Journal of the Neurological Sciences 198 (2002) 9 – 15

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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 In particular, MG-monomethyl-L-arginine hydrochloride

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 head-
ache (CTTH).
* affected by CTTH in comparison with those of age-matched
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: neuro@netemedia.net (P. Sarchielli).
(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
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
10 P. Sarchielli et al. / Journal of the Neurological Sciences 198 (2002) 9 –15
Moreover, on the NG day the AUC of CTTH patients was
significantly greater than that of healthy controls. In con-
trols, 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 res-
ponse 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 colla-
gen 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 mono-
phosphate (cGMP). Because of the similarities between
platelets and monoaminergic neurons (in particular seroto-
nergic neurons), variations in these blood elements have
been interpreted as a peripheral mirror of analogous varia-
tions 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 inves-
tigated 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 up-
regulation of the L-arginine/NO pathway, that was associa-
ted with a reduced content of serotonin and increased Ca2 +
levels in platelets.
We hypothesized that the occurrence of NO – cGMP-
mediated events is not too efficient in contrasting the intra-
cytosolic Ca2 + increase in platelets, which could be respon-
sible 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 patho-
logical condition.
The present research was aimed at assessing the colla-
gen-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 analge-
sic 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 acetami-
nophen (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 + caf-
feine: 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
Patients, Control subjects,
N = 25 N = 20
Males N=8 N=8
Females N = 17 N = 12
Age (years) 45.2 F 7.4 43.5 F 6.4
Duration of headache (years) 18.6 F 9.8
Duration of CTTH (years) 11 F 5
Analgesic misuse N = 16
No analgesic misuse N=9
 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-hydroxye-
thylpiperazine-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 ethyl-
ene 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, Haver-
ton, PA) with continuous stirring at 1200 rpm and then stimu-
lated 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).
3.3. NO formation in platelet cytosol
The platelet cytosol was prepared from 1 –2  1011 pla-
telets 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, assess-
ing 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 inves-
tigated 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
11
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 ether-
extracted 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 sul-
phonic 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 mono-
chromators 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 record-
ing was systematically corrected for the autofluorescence of
the cell suspension. Platelet cytosolic Ca2 + was calculated
from the experimental ratio of excitation fluorescence inten-
sities R = I335/I385 [12]. Data are expressed in nanometers
(nm).
3.6. Serotonin content and release from platelets
The platelet pellet was separated from platelet-poor-
plasma 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-hydroxye-
thylpiperazine-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 high-
performance liquid chromatography (HPLC) with electro-
chemical detection. The chromatographic system included: a
Kontron Instrument model 420 with a 5-Am particle diam-
eter, Tracer Analytics ODS and lLC4b amperometric detec-
tor. Phosphoric acid (0.1 M), with 0.1 mM EDTA adjusted to
pH 2.85 with 4 M KOH solution was the mobile phase. 3,4-
dihydroxybenzylamine was used as the internal standard.
Platelet 5HT levels were expressed in ng/109 platelets.
12 P. Sarchielli et al. / Journal of the Neurological Sciences 198 (2002) 9 –15

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 CTTH without C (c) Statistical
analgesic abuse (a) analgesic abuse (b) significance
Aggregation peak 27.3 F 5.2 33.0 F 6.4 40.6 F 4.8 a vs c: 0.0003
V (collagen 3 Ag) b vs c: 0.02
a vs b: 0.05
NO formation, fmol/min/mg 55.6 F 6.8 42.4 F 4.4 30.2 F 3.1 a vs c: 0.001
protein (collagen 3 Ag) b vs c: 0.005
a vs b: 0.02
cGMP formation, pmol/109 7.5 F 1.2 6.2 F 1.1 3.2 F 0.4 a vs b: 0.001
platelets (collagen 3 Ag) 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 deprotei-
nation. The sample (50 Al) was added to a solution containing
400 Al of boric acid, 490 Al of methanol, 50 Al of o-phthalal-
dehyde (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 aggre-
gation peaks due to 3 Ag/ml collagen, basal and collagen-
stimulated 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 con-
tent 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 CDH without C (c) Statistical
analgesic abuse (a) analgesic abuse (b) significance
Ca2 +, nM 184.3 F 12.9 159.3 F 19.9 130.3 F 8.9 a vs c: 0.008
(collagen 3 Ag) b vs c: 0.01
a vs b: n.s.
Serotonin, ng/109 340.1 F 32.4 463.5 F 45.8 596.7 F 42.9 a vs c: 0.0001
platelets basal content b vs c: 0.002
a vs b: 0.04
5HT secretion 410.1 F 34.5 448.4 F 39.7 666.4 F 42.9 a vs c: 0.0001
(collagen 3 Ag) b vs c: 0.0003
a vs b: 0.05
Glutamate, 63.9 F 4.5 54.2 F 6.4 45.2 F 2.3 a vs c: 0.001
nmol/109 platelets b vs c: 0.002
a vs b: 0.03
 P. Sarchielli et al. / Journal of the Neurological Sciences 198 (2002) 9 –15 13

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 signifi-
cantly 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.
14 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 (ANO-
VA: 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 analge-
sic abuse were also higher than those of controls (ANOVA:
p < 0.001). This increase was greater in patients with anal-
gesic 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 NO-
related and cGMP-mediated changes observed in platelets of
CTTH patients may be considered a physiological compen-
satory 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 sensitiza-
tion 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 signifi-
cant 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 pa-
tients 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 analge-
sic-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 accen-
tuated 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-
 P. Sarchielli et al. / Journal of the Neurological Sciences 198 (2002) 9 –15
ing sites of 5HT2A and an increase in the serotonin trans-
porter in frontal cortex membranes of male Wistar rats due
to chronic acetaminophen administration. They hypothe-
sized 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 involve-
ment 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 seroto-
nin, 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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