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nitric oxide pathway in chronic tension-type headache: relation with serotonin content and secretion and glutamate content
Journal of the Neurological Sciences 198 (2002) 9 – 15 www.elsevier.com/locate/jns
L-Arginine/nitric
oxide pathway in chronic tension-type headache: relation with serotonin content and secretion and glutamate content Paola Sarchielli a,*, Andrea Alberti a, Ardesio Floridi b, Virgilio Gallai a a
Interuniversity Center for the Study of Headache and Neurotransmitter Disorders, Perugia, Rome, Sassari, Bari, Naples, Florence—Unit of Perugia, Perugia, Italy b Department of Internal Medicine, Institute of Clinical and Applied Biochemistry, University of Perugia, Perugia, Italy Received 8 August 2001; received in revised form 1 November 2001; accepted 5 February 2002
Abstract Previous research of our group demonstrated an increase in L-arginine/nitric oxide (NO) pathway activity in patients with chronic daily headache (CDH) with a previous history of migraine, which was associated with a reduced platelet serotonin content and increased Ca2 + levels. In the present work, we assessed the variations in L-arginine/NO pathway activity and platelet cyclic guanosine 3V,5V-monophosphate (cGMP) levels in 25 patients affected by chronic tension-type headache (CTTH) (8 M, 17 F; age range: 34-54 years). The NO production, shown spectrophotometrically by stoichiometric transformation of oxyhemoglobin to methemoglobin due to NO synthase (NOS) activity, and inter platelet cGMP concentration, assessed with a RIA method, were determined in parallel to variations of aggregation response to 0.3 Ag/ml collagen. The intracellular platelet calcium concentrations were also determined using fluorescence polarisation spectrometry. Platelet serotonin content and collagen-induced secretion as well as glutamate content were also determined with high-performance liquid chromatography (HPLC). The above parameters were compared with those of an age-matched control group. A reduction in aggregation platelet response was found. The reduction in platelet aggregation was coupled with an increased NO and cGMP production ( p < 0.0002 and p < 0.001, respectively). A significant increase in cytosolic Ca2 + concentration was also detected compared to control individuals ( p < 0.001). This was accompanied by a reduced platelet content and collagen-induced secretion of serotonin and increased content of glutamate ( p < 0.0001, p < 0.0001 and p < 0.001, respectively). The above findings were more evident in patients with analgesic abuse. It can be hypothesized that the increased NOS activity shown in platelets of CTTH patients reflects an analogous central up-regulation of NOS activity in the spinal horn/trigeminal nucleus and supraspinal structures involved in the modulation of nociceptive input from myofascial cranial structures contributing to central sensitization. The increase in NOS activity seems to be associated with a hyposerotonergic status, particularly in patients with analgesic abuse, and this can contribute to central sensitization in CTTH patients. The increase in platelet glutamate content in the same patients suggests the implication of the above excitatory amino acid in spinal and supraspinal structures involved in head pain induction and maintenance. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Chronic tension-type headache; Platelets; Nitric oxide; Cyclic guanosine 3V, 5Vmonophosphate — cGMP; Calcium; Serotonin; Glutamate; Central sensitization
1. Introduction Recent findings suggest the involvement of nitric oxide (NO) in the central sensitization underlying chronic head pain and increased myofascial tenderness of pericranial muscle in patients affected by chronic tension-type headache (CTTH).
* Corresponding author. Neurological Clinic, University of Perugia, Via E. Dal Pozzo 79, 06126 Perugia, Italy. Tel.: +39-075-578-3871; fax: +39-075-578-3583. E-mail address: [email protected] (P. Sarchielli).
In particular, MG-monomethyl-L-arginine hydrochloride (L-NMMA) was demonstrated to be effective in reducing pain intensity, muscle hardness of the trapezius muscle and pericranial myofascial tenderness in patients affected by chronic tension-type headache (CTTH) [1,2]. Using the nitroglycerin (NG) model of experimental headache, Ashina et al. [3] also studied the intensity, quality and time profile of headache after infusion of this nitric oxide donor (0.5 Ag/kg per minute for 20 min) in patients affected by CTTH in comparison with those of age-matched healthy controls. In CTTH patients the area under the curve (AUC) defined by the intensity duration product on the NG day was significantly higher than on the placebo day.
0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 0 3 5 - 7
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Moreover, on the NG day the AUC of CTTH patients was significantly greater than that of healthy controls. In controls, the peak of pain intensity occurred 20 min after the start of infusion, whereas in CTTH patients 8 h after NG infusion. In the latter group, NO induced a biphasic response with an immediate and a delayed headache, as in migraine. The delayed NO-induced headache in chronic tension-type headache patients was similar to that suffered by the patients. Platelets have been proposed as a peripheral model for studying nitric oxide [4]. They, when stimulated by collagen produce NO, using L-arginine as a substrate [5]. A constitutive nitric oxide is present in platelets which is responsible for NO synthesis in the presence of high Ca2 + levels. NO induces the activation of guanylate cyclase and, therefore, the production of cyclic guanosine monophosphate (cGMP). Because of the similarities between platelets and monoaminergic neurons (in particular serotonergic neurons), variations in these blood elements have been interpreted as a peripheral mirror of analogous variations in central monoaminergic pathways. Platelets also contain glutamate and changes in platelet levels of this excitatory amino acid were interpreted to be suggestive of analogous changes in the central nervous system, at least in migraine. In a recent research using the platelet model, we investigated the variations in L-arginine/NO pathway activity and platelet cGMP levels in patients affected by chronic daily headache (CDH) [6], which evolved in all patients examined with a previous history of migraine. We found an upregulation of the L-arginine/NO pathway, that was associated with a reduced content of serotonin and increased Ca2 + levels in platelets. We hypothesized that the occurrence of NO – cGMPmediated events is not too efficient in contrasting the intracytosolic Ca2 + increase in platelets, which could be responsible for serotonin depletion from dense bodies, already shown by recent research in platelets of patients affected by CDH associated with analgesic abuse. No studies have been carried out until now to investigate NO synthase (NOS) activity in tension-type headache, even though the finding of the above mentioned analgesic effect of NOS inhibition in this pathological condition suggests the involvement of NO in central sensitization in this pathological condition. The present research was aimed at assessing the collagen-stimulated NO synthase (NOS) activity in the platelets of patients with CTTH. Moreover, cytosolic ionized calcium content and serotonin release and content were measured. Glutamate content in the platelets of the same patients was also investigated. Data were compared with those of a group of healthy age-matched control subjects. In the group of CTTH patients, values of the above variables from patients with simple or combination analgesic abuse (N = 16) were compared with those of patients without analgesic abuse (N = 9).
2. Patients and methods 2.1. Patients Twenty-five consecutive patients with CTTH attending the Headache Center of Perugia University were assessed. The diagnosis was made according to the IHS criteria [7]. To be included in the study they were required to have had headache at least 6 days per week for at least six months. In all patients CTTH evolved from an episodic tension-type headache. The mean duration of headache was 18.6 F 9.8 years. The criteria used for analgesic abuse were those of Silberstein et al. [8,9]: simple analgesic use > 1000 mg/ ASA/acetaminophen >5 days/week, combination analgesics >3 tablets/day >3 days/week. Twenty healthy age-matched control subjects were also assessed for the same parameters. Details of CTTH patients and control subjects are reported in Table 1. In the subgroup of CTTH patients with analgesic abuse (N = 16), the most frequently abused analgesic was acetaminophen (N = 13). Other analgesics abused were feldene: N = 8, diclofenac: N = 7, ketorolac: N = 5, and nimesulide: N = 6. Eight of the CTTH patients used more than one simple analgesic. Nine CTTH patients used combination analgesics (Optalidon-butalbital + propyphenazone + caffeine: N = 5; Difmetre-indomethacin + prochlorperazine + caffeine: N = 4), four of them in association with simple analgesics. No CTTH patients used drugs containing codeine. The monthly drug intake averaged 78.4 F 14.8 (mean F SD) tablets or suppositories. None of the CTTH patients took medications such as headache preventive drugs. The last time that the patients in the non-overuse group took medication for pain was at least 24 h previously. The patients in the overuse group were invited not to take symptomatic medication for at least 12 h before blood drawing (8 a.m.). All patients in the latter group took the last symptomatic drug before this time. All control subjects were headache-free for at least 2 months and none of them was taking any medication at the time of blood sampling nor had a personal or family history of migraine or suffered from tension-type headache.
Table 1 Characteristics of patients and control subjects
Males Females Age (years) Duration of headache (years) Duration of CTTH (years) Analgesic misuse No analgesic misuse
Patients, N = 25
Control subjects, N = 20
N=8 N = 17 45.2 F 7.4 18.6 F 9.8 11 F 5 N = 16 N=9
N=8 N = 12 43.5 F 6.4
P. Sarchielli et al. / Journal of the Neurological Sciences 198 (2002) 9 –15
3. Methods 3.1. Platelet separation Blood was drawn from the antecubital vein and collected in tubes containing 0.1 volumes of 3.8% citrate and then centrifuged at 200 g for 20 min at room temperature. Platelet-rich plasma (PRP)was removed and then centrifuged at 1000 g for 12 min at 0 jC to form a pellet. The platelets were washed once at room temperature in N-2-hydroxyethylpiperazine-N V-2-ethanesulfonic acid (HEPES) –Thyroid buffer (in mM: 129 NaCl, 2.8 KCl, 0.8 KH2 PO4, 0.8 MgCl2, 5.6 dextrose, 10 HEPES, pH= 7.4) containing 1 mM ethylene glycol-bis (h-aminoethyl ether)-N,N,N V,N V-tetraacetic acid (EGTA), with the addition of indomethacin (10 AM). The platelets were then passed through a Sepharose 2B-300 column preconditioned with the same medium. The platelets were counted using a Coulter counter (model ZN; Coulter Electronics, Hialeah, FL) and were adjusted to 3.5 108 platelets/ml by the addition of HEPES-buffer saline. 3.2. Platelet aggregation Platelet aggregation was performed using the method of Born and Cross [10]. A suspension of washed platelets (450 Al; 3.5 108 platelets/ml) was incubated at 37 jC for 2 min in an aggregometer (Chronolog 480 VS, Chronolog, Haverton, PA) with continuous stirring at 1200 rpm and then stimulated with collagen at concentrations of 0.3, 1, and 3 Ag/ml, which was purchased from Sigma (St Louis, MO, USA). The chart speed was 1 cm/min. Platelet impedance curves were analysed 7 min after the addition of the agonist. The aggregation amplitude was expressed as the platelet aggregation peak (5 V = 12 mm).
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stopped) by the addition of an equal volume of ice-cold trichloroacetic acid (10% wt/vol, final concentration). The incubation was performed both with and without L-NMMA at 10 AM. The measurement of cGMP was made from etherextracted supernatants of the samples. The assays of cGMP were performed using RIA kits (New England Nuclear). Data were expressed in pmol/109 platelets. 3.5. Platelet cytosolic Ca2+ concentration Platelet cytosolic Ca2 + was measured both in resting platelets and platelets stimulated with collagen (3 Ag/ml). Platelet cytosolic Ca2 + concentration was measured with the fluorescent indicator Fura-2 according to the method of Astarie-Dequeker et al. [11]. Platelets were loaded with 2 AM Fura 2AM for 40 min at 37 jC in the presence of plasma. They were then washed by centrifugation at 270 g for 15 min at 20 jC and resuspended at a density of 1 106 cells ml 1 in a medium containing (in mM): 145 NaCl, 5 KCl, 0.5 MgCl2, 10 N-2-hydroxyethylpiperazine-N V-2-ethan sulphonic acid (HEPES) and 5 glucose, pH = 7.4 at 37 jC, and 30 nM free Ca2 + concentration (that suppresses Ca2 + influx) adjusted with the required Ca2 + EGTA buffer. Fluorescence intensities were measured at 37 jC on a Fluorolog equipped with a 450-W Xenon lamp, two monochromators with a dual-mirror chopping mechanism that allows a rapid alternating excitation from 335- to 385-nm wavelengths with a fixed emission wavelength of 510 nm. Each platelet sample was checked for Fura-2 loading by recording the fluorescence spectra. The fluorescence recording was systematically corrected for the autofluorescence of the cell suspension. Platelet cytosolic Ca2 + was calculated from the experimental ratio of excitation fluorescence intensities R = I335/I385 [12]. Data are expressed in nanometers (nm).
3.3. NO formation in platelet cytosol 3.6. Serotonin content and release from platelets The platelet cytosol was prepared from 1 –2 1011 platelets by homogenization and sonication for 10 min with Soniprep and subsequent centrifugation at 150,000 g for 10 min. The NO production in the platelet cytosol obtained by sonication was evaluated spectrophotometrically, assessing the stoichiometric reaction of NO with oxyhemoglobin to yield methemoglobin and NO3 . Data were expressed in fmol/min/mg protein. In the same experiments with the aim to block cyclooxygenase activity, which can interfere with endogenous NO formation, platelet aggregation was investigated by adding to the platelet suspension indomethacin (10 AM) for 10 min at 37 jC, before measuring the aggregation. 3.4. Measurement of cyclic guanosine 3V,5V-monophosphate (cGMP) The incubation of gel-filtered platelets, both in the basal state and in the presence of collagen (0.3, 1, and 3 Ag/ml), was arrested after 7 min (the time at which the aggregation was
The platelet pellet was separated from platelet-poorplasma by centrifuging PRP at 7000 g for 10 min. It was washed twice with HEPES– Thyroid buffer (145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM N-2-hydroxyethylpiperazine-NV-2-ethansulfonic acid (HEPES) and 10 mM glucose, pH = 7.4). All platelets were resuspended at the concentration of 1 109 per ml and frozen at 80 jC until the assay. The samples were thawed at room temperature and sonicated just prior to determination. Platelet 5-HT was measured using reversed-phase highperformance liquid chromatography (HPLC) with electrochemical detection. The chromatographic system included: a Kontron Instrument model 420 with a 5-Am particle diameter, Tracer Analytics ODS and lLC4b amperometric detector. Phosphoric acid (0.1 M), with 0.1 mM EDTA adjusted to pH 2.85 with 4 M KOH solution was the mobile phase. 3,4dihydroxybenzylamine was used as the internal standard. Platelet 5HT levels were expressed in ng/109 platelets.
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Table 2 Summary of the aggregation peaks, NO formation and cGMP production (mean F SEM) in platelets of CTTH patients and control individuals (C) CTTH with analgesic abuse (a)
CTTH without analgesic abuse (b)
C (c)
Statistical significance
Aggregation peak V (collagen 3 Ag)
27.3 F 5.2
33.0 F 6.4
40.6 F 4.8
NO formation, fmol/min/mg protein (collagen 3 Ag)
55.6 F 6.8
42.4 F 4.4
30.2 F 3.1
cGMP formation, pmol/109 platelets (collagen 3 Ag)
7.5 F 1.2
6.2 F 1.1
3.2 F 0.4
a vs c: 0.0003 b vs c: 0.02 a vs b: 0.05 a vs c: 0.001 b vs c: 0.005 a vs b: 0.02 a vs b: 0.001 b vs c: 0.01 a vs b: 0.02
3.7. Platelet glutamate levels Glutamate levels were analyzed according to the method of Zecca and Ferrari [13] with some modifications. Briefly, the platelet pellet was separated from platelet-poor plasma by centrifuging PRP at 7000 g for 10 min. The pellet was resuspended in 1 ml of saline solution and sonicated for 5 min. Sulphosalicylic acid (30 mg/ml) was added for deproteination. The sample (50 Al) was added to a solution containing 400 Al of boric acid, 490 Al of methanol, 50 Al of o-phthalaldehyde (OPA) and 10 Al of the internal standard norvaline (C5H11NO2). Aliquots (20 Al) of supernatants were injected into a Millipore Waters HPLC system connected to a Perkin Elmer fluorescence spectrometer (purchased from Perkin Elmer, Beaconsfield, Buckinghamshire, England) and a reversed-phase column Supelcosil LC-18 (3 A, 15 cm 4.6 mm) (purchased from Sulpeco, Bellefonte, PA, USA). The wavelengths used to detect glutamate were 330 nm for excitation and 445 nm for emission, respectively. The retention time was 11V48U for glutamate and 30V65U for the internal standard. 3.8. Statistical analysis All values, both in unstimulated and stimulated platelets of CTTH patients and controls, were expressed as means F SEM. A non-parametric analysis of variance (ANOVA)
was carried out to compare the mean values of the aggregation peaks due to 3 Ag/ml collagen, basal and collagenstimulated NO formation and cGMP production, Ca2 + content and serotonin concentration in platelets of CTTH patients with the same values of the control group. The same analysis was performed to compare the mean values of the above mentioned parameters in the CHD patients with drug abuse with those of CTTH patients without drug abuse. Fisher’s least significant difference (LSD) was also used to compare the main effect means in ANOVA in the case of equal cell size. Five percent for two-sided tests was chosen as a minimum level of statistical significance. The Pearson correlation coefficient was also calculated between NO and cGMP values and serotonin content and release as well as glutamate content in the platelets of CTTH patients and controls.
4. Results Table 2 summarizes the findings on the aggregation peaks, NO formation and cGMP production of CTTH patients with and without analgesic abuse, and of control individuals. Table 3 shows the values of cytosolic Ca2 + , serotonin content and collagen-induced secretion and glutamate content in CTTH patients and control individuals.
Table 3 Summary of the values of cytosolic Ca2 + concentration, basal content and secretion of serotonin and glutamate concentration (mean F SEM) in platelets of CTTH patients and control individuals (C) CDH with analgesic abuse (a)
CDH without analgesic abuse (b)
C (c)
Statistical significance
Ca2 +, nM (collagen 3 Ag)
184.3 F 12.9
159.3 F 19.9
130.3 F 8.9
Serotonin, ng/109 platelets basal content
340.1 F 32.4
463.5 F 45.8
596.7 F 42.9
5HT secretion (collagen 3 Ag)
410.1 F 34.5
448.4 F 39.7
666.4 F 42.9
63.9 F 4.5
54.2 F 6.4
45.2 F 2.3
a vs c: 0.008 b vs c: 0.01 a vs b: n.s. a vs c: 0.0001 b vs c: 0.002 a vs b: 0.04 a vs c: 0.0001 b vs c: 0.0003 a vs b: 0.05 a vs c: 0.001 b vs c: 0.002 a vs b: 0.03
Glutamate, nmol/109 platelets
P. Sarchielli et al. / Journal of the Neurological Sciences 198 (2002) 9 –15
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Fig. 1. Correlation between serotonin secretion (ng/109 platelets) and NO formation (fmol/min/mg protein) in platelets of CTTH patients with and without analgesic abuse.
The aggregation peaks induced by collagen were significantly lower in CTTH patients than those of the control subjects (ANOVA: p < 0.002). This reduction was more accentuated in patients with analgesic abuse than in patients without analgesic abuse (Table 2). A slight but significant increase in the rate of NO formation in the cytosol of platelets before the addition of collagen was observed in CTTH (16.4 F 4.7), with respect to the control group (6.8 F 3.2) ( p < 0.01). The values tended to further increase after the addition of collagen and this increase was significantly greater in CTTH patients with analgesic abuse compared to that of patients without analgesic abuse (ANOVA: 0.0002) (Table 2). The
latter showed significantly greater values of NO production than healthy subjects. cGMP levels in unstimulated platelets of CTTH patients (1.4 F 0.8) were greater than those of control individuals (0.7 F 0.3) ( p < 0.05). Collagen induced a significant increase in platelet cGMP in CTTH patients, particularly in those with analgesic abuse and, to a lesser extent, in patients without analgesic abuse whose values were, in any case, significantly greater than those measured in platelets of control individuals (ANOVA: 0.001) (Table 2). In patients with CTTH, a slight but significant decrease in the basal content of serotonin was found compared to control individuals ( p < 0.0001). Collagen induced the secretion of
Fig. 2. Correlation between glutamate levels (nmol/109 platelets) and NO formation (fmol/min/mg protein) in platelets of control individuals, CTTH patients with and without analgesic abuse.
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P. Sarchielli et al. / Journal of the Neurological Sciences 198 (2002) 9 –15
serotonin both in controls and CTTH patients, but the values detected in the latter were lower than those found in control subjects, particularly in patients with analgesic abuse (ANOVA: p < 0.0001) (Table 3). At the same time, cytosolic Ca2 + concentration was significantly increased in CTTH patients with respect to age-matched healthy subjects, particularly in patients with analgesic abuse (ANOVA: p < 0.001) (Table 3). Glutamate values measured in platelets of CTTH patients with analgesic abuse were also higher than those of controls (ANOVA: p < 0.001). This increase was greater in patients with analgesic abuse compared to those without analgesic abuse ( p < 0.03) (Table 3). A significant negative correlation was found in CTTH patients between platelet NO production and 5HT secretion by platelets (R = 0.49, p < 0.001), whereas a significant positive value of the correlation coefficient (R = 0.78, p < 0.004) was calculated in the same patients (Figs. 1 and 2). A slight but significant negative correlation also emerged between platelet NO production and 5HT platelet content (R = 0.38, p < 0.05). No significant correlation emerged among the above parameters in control subjects.
5. Discussion The present research provides the first evidence of increased NOS activity in platelets of CTTH patients. As previously shown in patients affected by CDH evolved from a previous history of migraine without aura, we found an up-regulation of NOS activity in platelets of CTTH patients, that was associated that both basal and collagen-stimulated high cytosolic Ca2 + content. This latter is considered the key factor for NOS activation and NO formation also in these blood elements. The increased NOS activity in platelets of CTTH was also related to a serotonin depletion on the one hand, and to an increased glutamate content on the other hand, as shown by the significant negative and positive correlation observed between platelet serotonin secretion and glutamate content and NO formation, respectively. The above findings were more accentuated in CTTH patients with analgesic abuse compared with those without analgesic abuse. As in patients with ‘‘transformed migraine’’, the NOrelated and cGMP-mediated changes observed in platelets of CTTH patients may be considered a physiological compensatory mechanism counteracting the increase in cytosolic Ca2 + levels, that appears, however, to be scarcely efficient in limiting serotonin depletion by platelet dense bodies, particularly in patients with analgesic abuse. Platelets have been speculated to be a peripheral model for studying NO in headache patients [4]. The similarities between platelets and serotoninergic neurons have been emphasized by different authors and it can be prospected that the changes observed in platelets both in the serotonin
levels and NO production could be expressive of similar changes in the central pathways devoted to the modulation and processing of nociceptive input from myofascial cranial structures. It can therefore be hypothesized that the increased NOS activity in platelets of CTTH patients reflects an analogous central up-regulation of NOS activity in the same patients [14]. Evidence from experimental models of pain suggests that nitric oxide plays a crucial role in central sensitization. In particular, this emerged from the findings of the efficacy of NOS inhibitors in reducing spinal dorsal horn sensitization induced by a persisting peripheral painful input and conversely by the enhanced nociceptive response due to NO donors [15 – 17]. Involvement of NO in head pain and chronicity has been confirmed in patients with CTTH using the model of NGT-induced headache and the nonselective NOS inhibitor L-NMMA, respectively [1,2]. From the results of the present study, it may also be assumed that the NO and cGMP increase is associated with serotonin depletion not only in the periphery but also in supraspinal inhibitory pathways involved in head pain processing. This hyposerotonergic status may be related to central sensitization of headache in these patients but needs to be substantiated by further investigations [18]. In fact, the occurrence of a serotonin reduction in patients with CTTH is, at the moment, a matter of controversy. In contrast with the previous finding of a reduced platelet serotonin content after a cold pressor test in CTTH patients, there is recent evidence that does not demonstrate significant changes in plasma and platelet 5HT levels nor in the levels of the platelet 5HT transporter in CTTH patients [19,20]. In the present study, the reduction in serotonin content and secretion seems to be more accentuated in CTTH patients with both simple and combination analgesic abuse. These findings concur with the results of previous studies from Srikiatkhachorn and Anthony [21], demonstrating a reduced content of 5HT in platelets of patients with analgesic-induced headache, indicative of an analgesic-induced suppression of 5HT uptake that can interfere with the functioning pain modulatory system in the brainstem and other central sites. The greater reduction found in platelet serotonin levels in our CTTH patients confirms the findings of the above studies and appears to be strictly related to the increased production of NO. Depletion of serotonin from storage sites, more accentuated in patients with chronic analgesic abuse, has been suggested to be associated with a lowering of the and pain threshold and increased frequency of attacks. This concept is supported by the observation that a 1-month withdrawal of drugs involved in analgesic-induced headache results in an increase in blood serotonin levels accompanying the reduction in headache frequency [22]. Experimental data also confirm the impact of chronic analgesic consumption on the central serotonin system. Srikiatkhachorn et al. [23] showed a reduction in the bind-
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ing sites of 5HT2A and an increase in the serotonin transporter in frontal cortex membranes of male Wistar rats due to chronic acetaminophen administration. They hypothesized that chronic exposure to analgesics may lead to the loss of efficacy and produce analgesic-related headache chronicity. If the platelet model is also considered expressive of excitatory amino acid levels of the central nervous system, the increase in platelet glutamate content observed in our study in CTTH patients could be suggestive of the involvement of the above excitatory amino acid in this chronic pain condition [24]. This increase may be related to the higher platelet Ca2 + levels and together with the greater NO production and serotonin depletion, could contribute to central sensitization in these patients [25 –28]. Analgesic and anti-inflammatory drug abuse may further accentuate central sensitization and maintain chronic head pain as suggested by the more accentuated variations observed in the biochemical parameters assessed in the subgroup of patients with analgesic abuse. Because analogous changes, at least for NO and serotonin, have also been observed in patients with CDH evolved from a previous history of migraine, it can be speculated that the above findings more strictly reflect high headache frequency rather than the pathophysiology of the two chronic headache forms.
Acknowledgements We thank Ms. Nella Perugini for the technical assistance in carrying out the NO and cGMP assays.
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L-Arginine/nitric
oxide pathway in chronic tension-type headache: relation with serotonin content and secretion and glutamate content Paola Sarchielli a,*, Andrea Alberti a, Ardesio Floridi b, Virgilio Gallai a a
Interuniversity Center for the Study of Headache and Neurotransmitter Disorders, Perugia, Rome, Sassari, Bari, Naples, Florence—Unit of Perugia, Perugia, Italy b Department of Internal Medicine, Institute of Clinical and Applied Biochemistry, University of Perugia, Perugia, Italy Received 8 August 2001; received in revised form 1 November 2001; accepted 5 February 2002
Abstract Previous research of our group demonstrated an increase in L-arginine/nitric oxide (NO) pathway activity in patients with chronic daily headache (CDH) with a previous history of migraine, which was associated with a reduced platelet serotonin content and increased Ca2 + levels. In the present work, we assessed the variations in L-arginine/NO pathway activity and platelet cyclic guanosine 3V,5V-monophosphate (cGMP) levels in 25 patients affected by chronic tension-type headache (CTTH) (8 M, 17 F; age range: 34-54 years). The NO production, shown spectrophotometrically by stoichiometric transformation of oxyhemoglobin to methemoglobin due to NO synthase (NOS) activity, and inter platelet cGMP concentration, assessed with a RIA method, were determined in parallel to variations of aggregation response to 0.3 Ag/ml collagen. The intracellular platelet calcium concentrations were also determined using fluorescence polarisation spectrometry. Platelet serotonin content and collagen-induced secretion as well as glutamate content were also determined with high-performance liquid chromatography (HPLC). The above parameters were compared with those of an age-matched control group. A reduction in aggregation platelet response was found. The reduction in platelet aggregation was coupled with an increased NO and cGMP production ( p < 0.0002 and p < 0.001, respectively). A significant increase in cytosolic Ca2 + concentration was also detected compared to control individuals ( p < 0.001). This was accompanied by a reduced platelet content and collagen-induced secretion of serotonin and increased content of glutamate ( p < 0.0001, p < 0.0001 and p < 0.001, respectively). The above findings were more evident in patients with analgesic abuse. It can be hypothesized that the increased NOS activity shown in platelets of CTTH patients reflects an analogous central up-regulation of NOS activity in the spinal horn/trigeminal nucleus and supraspinal structures involved in the modulation of nociceptive input from myofascial cranial structures contributing to central sensitization. The increase in NOS activity seems to be associated with a hyposerotonergic status, particularly in patients with analgesic abuse, and this can contribute to central sensitization in CTTH patients. The increase in platelet glutamate content in the same patients suggests the implication of the above excitatory amino acid in spinal and supraspinal structures involved in head pain induction and maintenance. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Chronic tension-type headache; Platelets; Nitric oxide; Cyclic guanosine 3V, 5Vmonophosphate — cGMP; Calcium; Serotonin; Glutamate; Central sensitization
1. Introduction Recent findings suggest the involvement of nitric oxide (NO) in the central sensitization underlying chronic head pain and increased myofascial tenderness of pericranial muscle in patients affected by chronic tension-type headache (CTTH).
* Corresponding author. Neurological Clinic, University of Perugia, Via E. Dal Pozzo 79, 06126 Perugia, Italy. Tel.: +39-075-578-3871; fax: +39-075-578-3583. E-mail address: [email protected] (P. Sarchielli).
In particular, MG-monomethyl-L-arginine hydrochloride (L-NMMA) was demonstrated to be effective in reducing pain intensity, muscle hardness of the trapezius muscle and pericranial myofascial tenderness in patients affected by chronic tension-type headache (CTTH) [1,2]. Using the nitroglycerin (NG) model of experimental headache, Ashina et al. [3] also studied the intensity, quality and time profile of headache after infusion of this nitric oxide donor (0.5 Ag/kg per minute for 20 min) in patients affected by CTTH in comparison with those of age-matched healthy controls. In CTTH patients the area under the curve (AUC) defined by the intensity duration product on the NG day was significantly higher than on the placebo day.
0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 0 3 5 - 7
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Moreover, on the NG day the AUC of CTTH patients was significantly greater than that of healthy controls. In controls, the peak of pain intensity occurred 20 min after the start of infusion, whereas in CTTH patients 8 h after NG infusion. In the latter group, NO induced a biphasic response with an immediate and a delayed headache, as in migraine. The delayed NO-induced headache in chronic tension-type headache patients was similar to that suffered by the patients. Platelets have been proposed as a peripheral model for studying nitric oxide [4]. They, when stimulated by collagen produce NO, using L-arginine as a substrate [5]. A constitutive nitric oxide is present in platelets which is responsible for NO synthesis in the presence of high Ca2 + levels. NO induces the activation of guanylate cyclase and, therefore, the production of cyclic guanosine monophosphate (cGMP). Because of the similarities between platelets and monoaminergic neurons (in particular serotonergic neurons), variations in these blood elements have been interpreted as a peripheral mirror of analogous variations in central monoaminergic pathways. Platelets also contain glutamate and changes in platelet levels of this excitatory amino acid were interpreted to be suggestive of analogous changes in the central nervous system, at least in migraine. In a recent research using the platelet model, we investigated the variations in L-arginine/NO pathway activity and platelet cGMP levels in patients affected by chronic daily headache (CDH) [6], which evolved in all patients examined with a previous history of migraine. We found an upregulation of the L-arginine/NO pathway, that was associated with a reduced content of serotonin and increased Ca2 + levels in platelets. We hypothesized that the occurrence of NO – cGMPmediated events is not too efficient in contrasting the intracytosolic Ca2 + increase in platelets, which could be responsible for serotonin depletion from dense bodies, already shown by recent research in platelets of patients affected by CDH associated with analgesic abuse. No studies have been carried out until now to investigate NO synthase (NOS) activity in tension-type headache, even though the finding of the above mentioned analgesic effect of NOS inhibition in this pathological condition suggests the involvement of NO in central sensitization in this pathological condition. The present research was aimed at assessing the collagen-stimulated NO synthase (NOS) activity in the platelets of patients with CTTH. Moreover, cytosolic ionized calcium content and serotonin release and content were measured. Glutamate content in the platelets of the same patients was also investigated. Data were compared with those of a group of healthy age-matched control subjects. In the group of CTTH patients, values of the above variables from patients with simple or combination analgesic abuse (N = 16) were compared with those of patients without analgesic abuse (N = 9).
2. Patients and methods 2.1. Patients Twenty-five consecutive patients with CTTH attending the Headache Center of Perugia University were assessed. The diagnosis was made according to the IHS criteria [7]. To be included in the study they were required to have had headache at least 6 days per week for at least six months. In all patients CTTH evolved from an episodic tension-type headache. The mean duration of headache was 18.6 F 9.8 years. The criteria used for analgesic abuse were those of Silberstein et al. [8,9]: simple analgesic use > 1000 mg/ ASA/acetaminophen >5 days/week, combination analgesics >3 tablets/day >3 days/week. Twenty healthy age-matched control subjects were also assessed for the same parameters. Details of CTTH patients and control subjects are reported in Table 1. In the subgroup of CTTH patients with analgesic abuse (N = 16), the most frequently abused analgesic was acetaminophen (N = 13). Other analgesics abused were feldene: N = 8, diclofenac: N = 7, ketorolac: N = 5, and nimesulide: N = 6. Eight of the CTTH patients used more than one simple analgesic. Nine CTTH patients used combination analgesics (Optalidon-butalbital + propyphenazone + caffeine: N = 5; Difmetre-indomethacin + prochlorperazine + caffeine: N = 4), four of them in association with simple analgesics. No CTTH patients used drugs containing codeine. The monthly drug intake averaged 78.4 F 14.8 (mean F SD) tablets or suppositories. None of the CTTH patients took medications such as headache preventive drugs. The last time that the patients in the non-overuse group took medication for pain was at least 24 h previously. The patients in the overuse group were invited not to take symptomatic medication for at least 12 h before blood drawing (8 a.m.). All patients in the latter group took the last symptomatic drug before this time. All control subjects were headache-free for at least 2 months and none of them was taking any medication at the time of blood sampling nor had a personal or family history of migraine or suffered from tension-type headache.
Table 1 Characteristics of patients and control subjects
Males Females Age (years) Duration of headache (years) Duration of CTTH (years) Analgesic misuse No analgesic misuse
Patients, N = 25
Control subjects, N = 20
N=8 N = 17 45.2 F 7.4 18.6 F 9.8 11 F 5 N = 16 N=9
N=8 N = 12 43.5 F 6.4
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3. Methods 3.1. Platelet separation Blood was drawn from the antecubital vein and collected in tubes containing 0.1 volumes of 3.8% citrate and then centrifuged at 200 g for 20 min at room temperature. Platelet-rich plasma (PRP)was removed and then centrifuged at 1000 g for 12 min at 0 jC to form a pellet. The platelets were washed once at room temperature in N-2-hydroxyethylpiperazine-N V-2-ethanesulfonic acid (HEPES) –Thyroid buffer (in mM: 129 NaCl, 2.8 KCl, 0.8 KH2 PO4, 0.8 MgCl2, 5.6 dextrose, 10 HEPES, pH= 7.4) containing 1 mM ethylene glycol-bis (h-aminoethyl ether)-N,N,N V,N V-tetraacetic acid (EGTA), with the addition of indomethacin (10 AM). The platelets were then passed through a Sepharose 2B-300 column preconditioned with the same medium. The platelets were counted using a Coulter counter (model ZN; Coulter Electronics, Hialeah, FL) and were adjusted to 3.5 108 platelets/ml by the addition of HEPES-buffer saline. 3.2. Platelet aggregation Platelet aggregation was performed using the method of Born and Cross [10]. A suspension of washed platelets (450 Al; 3.5 108 platelets/ml) was incubated at 37 jC for 2 min in an aggregometer (Chronolog 480 VS, Chronolog, Haverton, PA) with continuous stirring at 1200 rpm and then stimulated with collagen at concentrations of 0.3, 1, and 3 Ag/ml, which was purchased from Sigma (St Louis, MO, USA). The chart speed was 1 cm/min. Platelet impedance curves were analysed 7 min after the addition of the agonist. The aggregation amplitude was expressed as the platelet aggregation peak (5 V = 12 mm).
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stopped) by the addition of an equal volume of ice-cold trichloroacetic acid (10% wt/vol, final concentration). The incubation was performed both with and without L-NMMA at 10 AM. The measurement of cGMP was made from etherextracted supernatants of the samples. The assays of cGMP were performed using RIA kits (New England Nuclear). Data were expressed in pmol/109 platelets. 3.5. Platelet cytosolic Ca2+ concentration Platelet cytosolic Ca2 + was measured both in resting platelets and platelets stimulated with collagen (3 Ag/ml). Platelet cytosolic Ca2 + concentration was measured with the fluorescent indicator Fura-2 according to the method of Astarie-Dequeker et al. [11]. Platelets were loaded with 2 AM Fura 2AM for 40 min at 37 jC in the presence of plasma. They were then washed by centrifugation at 270 g for 15 min at 20 jC and resuspended at a density of 1 106 cells ml 1 in a medium containing (in mM): 145 NaCl, 5 KCl, 0.5 MgCl2, 10 N-2-hydroxyethylpiperazine-N V-2-ethan sulphonic acid (HEPES) and 5 glucose, pH = 7.4 at 37 jC, and 30 nM free Ca2 + concentration (that suppresses Ca2 + influx) adjusted with the required Ca2 + EGTA buffer. Fluorescence intensities were measured at 37 jC on a Fluorolog equipped with a 450-W Xenon lamp, two monochromators with a dual-mirror chopping mechanism that allows a rapid alternating excitation from 335- to 385-nm wavelengths with a fixed emission wavelength of 510 nm. Each platelet sample was checked for Fura-2 loading by recording the fluorescence spectra. The fluorescence recording was systematically corrected for the autofluorescence of the cell suspension. Platelet cytosolic Ca2 + was calculated from the experimental ratio of excitation fluorescence intensities R = I335/I385 [12]. Data are expressed in nanometers (nm).
3.3. NO formation in platelet cytosol 3.6. Serotonin content and release from platelets The platelet cytosol was prepared from 1 –2 1011 platelets by homogenization and sonication for 10 min with Soniprep and subsequent centrifugation at 150,000 g for 10 min. The NO production in the platelet cytosol obtained by sonication was evaluated spectrophotometrically, assessing the stoichiometric reaction of NO with oxyhemoglobin to yield methemoglobin and NO3 . Data were expressed in fmol/min/mg protein. In the same experiments with the aim to block cyclooxygenase activity, which can interfere with endogenous NO formation, platelet aggregation was investigated by adding to the platelet suspension indomethacin (10 AM) for 10 min at 37 jC, before measuring the aggregation. 3.4. Measurement of cyclic guanosine 3V,5V-monophosphate (cGMP) The incubation of gel-filtered platelets, both in the basal state and in the presence of collagen (0.3, 1, and 3 Ag/ml), was arrested after 7 min (the time at which the aggregation was
The platelet pellet was separated from platelet-poorplasma by centrifuging PRP at 7000 g for 10 min. It was washed twice with HEPES– Thyroid buffer (145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM N-2-hydroxyethylpiperazine-NV-2-ethansulfonic acid (HEPES) and 10 mM glucose, pH = 7.4). All platelets were resuspended at the concentration of 1 109 per ml and frozen at 80 jC until the assay. The samples were thawed at room temperature and sonicated just prior to determination. Platelet 5-HT was measured using reversed-phase highperformance liquid chromatography (HPLC) with electrochemical detection. The chromatographic system included: a Kontron Instrument model 420 with a 5-Am particle diameter, Tracer Analytics ODS and lLC4b amperometric detector. Phosphoric acid (0.1 M), with 0.1 mM EDTA adjusted to pH 2.85 with 4 M KOH solution was the mobile phase. 3,4dihydroxybenzylamine was used as the internal standard. Platelet 5HT levels were expressed in ng/109 platelets.
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Table 2 Summary of the aggregation peaks, NO formation and cGMP production (mean F SEM) in platelets of CTTH patients and control individuals (C) CTTH with analgesic abuse (a)
CTTH without analgesic abuse (b)
C (c)
Statistical significance
Aggregation peak V (collagen 3 Ag)
27.3 F 5.2
33.0 F 6.4
40.6 F 4.8
NO formation, fmol/min/mg protein (collagen 3 Ag)
55.6 F 6.8
42.4 F 4.4
30.2 F 3.1
cGMP formation, pmol/109 platelets (collagen 3 Ag)
7.5 F 1.2
6.2 F 1.1
3.2 F 0.4
a vs c: 0.0003 b vs c: 0.02 a vs b: 0.05 a vs c: 0.001 b vs c: 0.005 a vs b: 0.02 a vs b: 0.001 b vs c: 0.01 a vs b: 0.02
3.7. Platelet glutamate levels Glutamate levels were analyzed according to the method of Zecca and Ferrari [13] with some modifications. Briefly, the platelet pellet was separated from platelet-poor plasma by centrifuging PRP at 7000 g for 10 min. The pellet was resuspended in 1 ml of saline solution and sonicated for 5 min. Sulphosalicylic acid (30 mg/ml) was added for deproteination. The sample (50 Al) was added to a solution containing 400 Al of boric acid, 490 Al of methanol, 50 Al of o-phthalaldehyde (OPA) and 10 Al of the internal standard norvaline (C5H11NO2). Aliquots (20 Al) of supernatants were injected into a Millipore Waters HPLC system connected to a Perkin Elmer fluorescence spectrometer (purchased from Perkin Elmer, Beaconsfield, Buckinghamshire, England) and a reversed-phase column Supelcosil LC-18 (3 A, 15 cm 4.6 mm) (purchased from Sulpeco, Bellefonte, PA, USA). The wavelengths used to detect glutamate were 330 nm for excitation and 445 nm for emission, respectively. The retention time was 11V48U for glutamate and 30V65U for the internal standard. 3.8. Statistical analysis All values, both in unstimulated and stimulated platelets of CTTH patients and controls, were expressed as means F SEM. A non-parametric analysis of variance (ANOVA)
was carried out to compare the mean values of the aggregation peaks due to 3 Ag/ml collagen, basal and collagenstimulated NO formation and cGMP production, Ca2 + content and serotonin concentration in platelets of CTTH patients with the same values of the control group. The same analysis was performed to compare the mean values of the above mentioned parameters in the CHD patients with drug abuse with those of CTTH patients without drug abuse. Fisher’s least significant difference (LSD) was also used to compare the main effect means in ANOVA in the case of equal cell size. Five percent for two-sided tests was chosen as a minimum level of statistical significance. The Pearson correlation coefficient was also calculated between NO and cGMP values and serotonin content and release as well as glutamate content in the platelets of CTTH patients and controls.
4. Results Table 2 summarizes the findings on the aggregation peaks, NO formation and cGMP production of CTTH patients with and without analgesic abuse, and of control individuals. Table 3 shows the values of cytosolic Ca2 + , serotonin content and collagen-induced secretion and glutamate content in CTTH patients and control individuals.
Table 3 Summary of the values of cytosolic Ca2 + concentration, basal content and secretion of serotonin and glutamate concentration (mean F SEM) in platelets of CTTH patients and control individuals (C) CDH with analgesic abuse (a)
CDH without analgesic abuse (b)
C (c)
Statistical significance
Ca2 +, nM (collagen 3 Ag)
184.3 F 12.9
159.3 F 19.9
130.3 F 8.9
Serotonin, ng/109 platelets basal content
340.1 F 32.4
463.5 F 45.8
596.7 F 42.9
5HT secretion (collagen 3 Ag)
410.1 F 34.5
448.4 F 39.7
666.4 F 42.9
63.9 F 4.5
54.2 F 6.4
45.2 F 2.3
a vs c: 0.008 b vs c: 0.01 a vs b: n.s. a vs c: 0.0001 b vs c: 0.002 a vs b: 0.04 a vs c: 0.0001 b vs c: 0.0003 a vs b: 0.05 a vs c: 0.001 b vs c: 0.002 a vs b: 0.03
Glutamate, nmol/109 platelets
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Fig. 1. Correlation between serotonin secretion (ng/109 platelets) and NO formation (fmol/min/mg protein) in platelets of CTTH patients with and without analgesic abuse.
The aggregation peaks induced by collagen were significantly lower in CTTH patients than those of the control subjects (ANOVA: p < 0.002). This reduction was more accentuated in patients with analgesic abuse than in patients without analgesic abuse (Table 2). A slight but significant increase in the rate of NO formation in the cytosol of platelets before the addition of collagen was observed in CTTH (16.4 F 4.7), with respect to the control group (6.8 F 3.2) ( p < 0.01). The values tended to further increase after the addition of collagen and this increase was significantly greater in CTTH patients with analgesic abuse compared to that of patients without analgesic abuse (ANOVA: 0.0002) (Table 2). The
latter showed significantly greater values of NO production than healthy subjects. cGMP levels in unstimulated platelets of CTTH patients (1.4 F 0.8) were greater than those of control individuals (0.7 F 0.3) ( p < 0.05). Collagen induced a significant increase in platelet cGMP in CTTH patients, particularly in those with analgesic abuse and, to a lesser extent, in patients without analgesic abuse whose values were, in any case, significantly greater than those measured in platelets of control individuals (ANOVA: 0.001) (Table 2). In patients with CTTH, a slight but significant decrease in the basal content of serotonin was found compared to control individuals ( p < 0.0001). Collagen induced the secretion of
Fig. 2. Correlation between glutamate levels (nmol/109 platelets) and NO formation (fmol/min/mg protein) in platelets of control individuals, CTTH patients with and without analgesic abuse.
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serotonin both in controls and CTTH patients, but the values detected in the latter were lower than those found in control subjects, particularly in patients with analgesic abuse (ANOVA: p < 0.0001) (Table 3). At the same time, cytosolic Ca2 + concentration was significantly increased in CTTH patients with respect to age-matched healthy subjects, particularly in patients with analgesic abuse (ANOVA: p < 0.001) (Table 3). Glutamate values measured in platelets of CTTH patients with analgesic abuse were also higher than those of controls (ANOVA: p < 0.001). This increase was greater in patients with analgesic abuse compared to those without analgesic abuse ( p < 0.03) (Table 3). A significant negative correlation was found in CTTH patients between platelet NO production and 5HT secretion by platelets (R = 0.49, p < 0.001), whereas a significant positive value of the correlation coefficient (R = 0.78, p < 0.004) was calculated in the same patients (Figs. 1 and 2). A slight but significant negative correlation also emerged between platelet NO production and 5HT platelet content (R = 0.38, p < 0.05). No significant correlation emerged among the above parameters in control subjects.
5. Discussion The present research provides the first evidence of increased NOS activity in platelets of CTTH patients. As previously shown in patients affected by CDH evolved from a previous history of migraine without aura, we found an up-regulation of NOS activity in platelets of CTTH patients, that was associated that both basal and collagen-stimulated high cytosolic Ca2 + content. This latter is considered the key factor for NOS activation and NO formation also in these blood elements. The increased NOS activity in platelets of CTTH was also related to a serotonin depletion on the one hand, and to an increased glutamate content on the other hand, as shown by the significant negative and positive correlation observed between platelet serotonin secretion and glutamate content and NO formation, respectively. The above findings were more accentuated in CTTH patients with analgesic abuse compared with those without analgesic abuse. As in patients with ‘‘transformed migraine’’, the NOrelated and cGMP-mediated changes observed in platelets of CTTH patients may be considered a physiological compensatory mechanism counteracting the increase in cytosolic Ca2 + levels, that appears, however, to be scarcely efficient in limiting serotonin depletion by platelet dense bodies, particularly in patients with analgesic abuse. Platelets have been speculated to be a peripheral model for studying NO in headache patients [4]. The similarities between platelets and serotoninergic neurons have been emphasized by different authors and it can be prospected that the changes observed in platelets both in the serotonin
levels and NO production could be expressive of similar changes in the central pathways devoted to the modulation and processing of nociceptive input from myofascial cranial structures. It can therefore be hypothesized that the increased NOS activity in platelets of CTTH patients reflects an analogous central up-regulation of NOS activity in the same patients [14]. Evidence from experimental models of pain suggests that nitric oxide plays a crucial role in central sensitization. In particular, this emerged from the findings of the efficacy of NOS inhibitors in reducing spinal dorsal horn sensitization induced by a persisting peripheral painful input and conversely by the enhanced nociceptive response due to NO donors [15 – 17]. Involvement of NO in head pain and chronicity has been confirmed in patients with CTTH using the model of NGT-induced headache and the nonselective NOS inhibitor L-NMMA, respectively [1,2]. From the results of the present study, it may also be assumed that the NO and cGMP increase is associated with serotonin depletion not only in the periphery but also in supraspinal inhibitory pathways involved in head pain processing. This hyposerotonergic status may be related to central sensitization of headache in these patients but needs to be substantiated by further investigations [18]. In fact, the occurrence of a serotonin reduction in patients with CTTH is, at the moment, a matter of controversy. In contrast with the previous finding of a reduced platelet serotonin content after a cold pressor test in CTTH patients, there is recent evidence that does not demonstrate significant changes in plasma and platelet 5HT levels nor in the levels of the platelet 5HT transporter in CTTH patients [19,20]. In the present study, the reduction in serotonin content and secretion seems to be more accentuated in CTTH patients with both simple and combination analgesic abuse. These findings concur with the results of previous studies from Srikiatkhachorn and Anthony [21], demonstrating a reduced content of 5HT in platelets of patients with analgesic-induced headache, indicative of an analgesic-induced suppression of 5HT uptake that can interfere with the functioning pain modulatory system in the brainstem and other central sites. The greater reduction found in platelet serotonin levels in our CTTH patients confirms the findings of the above studies and appears to be strictly related to the increased production of NO. Depletion of serotonin from storage sites, more accentuated in patients with chronic analgesic abuse, has been suggested to be associated with a lowering of the and pain threshold and increased frequency of attacks. This concept is supported by the observation that a 1-month withdrawal of drugs involved in analgesic-induced headache results in an increase in blood serotonin levels accompanying the reduction in headache frequency [22]. Experimental data also confirm the impact of chronic analgesic consumption on the central serotonin system. Srikiatkhachorn et al. [23] showed a reduction in the bind-
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ing sites of 5HT2A and an increase in the serotonin transporter in frontal cortex membranes of male Wistar rats due to chronic acetaminophen administration. They hypothesized that chronic exposure to analgesics may lead to the loss of efficacy and produce analgesic-related headache chronicity. If the platelet model is also considered expressive of excitatory amino acid levels of the central nervous system, the increase in platelet glutamate content observed in our study in CTTH patients could be suggestive of the involvement of the above excitatory amino acid in this chronic pain condition [24]. This increase may be related to the higher platelet Ca2 + levels and together with the greater NO production and serotonin depletion, could contribute to central sensitization in these patients [25 –28]. Analgesic and anti-inflammatory drug abuse may further accentuate central sensitization and maintain chronic head pain as suggested by the more accentuated variations observed in the biochemical parameters assessed in the subgroup of patients with analgesic abuse. Because analogous changes, at least for NO and serotonin, have also been observed in patients with CDH evolved from a previous history of migraine, it can be speculated that the above findings more strictly reflect high headache frequency rather than the pathophysiology of the two chronic headache forms.
Acknowledgements We thank Ms. Nella Perugini for the technical assistance in carrying out the NO and cGMP assays.
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