the reflex series continued for at least 2 h.Drugs were the nNOS inhibitor NPLA (Nω-propyl-L-arginine; TocrisBioscience, Bristol, United Kingdom, www.tocris.com), iNOS inhibitor

Fig. 2. Effects of NPLA on basal reflex. (A)With preceding intraperitoneal (i.p.)administration of different doses of NPLA or saline the reflex integral remainedunchanged within the time frame from -30 to -5 min. Data is presented as mean±S.E.M. Statistical analysis was performed with one-way repeated measures ANOVA.(B) Average reflex integral changes within 30 min after (i.p.) of saline or different dosesof NPLA are presented as box plots (dotted line: arithmetic mean). One-way ANOVA didnot show any significant differences between groups.

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1400 W (N-[[3-(Aminomethyl)phenyl]methyl]-ethanimidamidedihydrochloride; Tocris Bioscience, Bristol, United Kingdom, www.tocris.com), isotonic saline (0.9% sodium chloride) and α,β-meATP.All substances were resolved in isotonic saline solution. Therefore,saline served as vehicle control for both drugs, NPLA and 1400 W.

Two different experiments were conducted (Fig. 1). In the firstexperiment, NPLA or vehiclewere administered i.p. after stable baselinerecording prior to i.m. α,β-meATP infusion. Vehicle was isotonic saline(100 μl). Due to preceding pilot experiments, three different dosages ofNPLA (100 μl) were applied (0.5, 1 and 2 mg/kg). Six consecutive reflexseries were recorded before α,β-meATP infusion into semispinal neckmuscles. The jaw-opening reflex was then monitored for 90 min(Fig. 1C). In the second experiment, α,β-meATP was i.m. infused intoboth semispinal neck muscles after three stable baseline recordings.90 min subsequent to α,β-meATP administration, an i.p. injection ofsaline, 2 mg/kgNPLAor 2 mg/kg1400W(100 μl)wasperformed. Salinewas used as a vehicle for both NOS inhibitors. Reflex monitoringcontinued for at least 60 min (Fig. 1D). I.m. infusionof isotonic salineas avehicle forα,β-meATP did not affect the jaw-opening reflex (Ellrich andMakowska, 2007; Makowska et al., 2005a). These published experi-ments were not repeated in order to minimize the amount of animalsused in this study.

Onset latency, duration, and integral as the area under the curve ofthe jaw-opening reflex were analyzed in each single sweep. In recentstudies, reflex integral turned out to be the key parameter of reflexalteration in the present animal model (Ellrich and Makowska, 2007;Makowska et al., 2005a; Makowska et al., 2006; Makowska et al.,2005b; Reitz et al., 2009). Reflex integral, indeed, partly depends onduration of reflex. For the sake of clarity and readability, the presentmanuscript focuses on statistical analysis of reflex integral expressedas percentage changes from baseline. Arithmetic mean and standarderror of mean (mean±S.E.M.) were calculated. For the effect of i.p.applied NPLA or saline on the basal jaw-opening reflex, percentagechanges based upon average reflex integrals in the time window from−50 min to −40 min (Fig. 2). For the effect of preceding NPLA orsaline on ATP-induced facilitation, percentage changes based uponaverage reflexes in the time window from −15 min to −5 min(Fig. 3). For the effect of subsequent NPLA, 1400 W or saline, thereflex integrals from 80 min to 90 min were normalized to 100%changes from baseline. Differences between and within groupswere analyzed via two-way repeated measures analysis of variance(Two-way RM ANOVA, one factor repetition, F- and P-values). Factorswere substance and time. Data was also analyzed for interactionbetween these factors. Depending on data distribution, compari-son within groups was performed via one-way RM ANOVA (F- andP-values) or Friedman RM ANOVA on ranks (Χ2- and P-values).Comparison between groups was performed with one-way ANOVA(F- and P-values) or one-way ANOVA on ranks (Kruskal–Wallis, H-and P-values). Post-hoc comparisons were performed with theStudent–Newman–Keuls test (SNK, P-values). The level of signifi-cance was Pb0.05.

3. Results

In 39 mice electrical tongue stimulation elicited the jaw-openingreflex with a threshold intensity of 691±44 μA (mean±S.E.M). Teststimulus intensity was adjusted to 865±53 μA corresponding to 128±3% of the reflex threshold. There were no adverse events during andafter i.p. injection of saline, NPLA and 1400W within the monitoredparameters. Neither preceding nor subsequent i.p. injection of isotonicsaline caused visible or audible reaction in mice (n=11). Increaseddigastric muscle EMG was observed with preceding (0.5 mg/kg, n=1)and subsequent NPLA administration (n=1) immediately after injec-tion. Short-term increase in heart rate was observed in n=1mice withsubsequent 1400W.

3.1. Baseline effects of preceding NPLA (Fig. 2)

Time courses of reflex integrals from −50 min to −5 min wereanalyzed for the evaluation of basal effects of NPLA (Figs. 1, 2). In 24mice, different dosages of NPLA (0.5, 1, and 2 mg/kg) or saline (n=6each) were i.p. injected after stable baseline recordings (Figs. 1, 2).Drugs did not affect basal reflex within each group (One-Way RMANOVA, n.s., Fig. 2A). Mean values of percentage reflex integralchanges after i.p. administration of NPLA or saline in the time slot from−30 to −5 min were calculated and compared (One-Way ANOVA,Fig. 2B). There were no significant differences between groups.

3.2. Effects of preceding NPLA on α,β-meATP-induced reflex facilitation(Fig. 3)

In 20 mice, α,β-meATP was infused in semispinal neck muscles35 min after i.p. injection of saline or different dosages of NPLA(Figs. 1, 3). Reflex integrals in the time frame from −15 to 90 minwere tested for facilitatory effects of i.m. α,β-meATP within groups(one-way RM ANOVA, Fig. 3A). Reflex facilitation after i.m. α,β-meATP occurred with preceding saline or 0.5 mg/kg NPLA. Data wasaveraged in 30 min intervals (5–30, 35–60, 65–90 min) for each group(Fig. 3B). Two-way RM-ANOVA revealed an effect of time, substanceand interaction. There were no differences between groups in the first

Fig. 3. Dose-dependent reduction of facilitation with preceding NPLA. (A) Withpreceding intraperitoneal (i.p.) injection of saline or 0.5 mg/kg NPLA, a significant reflexfacilitation established after additional intramuscular (i.m.) infusion of α,β-meATP intosemispinal neck muscles. Preceding i.p. application of 1 and 2 mg/kg NPLA preventedthe α,β-meATP effect. Data is presented as mean±S.E.M. Statistical results of one-wayrepeated measures ANOVA and Friedman ANOVA are given. (B)Average reflex integralchanges within 30 min time frames after i.p. injection of saline or different doses ofNPLA and subsequent i.m. α,β-meATP are presented as box plots (dotted line:arithmetic mean). A two-way repeated measure ANOVA revealed significant effects forfactors time, substance and interaction. Student–Newman–Keuls post-hoc testsindicated significant differences (*: Pb0.05).

Fig. 4. Reversal of reflex facilitation with subsequent 1400 W. During established reflexfacilitation 90 min after intramuscular (i.m.) α,β-meATP administration, saline, 2 mg/kg NPLA, or 2 mg/kg 1400 W (n=5 each) were intraperitoneally applied (i.p.).(A) Whereas reflex facilitation remained unchanged with subsequent saline or NPLAinjection, integrals significantly decreased after application of 1400 W (FriedmanANOVA). Data is presented as mean±S.E.M. (B)Average reflex integral changes within20 min time frames after i.p. injection of saline, NPLA or 1400 W are presented as boxplots (dotted line: arithmetic mean). A one-way ANOVA revealed significant effectswithin the last time frame from 140 to 155 min after i.p. administration with lowerintegrals after 1400 W as compared to saline and NPLA. Student–Newman–Keuls post-hoc tests indicated significant differences (*: Pb0.05).

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30 min interval. Within 35–60 min, reflex integral of the group withpreceding 2 mg/kg NPLA differed from saline and 0.5 mg/kg NPLAgroups. At the 65–90 min interval, reflex integrals with precedingsaline or 2 mg/kg NPLA differed from all other groups.

3.3. Effects of subsequent saline, NPLA and 1400 W on facilitated reflex(Fig. 4)

Reflex integrals in the time frame from 100 min to 155 min wereanalyzed for effects of subsequently applied drugs on α,β-meATP-induced facilitation (Fig. 1, 4). In 15 mice, saline, 2 mg/kg NPLA or2 mg/kg 1400 W (n=5 each) were i.p. injected during establishedα,β-meATP-induced reflex facilitation. Within groups analysis (one-way RM ANOVA) showed no effect of saline or 2 mg/kg NPLA onestablished reflex facilitation. With subsequent 1400 W, reflexfacilitation partly dissolves (Fig. 4A). Reflex data were averaged into20 min intervals (100–115, 120–135, 140–155 min) for each group.Between groups analyses in the three time frames were conducted byone-way ANOVA (Fig. 4B). There were no differences between groups

within 100–115 min (F=0.3, n.s.) and 120–135 min (F=3.7, n.s.). Inthe time frame 140–155 min group data differed (F=5.6, Pb0.05)with significantly lower integrals after 1400 W as compared to salineand NPLA in the post hoc analysis. Moreover, the effect of 1400 W onfacilitated reflex levels was observed in one experiment for 2 h. Thelevel of inhibition was constant and not different from the 1400 Weffect after 1 h. In another experiment, 1 mg/kg L-NMMA was i.p.applied 1 h after 1400 W administration. Reversal of reflex facilitationto basal levels occurred after 60 min.

4. Discussion

The experiments demonstrate involvement of NOS isoenzymes inα,β-meATP-induced facilitation of neck muscle nociception inanesthetized mice. This facilitation is prevented by preceding nNOSinhibition and partly reversed by subsequent iNOS inhibition.

Electrical, thermal and mechanical stimulation of the craniofacialregion elicits the jaw-opening reflex (Chiang et al., 1991; Ellrich et al.,2001; Ellrich, 2004; Tambeli et al., 2002; Ulucan et al., 2003; Zhanget al., 1999). Sensory neurons in the spinal trigeminal nucleus receive

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convergent impulses from primary afferents (neck muscle, tongue)and project to digastric motoneurons (Kidokoro et al., 1968; Tsai et al.,1999). Noxious input from neck muscles leads to sustained reflexpotentiation evoked by α,β-meATP (Ellrich and Makowska, 2007;Makowska et al., 2006; Reitz et al., 2009). A,β-meATP exerts its effectsvia interaction with ionotropic, homomeric P2X3 and heteromericP2X2/3 receptors (Khakh, 2001) which in turn mediate nociception(Cockayne et al., 2005; North, 2004; Souslova et al., 2001), probablyvia group III muscle afferents (Ellrich and Makowska, 2007). Thisbrainstem reflex potentiation by additional noxious input from neckmuscles is due to heterosynaptic facilitation. Electrical stimulation ofafferent nerve fibers in tongue musculature reliably evokes the reflexvia a brainstem reflex network (Ellrich, 2004; Ellrich and Wesselak,2003). Nociceptive afferents from neck muscles gain access to thesame network as well. Thus, both afferents from tongue musculatureand neck muscles converge onto the same central reflex network.Therefore, additional excitatory input from neck muscle nociceptorsonto the reflex neuronal network facilitates the tongue-evoked reflexby heterosynaptic access. This sustained reflex facilitation persists forat least 4 h and remains unaffected by additional saline administration(Reitz et al., 2009). The present animal model is suggested to be atranslational model for the investigation of pathophysiologicalaspects of tension-type headache (Bendtsen and Jensen, 2006).

Systemic administration of unspecific NOS inhibitor L-NMMAdose-dependently prevents and reverses α,β-meATP-induced sus-tained facilitation of brainstem nociceptive processing (Ellrich et al.,2010). In the present study, preceding NPLA prevents the α,β-meATPeffect whereas 1400 W reverses it. This modulative effect of NOSinhibition most probably does not depend on mechanically inducedreflex alteration (volume). Both i.m. isotonic saline (Ellrich andMakowska, 2007; Makowska et al., 2005a) and i.p. NPLA do notchange basal jaw-opening reflex. This contradicts local effects ofvolume injection and suggests pharmacological actions of drugs.

NPLA is a slowly dissociable and cell-permeable NOS inhibitor. It ischaracterized as a highly selective and potent inhibitor of nNOS (IC50:57 nM) with 3158-fold and 149-fold selectivity over iNOS and eNOS,respectively (Zhang et al., 1997). Another study gives an IC50 of85 nM with 2459-fold and 24-fold relative selectivity over iNOS andeNOS, respectively (Cooper et al., 2000). The reversibility to inhibitnNOS was demonstrated in-vitro by application of excess argininewhich altered the ratio of arginine to NPLA following a newequilibrium condition with enhanced arginine binding and subse-quent partial restoration of NO formation rate (Cooper et al., 2000;Mayer et al., 1999). However, a recent literature search revealedmanystudies applying NPLA in different animal models but no pharmaco-kinetic data on half life under in-vivo conditions. This information wasconfirmed by personal communication with the distributor and thepharmaceutical developer of NPLA. Due to preceding pilot studies,NPLA dosages were set to 0.5, 1 and 2 mg/kg in the present study.Accordingly, similar NPLA dosages up to 10 mg/kg were applied in-vivo for selective nNOS inhibition in other studies (Sammut et al.,2007; Tanabe et al., 2009). NPLA effects in the present study weredose-dependent and did not show attenuation after 90 min ofrecording period. This might indicate a long half-life. Alternatively,nNOS is inhibited by NPLA just in the initial phase of reflex facilitationand is not involved in the maintenance of the effect.

1400 W is a slow, tight binding inhibitor of human iNOS. Inhibitedenzyme did not recover activity after 2 h (Garvey et al., 1997). Data onhalf life of 1400 W do not exist yet as reported by the distributor.1400 W is either an irreversible inhibitor or an extremely slowlyreversible inhibitor of human iNOS. In contrast, inhibition of humannNOS and eNOS is relatively weaker, rapidly reversible, andcompetitive with L-arginine, with Ki values of 2 μM and 50 μM,respectively. Thus, 1400 W is at least 5000-fold selective for iNOSversus eNOS. This selectivity is similar to that observed in rat aorticrings, in which 1400 W is greater than 1000-fold more potent against

rat iNOS than eNOS. Thus, potency and selectivity of 1400 Winhibition of iNOS both in-vitro and in-vivo is far greater than ofany previously described iNOS inhibitor. In-vivo, 1400 W dosages upto 3 mg/kg were applied for selective iNOS inhibition (Johannes et al.,2009; Rocha et al., 2002). The present study applied 2 mg/kg 1400 W.Molar concentrations of 1400 W and NPLA resembles in the presentstudy.

Subsequent 1400 W reduced facilitated jaw-opening reflex after1 h to about 35±21% whereas subsequent NPLA had no effect (109±19%) implying involvement of iNOS in the maintenance of the α,β-meATP effect. The level of iNOS inhibition effect remained constantwith prolonged recordings (2 h, n=1). Further reduction of the1400 W effect was observedwith subsequent L-NMMA administration(n=1). Two different options explain the incomplete reversal to basalreflex levels with 2 mg/kg 1400 W. Either a higher dosage of 1400 Wis required or iNOS and eNOS are both involved in the maintenanceeffect. Repetitive administration of 1400 W was not tested.

The role of NO and its catalyzing NOS isoenzymes in nociceptionand pain has been investigated in man (Ashina et al., 1999a; Ashinaet al., 1999c; Christiansen et al., 2008; Iversen et al., 1992; Iversenet al., 2008; Iversen and Olesen, 1996; Lassen et al., 1997) andexperimental animal models (Haley et al., 1992; Kitto et al., 1992;Koulchitsky et al., 2009; Meller and Gebhart, 1993; Moore et al.,1991). The majority of studies focused on the involvement of nNOS incentral nociception. Both, wild-type mice treated intrathecally withnNOS inhibitors and nNOS-knockout mice did not develop mechan-ical hypersensitivity after spinal nerve injury (Guan et al., 2007). Aputative role of nNOS in sensitization of dorsal root ganglia neuronswas concluded from experiments on inflammatory pain in eNOS-,iNOS- and nNOS-deficient mice (Boettger et al., 2007). Pronouncedreduction of thermal hyperalgesia in nNOS-deficient mice wasaccompanied by reduced immunoreactivity of calcitonin gene-relatedpeptide. This finding was supported by elevated nNOS expressionlevels in dorsal root ganglia whereas iNOS and eNOS expressionlevels remained unchanged. Reduced expression levels of spinalnNOS are documented in a vincristine-induced neuropathy model inmice (Kamei et al., 2005). It is assumed that nNOS could be a criticalfactor in chronic inflammatory pain (Chu et al., 2005). Systemicpharmacological inhibition of nNOS relieved mechanical allodynia-like responses after photochemically-induced spinal cord ischemia inrats (Hao and Xu, 1996). nNOS is also involved in antinociceptive anddefensive reactions induced by local glutamate N-methyl-D-aspartatereceptor activation in the brainstem (Miguel and Nunes-de-Souza,2006). NPLA might affect nNOS activity in the peripheral nervoussystem. Unspecific NOS inhibition affected the rat formalin testindicating contribution of NO to nociceptive processing in spinal cordand periphery (Haley et al., 1992). nNOS is constitutively expressedin skeletal muscles as well (Grozdanovic, 2001; Grozdanovic andBaumgarten, 1999).

Several studies demonstrated iNOS involvement in differentanimal pain models providing evidence for both peripheral andcentral nociceptive mechanisms. Intrathecal administration of theiNOS inhibitor 1400 W attenuated carrageenan-induced and forma-lin-induced hyperalgesia in rats (Bhat et al., 2008; Tang et al., 2007).iNOS and nNOS overexpression in peripheral and central nervoussystem was demonstrated in a murine model of mononeuropathyinduced by sciatic nerve chronic constriction injury (Martucci et al.,2008). After capsaicin injection into the rat masseter muscle, proteinexpression levels in the subnucleus caudalis of all three isoenzymeswere up-regulated (Lee et al., 2009). Pretreatment with NOS in-hibitors attenuated masseter hypersensitivity.

Taken together, observed prevention of the α,β-meATP effect viapreceding nNOS inhibition and its reversion can principally take placein different tissues such as neck muscle, peripheral nociceptiveneuron, and the central nervous system. Nociceptive events frommuscle and peripheral nervous system could explain the observation

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that nNOS inhibition only affects the induction of reflex facilitation byα,β-meATP in neck muscle nociception. Divergent roles of nNOS andiNOS in peripheral and central animal pain models suggestsorchestration of NOS isoenzymes at different sites and with differentimpact (Boettger et al., 2007; Lee et al., 2009; Martucci et al., 2008;Tanabe et al., 2009). Depotentiation via iNOS inhibition could occur inthe central nervous system but at which level remains speculative.

Prolonged noxious input from pericranial myofascial tissues mightcause central sensitization leading to chronic tension-type headache(Ashina, 2004; Ashina, 2007). As NO is implicated in pathophysio-logical mechanisms of central sensitization (Schmidtko et al., 2009),unspecific NOS inhibitor L-NMMA was tested in chronic tension-typeheadache patients (Ashina et al., 1999a; Ashina et al., 1999c). SinceL-NMMA reduced muscle hardness and headache intensity, tension-type headache patients might benefit from specific NOS isoenzymeinhibitors in clinical practice. Selective NOS inhibitors for clinicalstudies might be available in the near future. The nNOS inhibitorNXN-188 dihydrochloride is tested in phase 2 multicenter study inmigraneurs with aura (NCT00959751, http://clinicaltrials.gov/)pointing to new opportunities for the investigation of NOS isoen-zyme involvement in pathophysiological mechanisms of neck mus-cle nociception.

Acknowledgements

This research project was supported by grants of the GermanHeadache Consortium (01EM0516, project A3) and the LundbeckFoundation (R17/A1566). This project was also supported by a PhDstipend from the Medical Faculty, Aalborg University.

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