Tepper SJ, Rapoport A, Sheftell F.
The pathophysiology of migraine.
Neurolog. 2001 Sep;7(5):279-86.
"BACKGROUND: Migraine results from episodic changes in central nervous system physiologic function in hyperexcitable brain manifested by abnormal energy metabolism, lowered threshold for phosphene generation, and increased contingent negative variation. Human functional magnetic resonance imaging and magnetoencepholography data strongly suggest that aura is caused by cortical spreading depression. REVIEW SUMMARY: Brain hyperexcitability may be caused by low magnesium levels, mitochondrial abnormalities with abnormal phosphorylation of adenosine 5'-diphosphate, a dysfunction related to nitric oxide, or calcium channelopathy. Low magnesium can result in opening of calcium channels, increased intracellular calcium, glutamate release, and increased extracellular potassium, which may in turn trigger cortical spreading depression. Mitochondrial dysfunction has been suggested by a low phosphocreatine:Pi ratio and a possible response by migraine patients to riboflavin prophylaxis. Nitroglycerine administration results in a delayed migraine-like headache in migraine patients but not in control patients, and a nonspecific nitric oxide synthase inhibitor aborted migraine at 2 hours in the majority of tested migraine patients compared to controls. Many patients with familial hemiplegic migraine have a missense mutation in the P/Q calcium channel, so that this form of migraine, at least, is associated with a demonstrable calcium channelopathy. CONCLUSIONS: The generation of migraine occurs centrally in the brain stem, sometimes preceded by cortical spreading depression and aura. Activation of the trigeminovascular system stimulates perivascular trigeminal sensory afferent nerves with release of vasoactive neuropeptides, resulting in vasodilation and transduction of central nociceptive information. There is then a relay of pain impulses to central second- and third-order neurons and activation of brain stem autonomic nuclei to induce associated symptoms." [Abstract]

Ashina M.
Nitric oxide synthase inhibitors for the treatment of chronic tension-type headache.
Expert Opin Pharmacother. 2002 Apr;3(4):395-9.
"Chronic tension-type headache may be caused by prolonged painful input from pericranial myofacial tissues, for example tender points, resulting in central sensitisation (increased excitability of neurons in the central nervous system). Animal studies have shown that sensitisation of pain pathways may be caused by or associated with the activation of neuronal nitric oxide synthase and the generation of nitric oxide. Furthermore, it has been shown that nitric oxide synthase inhibitors reduce central sensitisation in animal models of persistent pain. On the basis of this information, the analgesic effect of the nitric oxide synthase inhibitor L-N(G) methyl arginine hydrochloride was investigated. This drug significantly reduced headache and myofacial factors in patients with chronic tension-type headache. These studies show that nitric oxide plays a crucial role in the pathophysiology of tension-type headache. The analgesic effect of nitric oxide synthase inhibition in patients with chronic tension-type headache is probably due to a reduction in central sensitisation at the level of the spinal dorsal horn, trigeminal nucleus or both. Furthermore, inhibition of nitric oxide synthase may become a novel principle in the future treatment of chronic headache." [Abstract]

Thomsen LL, Olesen J.
Nitric oxide in primary headaches.
Curr Opin Neurol. 2001 Jun;14(3):315-21.
"The molecular mechanisms that underlie the primary headaches-migraine, cluster headache and tension-type headache-have not yet been clarified. On the basis of studies in headache induced by intravenous infusions of glyceryl trinitrate (an exogenous nitric oxide donor) and histamine (which liberates nitric oxide from vascular endothelium), it has been suggested that nitric oxide is a likely candidate responsible molecule. The present review deals with the biology of this small messenger molecule, and the updated scientific evidence that suggests a key role for this molecule in primary headaches. This evidence suggests that the release of nitric oxide from blood vessels, perivascular nerve endings or from brain tissue is an important molecular trigger mechanism in spontaneous headache pain. Pilot trials have shown efficacy of a nitric oxide synthase inhibitor in both migraine attacks and chronic tension-type headache. These observations suggest new approaches to the pharmacological treatment of headache." [Abstract]

Read SJ, Smith MI, Hunter AJ, Parsons AA.
Enhanced nitric oxide release during cortical spreading depression following infusion of glyceryl trinitrate in the anaesthetized cat.
Cephalalgia. 1997 May;17(3):159-65.
"Intravenous infusion of glyceryl trinitrate (GTN) into migraineurs induces an immediate headache followed by migraine. We studied the effect of GTN (0.25 microgram kg-1 min-1) on local cerebrovascular laser Doppler flux (rCBFLDF), artery diameter and NO concentration (selective NO microelectrode) in the pial middle cerebral artery perfusion territory of the anaesthetized cat, at rest and during cortical spreading depression (SD). GTN infusion induced a significant increase in pial artery diameter, rCBFLDF, and NO concentration. Following termination of infusion, NO concentrations remained significantly elevated above controls for 60 min, other parameters returned to baseline within 10 min (p < 0.05, ANOVA, post hoc Dunnett's multiple comparison procedure). Two hours after termination of infusion KCl-evoked SD was initiated. GTN-treated animals exhibited significantly (p < 0.05, Kruskal-Wallis) elevated SD-induced NO release compared to controls. All other parameters remained unaffected. Our results demonstrate that GTN induces a prolonged increase in local NO concentrations and enhances SD-induced NO release." [Abstract]

Wahl M, Schilling L, Parsons AA, Kaumann A.
Involvement of calcitonin gene-related peptide (CGRP) and nitric oxide (NO) in the pial artery dilatation elicited by cortical spreading depression.
Brain Res. 1994 Feb 21;637(1-2):204-10.
"The aim of the present study was to examine whether the initial transient arterial dilatation during cortical spreading depression (CSD) was mediated by the release of calcitonin gene-related peptide (CGRP) and/or nitric oxide (NO). This question is of interest as the initial phase of CSD appears to be a model of events occurring during functional hyperemia and during the first period of classic migraine. Using an open cranial window technique, pial arterial diameter in the parietal cortex of cats was recorded with an image splitting method. Employing micropuncture technique, perivascularly applied CGRP8-37 did not alter the resting diameter of pial arteries but antagonized concentration dependently (5 x 10(-9)-10(-6) M) the dilatation (35%) due to 5 x 10(-8) M CGRP. NG-Nitro-L-Arginine (NOLAG, 10(-4) M) also had no effect on resting diameter of pial arteries, indicating that their resting tone is neither mediated by a continuous release of CGRP nor of NO. CSD was triggered by a remote intracortical injection of KCl (150 mM) and recorded by a microelectrode placed adjacent to the artery under investigation. CSD elicited a transient negative DC shift which was accompanied by a peak dilatation of 44 +/- 5.2% (S.E.M.). This dilatation was reduced by approximately 50% during topical application of 10(-7) M CGRP8-37 and 10(-4) M NOLAG each. A 75% inhibition of the CSD-induced dilatation was found during simultaneous application of both compounds. These data indicate that the initial dilatation during CSD is mediated, at least in part, by a release of CGRP and NO." [Abstract]

Obrenovitch TP, Urenjak J, Wang M.
Nitric oxide formation during cortical spreading depression is critical for rapid subsequent recovery of ionic homeostasis.
J Cereb Blood Flow Metab. 2002 Jun;22(6):680-8.
"Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex. Cortical spreading depression promotes lesion progression in experimental stroke, and may contribute to the initiation of migraine attacks. The purpose of this study was to investigate the roles of the marked increase of nitric oxide (NO) formation that occurs with CSD. Microdialysis electrodes were implanted in the cortex of anesthetized rats to perform the following operations within the same region: (1) elicitation of CSD by perfusion of high K+ medium; (2) recording of CSD elicitation; (3) application of the NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME); and (4) recording of dialysate pH changes. The primary effect of l-NAME (0.3 to 3.0 mmol/L in the perfusion medium) was a marked widening of individual CSD wave, resulting essentially from a delayed initiation of the repolarization phase. This change was due to NO synthase inhibition because it was not observed with the inactive isomer d-NAME, and was reversed by l-arginine. This effect did not appear to be linked to the suppression of a sustained, NO-mediated vascular change associated with the superposition of NO synthase inhibition on high levels of extracellular K+. The delayed initiation of repolarization with local NO synthase inhibition may reflect the suppression of NO-mediated negative feedback mechanisms acting on neuronal or glial processes involved in CSD genesis. However, the possible abrogation of a very brief, NO-mediated vascular change associated with the early phase of CSD cannot be ruled out." [Abstract]

Wang M, Obrenovitch TP, Urenjak J.
Effects of the nitric oxide donor, DEA/NO on cortical spreading depression.
Neuropharmacology. 2003 Jun;44(7):949-57.
"Cortical spreading depression (CSD) is a transient disruption of local ionic homeostasis that may promote migraine attacks and the progression of stroke lesions. We reported previously that the local inhibition of nitric oxide (NO) synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) delayed markedly the initiation of the recovery of ionic homeostasis from CSD. Here we describe a novel method for selective, controlled generation of exogenous NO in a functioning brain region. It is based on microdialysis perfusion of the NO donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO). As DEA/NO does not generate NO at alkaline pH, and as the brain has a strong acid-base buffering capacity, DEA/NO was perfused in a medium adjusted at alkaline (but unbuffered) pH. Without DEA/NO, such a microdialysis perfusion medium did not alter CSD. DEA/NO (1, 10 and 100 microM) had little effect on CSD by itself, but it reversed in a concentration-dependent manner the effects of NOS inhibition by 1 mM L-NAME. These data demonstrate that increased formation of endogenous NO associated with CSD is critical for subsequent, rapid recovery of cellular ionic homeostasis. In this case, the molecular targets for NO may be located either on brain cells to suppress mechanisms directly involved in CSD genesis, or on local blood vessels to couple flow to the increased energy demand associated with CSD." [Abstract]

Fabricius M, Akgoren N, Lauritzen M.
Arginine-nitric oxide pathway and cerebrovascular regulation in cortical spreading depression.
Am J Physiol. 1995 Jul;269(1 Pt 2):H23-9.
"Nerve cells release nitric oxide (NO) in response to activation of glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype. We explored the hypothesis that NO influences the changes of cerebral blood flow (CBF) during cortical spreading depression (CSD), which is known to be associated with NMDA receptor activation. CBF was monitored in parietal cortex by laser-Doppler flowmetry in halothane-anesthetized rats. Under control conditions, CSD induced regular changes of CBF, which consisted of four phases: a brief hypoperfusion before the direct current (DC) shift; a marked CBF rise during the DC shift; followed by a smaller, but protracted increase of CBF; and a prolonged CBF reduction (the oligemia). NO synthase inhibition by intravenous and/or topical application of NG-nitro-L-arginine enhanced the brief initial hypoperfusion, but the CBF increases and the oligemia were unchanged. L-Arginine prevented the development of the prolonged oligemia after CSD but had no influence on the marked rise of CBF during CSD. Animals treated with L-arginine recovered the reduced vascular reactivity to hypercapnia after CSD much faster than control rats. Functional denervation of cortical and pial arterioles by tetrodotoxin accentuated the pre-CSD hypoperfusion and the oligemia but did not affect the CBF increases. The results suggest that NO is important for the changes of cerebrovascular regulation following CSD. The observations may have clinical importance, since CBF changes during migraine may be triggered by CSD." [Abstract]

Read SJ, Hirst WD, Upton N, Parsons AA.
Cortical spreading depression produces increased cGMP levels in cortex and brain stem that is inhibited by tonabersat (SB-220453) but not sumatriptan.
Brain Res. 2001 Feb 9;891(1-2):69-77.
"Migraine headache is proposed to be mediated by nitric oxide (NO). Suitable mechanisms for eliciting increases in brain NO concentration in migraineurs have not yet been identified, although, animal models highlight cortical spreading depression (CSD) as a potential candidate. These studies have focused on CSD-associated NO release at highly acute time points (min-hours) and have not employed markers of NO metabolism with direct clinical application e.g. cGMP. The current study evaluated changes in plasma cGMP concentrations 3 h, 24 h and 3 days post-CSD and compared these to cortical and brainstem cGMP concentrations at 3 days. Moreover, this study also examined the effect of sumatriptan, a clinically effective antimigraine agent, and tonabersat (SB-220453) a potential novel antimigraine agent, on any observed changes in cGMP. Following pre-treatment with vehicle (n=3), sumatriptan (300 microg kg(-1) i.v, n=3) or tonabersat (SB-220453 10 mg kg(-1) i.p., n=3), CSD was evoked in anaesthetised rats by a 6-min KCl application to the parietal cortex. In the vehicle-treated group a median of eight depolarisations, were observed. Sumatriptan had no effect on the number of depolarisations, whereas tonabersat significantly reduced the number of events (median=2). No depolarisation events were observed throughout the recording period in the sham group. Following KCl application plasma cGMP concentrations were reduced up to 24 h post-CSD, but not significantly different from sham animals at 3 days. CSD in vehicle-treated animals produced a highly significant elevation in cGMP concentration in the brain stem 3 days after application of KCl. cGMP concentration increased 2.3-fold from 68+/-8 fmol/mg in sham animals (n=3) to 158+/-28 fmol/mg in the vehicle group. This increase in brain stem cGMP was abolished by tonabersat pre-treatment but not by sumatriptan." [Abstract]

Smith MI, Read SJ, Chan WN, Thompson M, Hunter AJ, Upton N, Parsons AA.
Repetitive cortical spreading depression in a gyrencephalic feline brain: inhibition by the novel benzoylamino-benzopyran SB-220453.
Cephalalgia. 2000 Jul;20(6):546-53.
"Transient cortical depolarization is implicated in the pathology of migraine. SB-220453 is a potent anti-convulsant which inhibits neurogenic inflammation and cortical spreading depression (SD)-evoked nitric oxide release via a novel but unknown mechanism. This study further investigates the effects of SB-220453 on generation and propagation of repetitive SD in the anaesthetized cat. Vehicle or SB-220453 1, 3 or 10 mg/kg was administered intraperitoneally 90 min prior to induction of SD in the suprasylvian gyrus (SG). Changes in d.c. potential were recorded in the SG and the adjacent marginal gyrus (MG). In vehicle-treated animals (n = 7), a brief exposure (6 min) to KCl induced a median (25-75% range) number of five (four to six) and three (two to four) depolarizations over a duration of 55 min (32-59 min) and 51 min (34-58 min) in the SG and MG, respectively. SB-220453 produced dose-related inhibition of the number of events and period of repetitive SD activity. SB-220453 also reduced SD-induced repetitive pial vasodilatation but had no effect on resting haemodynamics. However, when SD events were observed in the presence of SB-220453, it had no effect on metabolic coupling. These results show that SB-220453 produces marked inhibition of repetitive SD in the anaesthetized cat. SB-220453 may therefore have therapeutic potential in treatment of SD-like activity in migraine." [Abstract]

Knyihar-Csillik E, Tajti J, Chadaide Z, Csillik B, Vecsei L.
Functional immunohistochemistry of neuropeptides and nitric oxide synthase in the nerve fibers of the supratentorial dura mater in an experimental migraine model.
Microsc Res Tech. 2001 May 1;53(3):193-211.
"The supratentorial cerebral dura of the albino rat is equipped with a rich sensory innervation both in the connective tissue and around blood vessels, which includes nociceptive axons and their terminals; these display intense calcitonin gene-related peptide (CGRP) immunoreactivity. Stereotactic electrical stimulation of the trigeminal (Gasserian) ganglion, regarded as an experimental migraine model, caused marked increase and disintegration of club-like perivascular CGRP-immunopositive nerve endings in the dura mater and induced an apparent increase in the lengths of CGRP-immunoreactive axons. Intravenous administration of sumatriptan or eletriptan, prior to electrical stimulation, prevented disintegration of perivascular terminals and induced accumulation of CGRP in terminal and preterminal portions of peripheral sensory axons. Consequently, immunopositive terminals and varicosities increased in size; accumulation of axoplasmic organelles resulted in the "hollow" appearence of numerous varicosities. Since triptans exert their anti-migraine effect by virtue of agonist action on 5-HT(1D/B) receptors, we suggest that these drugs prevent the release of CGRP from perivascular nerve terminals in the dura mater by an action at 5-HT(1D/B) receptors. Nitroglycerine (NitroPOHL), given subcutaneously to rats, induces increased beading of nitric oxide synthase (NOS)-immunoreactive nerve fibers in the supratentorial cerebral dura mater, and an apparent increase in the number of NOS-immunoreactive nerve fibers in the dural areas supplied by the anterior and middle meningeal arteries, and the sinus sagittalis superior. Structural alterations of nitroxidergic axons innervating blood vessels of the dura mater support the idea that nitric oxide (NO) is involved in the induction of headache, a well-known side effect of coronary dilator agents." [Abstract]

Strecker T, Messlinger K.
[Neuropeptide release in the dura mater encephali in response to nitric oxide--relevance for the development of vascular headaches?]
Schmerz. 2003 Jun;17(3):179-84.
"Nitric oxide (NO) and calcitonin gene-related peptide (CGRP), potent vasodilators in the meninges,may be involved in the pathophysiology of vascular headaches such as migraine pain. NO donators can provoke headache attacks in migraineurs and increased levels of CGRP have been found in the venous outflow from the head during migraine attacks. We therefore examined the effect of both NO and CGRP on dural blood, a process which may parallel nociceptive processes in the meninges. 1.Arterial blood flow was measured in the exposed dura mater encephali of the rat using laser Doppler flowmetry. Local application of different NO donors (SNAP,NONOate, and NOC-12) caused dose-dependent increases in meningeal blood flow. CGRP(8-37) at 10(-4) M did not significantly change the basal flow but attenuated increases in blood flow caused by the NO donors at concentrations of 10(-5)-10(-3) M.2. In another series of experiments, the hemisected skulls of adult Wistar rats, complete with intact dura mater, were filled with oxygenated synthetic interstitial fluid (SIF) and the CGRP content of this fluid was assessed every 5 min. When the NO donator NONOate, at concentrations of 10(-5)-10(-3) M, was added to the SIF, or when the SIF was bubbled with NO gas (1000 ppm in N(2) atmosphere) instead of carbogen, CGRP release increased in a concentration-dependent manner. We conclude that the vasodilatory effect of NO that causes increased meningeal blood flow is in part the result of both stimulating the release of CGRP and promoting the vasodilatory action of CGRP. Since NO donors such as nitroglycerin are known to provoke headache and CGRP is released during migraine pain, the NO-stimulated CGRP release may be relevant for the development of vascular headaches that are accompanied by meningeal hyperaemia." [Abstract]

Strecker T, Dux M, Messlinger K.
Nitric oxide releases calcitonin-gene-related peptide from rat dura mater encephali promoting increases in meningeal blood flow.
J Vasc Res. 2002 Nov-Dec;39(6):489-96.
"Nitric oxide (NO) and calcitonin-gene-related peptide (CGRP) are implicated in the pathophysiology of vascular headaches. We studied the interaction of these two vasodilatory mediators in an animal model and suggest that NO may increase meningeal blood flow not only by its direct vasodilatory action but also by stimulating CGRP release. First, CGRP release from the rat cranial dura mater was measured in vitro using an enzyme immunoassay. Hemisected skulls with adhering dura mater were filled with synthetic interstitial fluid and stimulated with the NO donor diethylamine-NONOate (10(-5)-10(-3) M) or with NO gas (1,000 ppm), which caused concentration-dependent increases in CGRP release up to 166.8%. Second, meningeal blood flow was recorded in vivo in the exposed dura mater using laser Doppler flowmetry. Topical application of the NO donors NONOate, S-nitroso-N-acetylpenicillamine and N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethenamine (10(-5)-10(-3) M) caused concentration-dependent increases in blood flow. These increases were significantly reduced by local preliminary application of the CGRP receptor antagonist CGRP(8-37) (10(-4) M). We conclude that NO stimulates the release of CGRP from dural afferents. The blood-flow-increasing effect of NO seems to be partly mediated by CGRP. The interaction of NO and CGRP may be relevant for the development of vascular headaches." [Abstract]

Strecker T, Dux M, Messlinger K.
Increase in meningeal blood flow by nitric oxide--interaction with calcitonin gene-related peptide receptor and prostaglandin synthesis inhibition.
Cephalalgia. 2002 Apr;22(3):233-41.
"This study addresses possible interactions of the vasodilators nitric oxide (NO), calcitonin gene-related peptide (CGRP) and prostaglandins, which may be implicated in the generation of vascular headaches. Local application of the NO donator diethylamine-NONOate (NONOate) to the exposed dura mater encephali of the rat caused dose-dependent increases in meningeal blood flow recorded by laser Doppler flowmetry. Pre-application of the CGRP receptor antagonist CGRP8-37 significantly attenuated the evoked blood flow increases, while the cyclooxygenase inhibitors acetylsalicylic acid and metamizol were only marginally effective. Stimulation of rat dura mater with NONOate in vitro caused increases in CGRP release. NADPH-diaphorase activity indicating NO production was restricted to the endothelium of dural arterial vessels. We conclude that increases in meningeal blood flow caused by NO depend partly on the release and vasodilatory action of CGRP from dural afferents, while prostaglandins are not significantly involved." [Abstract]

Gallai V, Alberti A, Gallai B, Coppola F, Floridi A, Sarchielli P.
Glutamate and nitric oxide pathway in chronic daily headache: evidence from cerebrospinal fluid.
Cephalalgia. 2003 Apr;23(3):166-74.
"A central sensitization has been advocated to explain chronic daily headache (CDH) due to sustained peripheral sensitization of allogenic structures responsible for sustained trigeminovascular system activation. Several mechanisms have been suggested to underlie central sensitization, but have been poorly investigated in CDH. They involve N-methyl-D-aspartate (NMDA) receptor activation and nitric oxide (NO) production and supersensitivity and increased and maintained production of sensory neuropeptides. The present study supports the above pathogenic mechanisms demonstrating a significant increase in glutamate and nitrite levels in the CSF of CDH patients, without a significant difference between patients without and those with analgesic overuse headache (P < 0.0001 and P < 0.002). The increase in CSF nitrites was accompanied by a significant rise in the CSF values of cyclic guanosine monophosphate (cGMP) in patients in comparison with controls (P < 0.0001). A statistically significant correlation emerged between visual analogic scale (VAS) values and glutamate, nitrites and cGMP. Although substance P (SP) and calcitonin gene-related peptide (CGRP), and to a lesser extent neurokinin A, were significantly increased in CSF compared with control subjects, their values did not correlate with glutamate, nitrites and cGMP levels in CSF in the patient group. The present study confirms the involvement of glutamate-NO-cGMP-mediated events underlying chronic head pain that could be the target of a new therapeutic approach which should be investigated." [Abstract]

Eltorp CT, Jansen-Olesen I, Hansen AJ.
Release of calcitonin gene-related peptide (CGRP) from guinea pig dura mater in vitro is inhibited by sumatriptan but unaffected by nitric oxide.
Cephalalgia. 2000 Nov;20(9):838-44.
"Migraine attacks can be provoked by administration of nitroglycerin, suggesting a role for nitric oxide (NO). The fact that release of the neuropeptide CGRP from trigeminal sensory nerves occurs during the pain phase of migraine and that NO can augment transmitter release prompted us to study CGRP release from the in situ dura mater in guinea pig skulls. Release of CGRP by capsaicin or by high potassium concentration was concentration-dependent and counteracted in calcium-free medium. The anti-migraine compound, sumatriptan, inhibited CGRP release via the 5-HT1-receptor. The NO donors, nitroglycerin, sodium nitroprusside and S-nitroso-N-acetylpenicillamine did not influence CGRP release, alone or together with the stimulants. We concluded that the skull preparation is well suited for scrutinizing CGRP release from dura mater. The fact that sumatriptan inhibits CGRP release as in migraine patients suggests a use for the present preparation in headache research." [Abstract]

Ashina M, Bendtsen L, Jensen R, Schifter S, Olesen J.
Calcitonin gene-related peptide levels during nitric oxide-induced headache in patients with chronic tension-type headache.
Eur J Neurol. 2001 Mar;8(2):173-8.
"It has been proposed that nitric oxide (NO) induced headache in primary headaches may be associated with release of calcitonin gene-related peptide (CGRP). In the present study we aimed to investigate plasma levels of CGRP during headache induced by the NO donor glyceryl trinitrate (GTN) in 16 patients with chronic tension-type headache and 16 healthy controls. The subjects were randomly allocated to receive 0.5 microg/kg/min GTN or placebo over 20 min on two headache-free days. Blood samples were collected at baseline, 10, 20 and 60 min after start of infusion. Both patients and controls developed significantly stronger immediate headache on the GTN day than on the placebo day and the headache was significantly more pronounced in patients than in controls. There was no difference between the area under the CGRP curve (AUCCGRP) on GTN vs. placebo day in either patients (P=0.65) or controls (P=0.48). The AUCCGRP recorded on the GTN day did not differ between patients and controls (P=0.36). Both in patients and controls, CGRP levels changed significantly over time, on both the GTN and placebo days (P < 0.05). The present study indicates that NO-induced immediate headache is not associated with release of CGRP." [Abstract]

Messlinger K, Suzuki A, Pawlak M, Zehnter A, Schmidt RF.
Involvement of nitric oxide in the modulation of dural arterial blood flow in the rat.
Br J Pharmacol. 2000 Apr;129(7):1397-404.
"1. Nitric oxide (NO) has been proposed to be a key molecule in the pathogenesis of migraine pain and other headaches that are linked to vascular disorders. Several lines of evidence indicate that the meningeal vascularization is crucially involved in the generation of these headaches. In an experimental model in the rat a dominating role of calcitonin gene-related peptide (CGRP) in causing neurogenic vasodilatation and increased blood flow has been shown. The aim of the present study was to clarify the role of NO in this model with regard to the meningeal blood flow. 2. The blood flow in and around the medial meningeal artery (dural arterial flow) was recorded in the exposed parietal dura mater encephali of barbiturate anaesthetized rats using laser Doppler flowmetry. Local electrical stimulation of the dura mater (pulses of 0.5 ms delivered at 7.5 - 17.5 V and 5 or 10 Hz for 30 s) caused temporary increases in dural arterial flow for about 1 min that reached peaks of 1.6 - 2.6 times the basal flow. The effects of NO synthase (NOS) inhibitors on the basal flow and the electrically evoked increases in flow were examined. 3. Systemic (i. v.) administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) at cumulative doses of 10 and 50 mg kg(-1) lowered the basal flow to 87 and 72%, respectively, of the control and reduced the evoked increases in blood flow to 82 and 44% on an average. Both these effects could partly be reversed by 300 mg kg(-1) L-arginine. The systemic arterial pressure was increased by L-NAME at both doses. Injection of the stereoisomer D-NAME at same doses did not change basal flow and evoked increases in flow. 4. 4. Topical application of L-NAME (10(-4) - 10(-2) M) was effective only at the highest concentration, which caused lowering of the basal blood flow to 78% of the control; the evoked increases in flow were not changed. Topical application of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NOS, at concentrations of 10(-4) - 10(-2) M lowered the basal flow to 89, 87.5 and 85%, respectively, but did not significantly change the evoked flow increases. Same concentrations of 7-nitroindazole monosodium salt (7-NINA), a specific inhibitor of the neuronal NOS, had no significant effects on basal flow and evoked increases in flow. 5. It is concluded that NO is involved in the maintenance of the basal level of dural arterial blood flow as well as in the electrically evoked flow increases, which have been shown to be mainly mediated by CGRP released from dural afferent fibres. The most important source of NO is probably the endothelium of dural arterial vessels. The synergistic effect of NO and CGRP on the stimulated blood flow may be in part due to a NO mediated facilitation of the CGRP release." [Abstract]

Akerman S, Williamson DJ, Kaube H, Goadsby PJ.
Nitric oxide synthase inhibitors can antagonize neurogenic and calcitonin gene-related peptide induced dilation of dural meningeal vessels.
Br J Pharmacol. 2002 Sep;137(1):62-8.
"1. The detailed pathophysiology of migraine is beginning to be understood and is likely to involve activation of trigeminovascular afferents. 2. Clinically effective anti-migraine compounds are believed to have actions that include peripheral inhibition of calcitonin gene-related peptide (CGRP) release from trigeminal neurones, or preventing dural vessel dilation, or both. CGRP antagonists can block both neurogenic and CGRP-induced dural vessel dilation. 3. Nitric oxide (NO) can induce headache in migraine patients and often triggers a delayed migraine. The initial headache is thought to be caused via a direct action of the NO-cGMP pathway that causes vasodilation by vascular smooth muscle relaxation, while the delayed headache is likely to be a result of triggering trigeminovascular activation. Nitric oxide synthase (NOS) inhibitors are effective in the treatment of acute migraine. 4. The present studies used intravital microscopy to examine the effects of specific NOS inhibitors on neurogenic dural vasodilation (NDV) and CGRP-induced dilation. 5. The non-specific and neuronal NOS (nNOS) inhibitors were able to partially inhibit NDV, while the non-specific and endothelial NOS (eNOS) inhibitors were able to partially inhibit the CGRP induced dilation. 6. There was no effect of the inducible NOS (iNOS) inhibitor. 7. The data suggest that the delayed headache response triggered by NO donors in humans may be due, in part, to increased nNOS activity in the trigeminal system that causes CGRP release and dural vessel dilation. 8. Further, eNOS activity in the endothelium causes NO production and smooth muscle relaxation by direct activation of the NO-cGMP pathway, and may be involved in the initial headache response." [Abstract]

Ashina M, Lassen LH, Bendtsen L, Jensen R, Olesen J.
Effect of inhibition of nitric oxide synthase on chronic tension-type headache: a randomised crossover trial.
Lancet. 1999 Jan 23;353(9149):287-9.
"BACKGROUND: Studies in animals have shown that nitric oxide plays an important part in central sensitisation and that inhibitors of nitric oxide synthase (NOS) decrease sensitisation in models of persistent pain. The efficacy of inhibitors of NOS has not been tested in patients with tension-type chronic headache. We aimed to show whether N(G)-monomethyl-L-arginine hydrochloride (L-NMMA), an inhibitor of NOS, is effective in relieving pain in such patients. METHODS: We undertook a randomised double-blind, crossover trial of 16 patients with chronic-tension-type headache. Patients were assigned intravenous infusion of 6 mg/kg L-NMMA or placebo on 2 days separated by at least 1 week in a randomised order. Headache intensity was measured on a 100 mm visual analogue scale, and on a verbal rating scale at baseline and at 30 min, 60 min, and 120 min after start of treatment. The primary endpoint was reduction of pain intensity on the visual analogue scale by the active treatment compared with placebo. FINDINGS: L-NMMA reduced pain intensity on the visual analogue scale significantly more than placebo: 120 min after start of treatment, the mean pain score was decreased from 49 to 33 with L-NMMA and from 44 to 40 with placebo (p=0.01). Pain intensity on the verbal rating scale was also significantly lower for treatment with L-NMMA than for treatment with placebo (p=0.02). INTERPRETATION: Inhibition of NOS had an analgesic effect in chronic tension-type headache. Further tests are required before clinical application." [Abstract]

Lassen LH, Ashina M, Christiansen I, Ulrich V, Grover R, Donaldson J, Olesen J.
Nitric oxide synthase inhibition: a new principle in the treatment of migraine attacks.
Cephalalgia. 1998 Jan;18(1):27-32.
"Glyceryl trinitrate, an exogenous nitric oxide (NO) donor, and histamine, which causes NO formation in vascular endothelium, have been shown to trigger migraine attacks. However, it remains uncertain whether NO is involved in the subsequent phase of migraine attacks. To answer this question we studied the effect of L-NGmethylarginine hydrochloride (546C88), a NO-synthase inhibitor, on spontaneous migraine attacks. In a double-blind study design, 18 patients with migraine without aura randomly received 546C88 (6 mg/kg) or placebo (5% dextrose) i.v. given over 15 min for a single migraine attack (546C88:placebo, 15:3). Furthermore, 11 placebo-treated patients from previous double-blind trials with almost identical design were added to the placebo group in the statistical evaluation. Two hours after the infusion, 10 of 15 L-NGmethylarginine hydrochloride-treated patients experienced headache relief compared to 2 of 14 placebo-treated patients (p = 0.01). Symptoms such as phono- and photophobia were also significantly improved. A similar trend for nausea was not significant. We conclude that NO may be involved in the pain mechanisms throughout the course of spontaneous migraine attacks." [Abstract]

van der Kuy PH, Merkus FW, Lohman JJ, ter Berg JW, Hooymans PM.
Hydroxocobalamin, a nitric oxide scavenger, in the prophylaxis of migraine: an open, pilot study.
Cephalalgia. 2002 Sep;22(7):513-9.
"Drugs which directly counteract nitric oxide (NO), such as endothelial receptor blockers, NO-synthase inhibitors, and NO-scavengers, may be effective in the acute treatment of migraine, but are also likely to be effective in migraine prophylaxis. In the underlying pilot study the prophylactic effect of the NO scavenger hydroxocobalamin after intranasal administration in migraine was evaluated. Twenty patients, with a history of migraine of > 1 year and with two to eight migraine attacks per month, were included in an open trial. A baseline period was followed by an active treatment period of 3 months with 1 mg intranasal hydroxocobalamin daily. Patients were instructed to complete a diary in which details of each attack were described. A reduction in migraine attack frequency of >/ or = 50% was seen in 10 of 19 patients, which corresponds to 53% of the patients (responders). A reduction of > or = 30% was noted in 63% of the patients. The mean attack frequency in the total study population showed a reduction from 4.7 +/- 1.7 attacks per month to 2.7 +/- 1.6 (P < 0.001). For the responders the migraine attack frequency was reduced from 5.2 +/- 1.9 (baseline) to 1.9 +/- 1.3 attacks per month (P < 0.005), while for those who did not respond a non-significant reduction was found: 4.1 +/- 1.4 to 3.7 +/- 1.5 (P > 0.1). A reduction was also observed for the total duration of the migraine attacks per month, the total number of migraine days per month and the number of medication doses for acute treatment used per month. This is the first prospective, open study indicating that intranasal hydroxocobalamin may have a prophylactic effect in migraine. As a percentage of responders in prophylactic trials of > 35-40% is unlikely to be a placebo effect, a double-blind study is warranted." [Abstract]

Read SJ, Parsons AA.
Sumatriptan modifies cortical free radical release during cortical spreading depression. A novel antimigraine action for sumatriptan?
Brain Res. 2000 Jul 7;870(1-2):44-53.
"Increases in concentration of brain NO are proposed to initiate and mediate migraine headache. Triggered by focal depolarisation, spreading depression (SD) represents a suitable mechanism for eliciting widespread release of nitric oxide. The current study examines the effect of sumatriptan, a 5-HT(1B/1D) agonist and effective antimigraine therapy, on free radical release (nitric oxide and superoxide) in SD in the simple and complex cortices of the rat and cat. Following initiation of SD, sumatriptan pretreatment (300 microg kg(-1) i.v., 15 min prior to SD) modulated all phases of nitric oxide release associated with each SD in both cats and rats. As a result, superoxide levels were observed to significantly (ANOVA, post hoc LSD) increase versus vehicle treated animals (saline 1 ml kg(-1) i.v. 15 min prior to SD) during specific phases of each SD depolarisation. Averaged over all SD depolarisations, mean peak SD nitric oxide levels per depolarisation were 0.73+/-0.23 microM (n=29) in cats, and 0.42+/-0.09 microM (n=34) in rats. Sumatriptan significantly (Students t-test, P<0.05, two tailed hypothesis, P<0.05) modulated this increase in cortical nitric oxide concentrations to 0.32+/-0.06 microM (n=25) and 0. 22+/-0.07 microM (n=37) in cats and rats. Sumatriptan appears to decrease the amplitude of nitric oxide release but enhances extracellular superoxide concentrations in both lissencephalic and gyrencephalic cortices during SD." [Abstract]

Ikeda Y, Jimbo H, Shimazu M, Satoh K.
Sumatriptan scavenges superoxide, hydroxyl, and nitric oxide radicals: in vitro electron spin resonance study.
Headache. 2002 Oct;42(9):888-92.
"BACKGROUND: The molecular mechanisms of migraine have not yet been clarified. Oxygen free radicals have been implicated in the genesis of many pathological processes, including migraine. Sumatriptan succinate is known to be a very effective drug for acute relief of migraine attack. OBJECTIVE: To investigate the direct scavenging activities of sumatriptan for superoxide, hydroxyl, and nitric oxide (NO) radicals using electron spin resonance (ESR) spectroscopy. METHODS: Measurement of superoxide and hydroxyl radical scavenging activities was performed by ESR using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap. NO was generated from 1-hydroxy-2-oxo-3-(N-3-methyl-3-aminopropyl)-3-methyl-1-triazene and analyzed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl produced from the reaction between 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and NO. RESULTS: The ESR study demonstrated that sumatriptan scavenged superoxide, hydroxyl, and NO in a dose-dependent manner. CONCLUSIONS: Sumatriptan has direct scavenging activity on free radicals and NO. Acute migraine drugs with antioxidant properties may provide heretofore unheralded benefits via this mechanism." [Abstract]

Read SJ, Manning P, McNeil CJ, Hunter AJ, Parsons AA.
Effects of sumatriptan on nitric oxide and superoxide balance during glyceryl trinitrate infusion in the rat. Implications for antimigraine mechanisms.
Brain Res. 1999 Nov 13;847(1):1-8.
"Infusion of glyceryl trinitrate (GTN) into patients with migraine precipitates the onset of a migraine attack several hours after completion of the infusion. Using an infusion of GTN into anaesthetised rats, this study investigates the relationship of regional cerebral blood flux rCBF(ldf), cortical nitric oxide (NO) and cortical superoxide concentrations and the effect of sumatriptan on each variable. In saline treated animals, a 30 min infusion of GTN (2 microgram kg(-1) min(-1), i.v.) was found to markedly increase cortical rCBF(ldf) (133+/-3% of baseline) and NO concentrations (141+/-13% of baseline). Superoxide levels exhibited an inverse relationship to NO levels, decreasing below basal to 48+/-14% of baseline. It is hypothesised that high NO levels during GTN infusion may decrease the detectable superoxide due to "leeching" of the superoxide into low level peroxynitrite formation. In the presence of sumatriptan, a decrease below baseline in cortical rCBF(ldf) (82+/-5% of baseline) and NO concentration (64+/-13% of baseline) was observed throughout GTN infusion, although superoxide levels significantly increased above baseline by 105+/-14 nM (p<0.05, ANOVA post hoc LSD test). The mechanism for this action of sumatriptan is unknown but may include; modulation of cell redox state, NO scavenging or direct manipulation of superoxide release." [Abstract]

Iversen HK, Olesen J.
Headache induced by a nitric oxide donor (nitroglycerin) responds to sumatriptan. A human model for development of migraine drugs.
Cephalalgia. 1996 Oct;16(6):412-8.
"Experimental "vascular" headache in humans may be used in characterizing new migraine drugs. The effects of sumatriptan on nitroglycerin-(NTG)-induced headache and arterial responses were therefore studied. Following a double-blind randomized crossover design, 10 healthy volunteers received sumatriptan 6 mg s.c. or placebo succeeded by 20 min NTG (0.12 microgram/kg/min) infusion. Headache was rated on a 10 points scale. Temporal and radial artery diameters and velocity in the middle cerebral artery (MCA) were measured with ultrasound. Sumatriptan reduced the NTG-induced headache, median score 1.5 versus 4 after placebo (p < 0.01) and decreased temporal and radial artery diameters 75 +/- 3 and 86 +/- 3% of baseline respectively (p < 0.05). Blood velocity in the MCA was unaffected. The NTG model may prove to be a valuable tool in the development of future migraine drugs. The results suggest that NTG headache in non-migraineurs may share mechanisms with migraine headache." [Abstract]

Akerman S, Williamson DJ, Kaube H, Goadsby PJ.
The effect of anti-migraine compounds on nitric oxide-induced dilation of dural meningeal vessels.
Eur J Pharmacol. 2002 Oct 4;452(2):223-8.
"Migraine is characteristically accompanied by a throbbing quality of head pain thought to involve trigeminovascular afferents. Administration of nitric oxide (NO) donors provides the most reliable model of migraine induction in humans. The present studies used intravital microscopy to monitor the effect of local meningeal nerve stimulation and NO on dural blood vessels and any modulation of that effect by anti-migraine compounds. NO caused an immediate and reproducible dilation of meningeal blood vessels that was partially blocked by sumatriptan and indomethacin, while flunarizine and histamine H(1) and H(2) receptor antagonists were unable to block the dilation. Indomethacin also inhibited the neurogenic dilation while flunarizine did not. The present studies demonstrate that NO is unlikely to interact with histamine to produce its dilatory response. Sumatriptan and indomethacin inhibit the NO response by inhibiting trigeminal activation and calcitonin gene-related peptide (CGRP) release. Flunarizine does not modify either the neurogenic vasodilator response or the NO meningeal dilator response at least acutely." [Abstract]

Ayajiki K, Okamura T, Toda N.
Flunarizine, an anti-migraine agent, impairs nitroxidergic nerve function in cerebral arteries.
Eur J Pharmacol. 1997 Jun 18;329(1):49-53.
"Flunarizine is an anti-migraine agent that blocks the Ca2+ entry across cell membrane. In order to obtain a clue of mechanisms underlying the migraine headache, modifications by flunarizine of the response to nitric oxide (NO), a cerebral vasodilator and algogenic agent, derived from perivascular nerves were evaluated. Relaxations due to nerve stimulation by electrical pulses (5 Hz) and nicotine (10(-4) M) in canine cerebral arterial strips were attenuated by treatment with flunarizine dose-dependently, whereas the responses to exogenous NO (10(-7)-10(-6) M) and nitroprusside (10(-8)-10(-6) M) were unaffected. The inhibition by the Ca2+ entry blocker of the response to electrical nerve stimulation and nicotine was obtained in a concentration (10(-6) M) that did not significantly relax the arterial strips. NO derived from perivascular nerve may be one of the factors involved in the genesis of migraine attack, which is expected to be relieved by a reduction of neural NO synthase activity associated with a decreased Ca2+ influx by flunarizine during nerve activation." [Abstract]

Nattero G, Mengozzi G, Inconis T, Paradisi L.
Nitric oxide, endothelin-1, and transcranial Doppler in migraine. Findings in interictal conditions and during migraine attack.
Headache. 1996 May;36(5):307-11.
"The role of vascular phenomena taking place during an attack of migraine are poorly understood. The aim of this study was to measure systemic levels of nitric oxide and endothelin-1, two of the most potent vasoactive mediators known, and to assess vasomotor responses through transcranial Doppler ultrasound monitoring in patients suffering from migraine without aura, both during the headache event and in headache-free periods as well as after pharmacologically induced pain relief. Seven patients (mean age 31.3 years, range 24 to 49 years), five women and two men, were enrolled in the pilot study. Transcranial Doppler recordings were performed according to conventional procedure. Endothelin-1 concentrations were measured by means of radioimmunoassay, whereas nitric oxide levels were estimated using electron paramagnetic resonance spectroscopy. Ultrasound evaluation did not show significant changes during migraine attacks compared to the interictal condition. Nitric oxide levels showed only slight differences between basal and attack conditions (0.85 +/- 0.46 versus 1.56 +/- 0.88, expressed as arbitrary units), and were raised after pharmacological intervention (2.91 +/- 1.93, P < 0.05). Plasma endothelin-1 concentrations decreased during migraine attacks with respect to interictal conditions (3.99 +/- 1.21 pg/mL versus 4.23 +/- 1.19), and returned to basal values (4.44 +/- 1.08 pg/mL) after relief of pain. Coupling the measurements of systemic levels of nitric oxide and endothelin-1 with transcranial Doppler velocity results will provide useful information on the hemodynamic changes of cerebral blood flow regulation in migraineurs, thereby adding new insights into the mechanisms of the migraine attack." [Abstract]

Pardutz A, Krizbai I, Multon S, Vecsei L, Schoenen J.
Systemic nitroglycerin increases nNOS levels in rat trigeminal nucleus caudalis.
Neuroreport. 2000 Sep 28;11(14):3071-5.
"Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraineurs a delayed attack of unknown mechanisms. Subcutaneous nitroglycerin (10 mg/kg) produced a significant increase of nitric oxide synthase (NOS)- and c-fos-immunoreactive neurons in the cervical part of trigeminal nucleus caudalis in rats after 4 h. This effect was not observed in the thoracic dorsal horn. Similar increase of NOS and c-fos was obtained in the brain stem after a somatic nociceptive stimulus, i.e. on the side of the formalin injection in the lip. Nitric oxide is thus able to increase NOS availability in second order nociceptive trigeminal neurons, which may be relevant for central sensitization and the understanding of its effect in migraine." [Abstract]

Knyihar-Csillik E, Vecsei L.
Effect of a nitric oxide donor on nitroxergic nerve fibers in the rat dura mater.
Neurosci Lett. 1999 Jan 29;260(2):97-100.
"Nitroglycerine, given subcutaneously to rats (10 mg/kg body weight) induces increased beading of nitric oxide synthase immunoreactive (NOS-IR) nerve fibers in the supratentorial cerebral dura mater, and an apparent increase in the number of NOS-IR nerve fibers in the dural areas supplied by the anterior and middle meningeal arteries, and the sinus sagittalis superior. Structural alterations of nitroxergic axons innervating blood vessels of the dura mater support the idea that nitric oxide is involved in the induction of headache also by a primary peripheral action, a well-known side effect of coronary dilator agents." [Abstract]

Hoskin KL, Bulmer DC, Goadsby PJ.
Fos expression in the trigeminocervical complex of the cat after stimulation of the superior sagittal sinus is reduced by L-NAME.
Neurosci Lett. 1999 May 14;266(3):173-6.
"Primary neurovascular headaches, such as migraine and cluster headache probably involve activation of trigeminovascular pain structures projecting to the trigeminocervical complex of neurons in the caudal brain stem and upper cervical spinal cord. It has recently been demonstrated that blockade of the synthesis of nitric oxide (NO) by an NO synthesis inhibitor can abort acute migraine attacks and thus it is of interest to determine whether there is an influence of NO generation on trigeminocervical neurons. Cats were anaesthetised with alpha-chloralose (60 mg/kg, i.t.). supplemental 20 mg/kg, intravenously (i.v.)) and halothane for surgery (0.5-3% by inhalation). A circular midline craniotomy was performed to isolate the superior sagittal sinus (SSS) for electrical stimulation (0.3 Hz, 150 V, 250 micros duration for 2 h). Two groups were compared, one stimulated after administration of vehicle and the other stimulated after administration of N(G)-nitro-L-arginine methylester (L-NAME: 100 mg/kg, i.v.). After stimulation of the SSS Fos immunoreactivity was observed in lamina I/IIo of the trigeminal nucleus caudalis and dorsal horns of C1 and C2 to a median total of 136 cells (range 122-146). After L-NAME treatment Fos expression was significantly reduced to 40 cells (24-54; P < 0.02). In conclusion, inhibition of NO synthesis L-NAME markedly reduces Fos expression in the trigeminocervical complex of the cat. These data taken together with the clinical observations of the effect of NO synthesis blockade in migraine suggest a role for NO generation in mediating nociceptive transmission in acute migraine." [Abstract]

De Col R, Koulchitsky SV, Messlinger KB.
Nitric oxide synthase inhibition lowers activity of neurons with meningeal input in the rat spinal trigeminal nucleus.
Neuroreport. 2003 Feb 10;14(2):229-32.
"Nitric oxide is thought to control transmitter release and neuronal activity in the spinal dorsal horn and the spinal trigeminal nucleus, where nociceptive information from extra- and intracranial tissues is processed. Extracellular impulse activity was recorded from neurons in the rat spinal trigeminal nucleus with afferent input from the cranial dura mater. In contrast to the inactive isomer D-NAME, infusion of the nitric oxide synthase inhibitor L-NAME (20 mg/kg) significantly reduced neuronal activity and increased systemic blood pressure. It is concluded that nitric oxide production contributes to the ongoing activity of sensitized neurons in the spinal trigeminal nucleus. The results suggest that nitric oxide may be involved in the generation and maintenance of primary headaches such as migraine." [Abstract]

Jones MG, Lever I, Bingham S, Read S, McMahon SB, Parsons A.
Nitric oxide potentiates response of trigeminal neurones to dural or facial stimulation in the rat.
Cephalalgia. 2001 Jul;21(6):643-55.
"Infusing glyceryl trinitrate as a donor molecule, we have used electrophysiological and c-fos immunostaining techniques to study the effects of nitric oxide on neurones in the nucleus trigeminalis caudalis. Following infusion of glyceryl trinitrate, responses of neurones to electrical stimulation of periorbital cutaneous afferents were potentiated and threshold for activation of neurones by stimulation of dural afferents was reduced. Expression of c-fos was unchanged by glyceryl trinitrate compared to saline controls. Intradermal injection of capsaicin in the periorbital area increased c-fos expression in nucleus trigeminalis caudalis; this was significantly potentiated by glyceryl trinitrate. These results suggest that, in the anaesthetized rat, glyceryl trinitrate alone may not acutely activate the trigeminovascular system to a significant degree at doses that cause headache and later trigger migraine headache in migraineurs. Nevertheless, it is susceptible to exogenous nitric oxide in that activation of trigeminal neurones through cutaneous or dural pathways is potentiated. This may in some measure underlie the pathogenesis of migraine headache." [Abstract]

Behrends S, Knyihar-Csillik E, Kempfert J, Scholz H, Csillik B, Vecsei L.
Glyceryl trinitrate treatment up-regulates soluble guanylyl cyclase in rat dura mater.
Neuroreport. 2001 Dec 21;12(18):3993-6.
"Nitric oxide (NO) is a key molecule in vascular headaches and the dura mater has been implicated as a tissue where vascular headache develops. Here we demonstrate expression, enzyme activity and cellular distribution of the intracellular receptor for NO, soluble guanylyl cyclase (sGC), in rat dura mater. Subcutaneous treatment of rats with the NO-donor glyceryl trinitrate (GTN) induced an increase of sGC expression and activity in dural blood vessels after 20-30 min. It has previously been shown that GTN induces headache in normal subjects after 20-30 min. Our findings suggest that an up-regulation of the NO target enzyme contributes to the pathogenesis of GTN-induced headache explaining the subacute rather than acute onset of symptoms." [Abstract]

Nemade RV, Lewis AI, Zuccarello M, Keller JT.
Immunohistochemical localization of endothelial nitric oxide synthase in vessels of the dura mater of the Sprague-Dawley rat.
Neurosci Lett. 1995 Sep 1;197(1):78-80.
"Nitric oxide (NO) and the dura mater are implicated in the pathogenesis of vascular headache. Many studies have demonstrated the participation of NO in headache; however, few studies have identified NO in the dura mater. In this study, nine Sprague-Dawley rats were examined with immunohistochemistry using two different endothelial nitric oxide synthase (eNOS) monoclonal antibodies, H32 and ECNOS. eNOS was successfully localized to the endothelium of the middle meningeal artery. To the best of our knowledge, this is the first study to report NOS immunopositive endothelial cells in the blood vessels of the rat dura mater. The authors propose that NO plays an active role in dural vasodilation, contributing to the pathogenesis of vascular headache; in the future, NO inhibitors could serve as pharmacological agents to treat vascular headache." [Abstract]

Valenzuela RF, Donoso MV, Mellado PA, Huidobro-Toro JP.
Migraine, but not subarachnoid hemorrhage, is associated with differentially increased NPY-like immunoreactivity in the CSF.
J Neurol Sci. 2000 Feb 15;173(2):140-6.
"To test whether migraine and subarachnoid hemorrhage (SAH) are associated with increased sympathetic tone, we compared the neuropeptide Y-like (NPY-LI) and chromogranin A-like immunoreactivities (LI) of cerebrospinal fluid (CSF) from migraneurs and SAH patients with those from control subjects. Increased sympathetic tone was expected to produce higher co-release of these co-stored peptides and concordant changes in their CSF levels. In addition, we investigated a possible disturbed nitric oxide homeostasis by measuring CSF nitrites (NO). More than 70% of CSF NPY-LI corresponded to the chromatographic peak (HPLC) for the intact molecule in all three groups. Migraneurs had 64% higher CSF NPY-LI, but no significant difference in CSF chromogranin A-LI, as compared to controls. In contrast, SAH patients had 74% less CSF chromogranin A-LI and a trend to lower NPY-LI, as compared to controls. No differences in CSF NO were detected among groups. These results argue against an increased sympathetic tone in patients with either migraine or SAH, and suggest that the higher CSF NPY-LI of migraneurs probably originates from central neurons. Furthermore, our findings in SAH patients argue in favor of a decreased sympathetic tone; this could be a homeostatic response to counterbalance vasoconstriction mediated by other mechanisms." [Abstract]

Reuter U, Chiarugi A, Bolay H, Moskowitz MA.
Nuclear factor-kappaB as a molecular target for migraine therapy.
Ann Neurol. 2002 Apr;51(4):507-16.
"Nitric oxide (NO) generated from inducible NO synthase (iNOS) participates in immune and inflammatory responses in many tissues. The NO donor glyceryl trinitrate (GTN) provokes delayed migraine attacks when infused into migraineurs and also causes iNOS expression and delayed inflammation within rodent dura mater. Sodium nitroprusside, an NO donor as well, also increases iNOS expression. Because inflammation and iNOS are potential therapeutic targets, we examined transcriptional regulation of iNOS following GTN infusion and the consequences of its inhibition within dura mater. We show that intravenous GTN increases NO production within macrophages. L-N(6)-(1-iminoethyl)lysine, a selective iNOS inhibitor, attenuates the NO signal, emphasizing the importance of enzymatic activity to delayed NO production. iNOS expression is preceded by significant nuclear factor kappa B (NF-kappaB) activity, as reflected by a reduction in the inhibitory protein-kappa-Balpha (IkappaBalpha) and activation of NF-kappaB after GTN infusion. IkappaBalpha degradation, NF-kappaB activation, and iNOS expression were attenuated by parthenolide (3mg/kg), the active constituent of feverfew, an anti-inflammatory drug used for migraine treatment. These findings suggest that GTN promotes NF-kappaB activity and inflammation with a time course consistent with migraine attacks in susceptible individuals. We conclude, based on results with this animal model, that blockade of NF-kappaB activity provides a novel transcriptional target for the development of anti-migraine drugs." [Abstract]

Lea RA, Curtain RP, Shepherd AG, Brimage PJ, Griffiths LR.
No evidence for involvement of the human inducible nitric oxide synthase (iNOS) gene in susceptibility to typical migraine.
Am J Med Genet. 2001 Jan 8;105(1):110-3. [Abstract]

Christiansen I, Iversen HK, Olesen J.
Induction of nitrate tolerance is not a useful treatment in cluster headache.
Cephalalgia. 2000 Jun;20(5):445-54. [Abstract]

Christiansen I, Iversen HK, Olesen J.
Headache characteristics during the development of tolerance to nitrates: pathophysiological implications.
Cephalalgia. 2000 Jun;20(5):437-44.
"Recent studies suggest that nitric oxide (NO) plays an important role in nitrate-induced headache and in spontaneous migraine attacks. Organic nitrates act as prodrugs for NO and headache is a predominant adverse effect of nitrates but often disappears during continuous treatment. Insight into tolerance to headache could lead to insight into vascular headache mechanisms in general. The specific aim of the present study was therefore to characterize the headache and accompanying symptoms during continuous nitrate administration until a state of tolerance to headache had developed. 5-isosorbide-mononitrate (5-ISMN) 30 mg three times daily was administered orally for 7 days in 11 healthy subjects in a double-blind, randomized placebo controlled cross-over design. Wash-out between periods was 14 days or more. Haemodynamic data from the present study were compared to the observed changes of headache over time. Headache during 5-ISMN was longer lasting and more severe compared to placebo (P<0.004). In 10 subjects the headache fulfilled the pain sub-criteria for migraine and in five subjects all diagnostic criteria for migraine without aura were fulfilled. Conversely, 20 min of intravenous infusion of glyceryl trinitrate caused a milder headache and no migraine. The present results therefore suggest that NO may elicit a migraine attack in many healthy subjects if a high enough dose is given for several hours. A close temporal association between the disappearance of headache and the attenuation of the 5-ISMN induced dilatation of the superficial temporal artery was observed. In contrast, tolerance in the middle cerebral artery already appeared after 24 h, which was earlier than the development of tolerance to headache. If vasodilatation is the cause of headache the results point to extracerebral arteries. However, cytotoxic and pain modulating central nervous system effects of NO, the time courses of which are unknown, may also play a role, involving both intra- and extracranial arteries." [Abstract]

Stepien A, Chalimoniuk M.
Level of nitric oxide-dependent cGMP in patients with migraine.
Cephalalgia. 1998 Nov;18(9):631-4.
"It is believed that nitric oxide (NO) plays a significant role in migraine attacks. This molecule is formed due to the conversion of L-arginine into L-citrulline. The target receptor for NO is ferrum in the heme group of cytoplasmic guanyl cyclase, the enzyme catalyzing cyclic guanosine monophosphate (cGMP) formation. To confirm this hypothesis, cGMP and nitrite level in the blood serum were measured in patients with migraine. The group under study included 37 subjects suffering from migraine with and without aura and 40 normal control subjects. The cGMP was measured during a migraine attack and 60 min following the administration of sumatriptan 6 mg subcutaneously. A statistically significant increase in cGMP level was observed in patients during a migraine attack compared to the controls. This level decreased after the administration of sumatriptan, but it was still higher than in the controls. No correlation was found between the increased cGMP level and pain intensification with clinical symptoms of migraine. The results suggest the participation of biochemical changes in migraine pathogenesis in the L-arginine-NO-cGMP pathway." [Abstract]

Gallai V, Floridi A, Mazzotta G, Codini M, Tognoloni M, Vulcano MR, Sartori M, Russo S, Alberti A, Michele F, Sarchielli P.
L-arginine/nitric oxide pathway activation in platelets of migraine patients with and without aura.
Acta Neurol Scand. 1996 Aug;94(2):151-60.
"Nitric oxide (NO) in platelets has been proposed as a promising tool for studying NO variations in migraine. In the present research the platelet response to collagen and the basal and collagen-induced production of NO and cGMP in platelet cytosol were assessed in migraine patients (25 with aura and 35 without aura) both interictally and ictally, and compared with the same parameters in 30 age-matched control subjects. A reduced responsiveness to collagen was found in migraine patients, particularly those with aura, and this was more marked during attacks (ANOVA interictal periods: p < 0.01, attacks: p < 0.02) The basal and collagen-stimulated production of NO and cGMP in the platelet cytosol was significantly higher in migraine patients with aura assessed in interictal periods than in control subjects, and this production was further increased during attacks (interictal period: NO ANOVA: p < 0.001, ictal period: p < 0.01; cGMP: interictal period p < 0.01, ictal period: p < 0.02). The increase in platelet NO and cGMP production was also evident, though to a lesser extent, in migraine patients without aura. The present research supports the hypothesis of an activation of the L-arginine/NO pathway in migraine patients, especially those with aura, and confirms the findings of a previous study of increased levels of L-arginine in platelets of migraine patients studied in headache free-periods, and decreased collagen aggregation in whole blood." [Abstract]

Shimomura T, Murakami F, Kotani K, Ikawa S, Kono S.
Platelet nitric oxide metabolites in migraine.
Cephalalgia. 1999 May;19(4):218-22.
"Nitric oxide (NO) is a candidate as a causative molecule in migraine. We determined nitrite, total nitrate/nitrite, and cyclic guanosine 3',5'-monophosphate (cGMP) concentrations in platelets from 30 migraine without aura (MwoA) patients and 17 migraine with aura (MwA) patients. All migraine patients were studied during their migraine attacks. The control group consisted of 28 healthy volunteers. Concentrations of platelet nitrite and total nitrate/nitrite were determined using simple and sensitive nitrate/nitrite fluorometric assay techniques. High concentrations of platelet nitrite and total nitrate/nitrite were found in patients with MwoA and MwA when compared with healthy controls. High concentrations of platelet cGMP were also found in patients with MwoA and MwA. The levels of platelet total nitrate/nitrite significantly decreased in headache-free periods after treatment with oral propranolol. These findings suggest that NO is produced in platelets during migraine attacks. It may also be related to the migrainous pain and the changes in cerebral blood flow experienced during migraine attacks. These data may provide new strategies for the treatment of migraine." [Abstract]

D'Amico D, Ferraris A, Leone M, Catania A, Carlin A, Grazzi L, Bussone G.
Increased plasma nitrites in migraine and cluster headache patients in interictal period: basal hyperactivity of L-arginine-NO pathway?
Cephalalgia. 2002 Feb;22(1):33-6.
"Nitrite concentrations in plasma were investigated in a population of migraine and cluster headache patients and a group of healthy non-headache controls. A hundred migraine patients and 69 cluster headache patients in the interictal period, and 112 controls, were studied. Significantly higher nitrite concentrations were found in migraine patients, with and without aura, and cluster headache patients, in remission and cluster phase, than in controls. These findings suggest that a basal dysfunction in the L-arginine-NO pathway may be involved in the peripheral mechanisms predisposing subjects with neurovascular headaches to individual attacks." [Abstract]

Shukla R, Barthwal MK, Srivastava N, Nag D, Seth PK, Srimal RC, Dikshit M.
Blood nitrite levels in patients with migraine during headache-free period.
Headache. 2001 May;41(5):475-81.
"OBJECTIVE: To investigate blood nitrite levels after migraine attacks and to assess whether or not the change in nitric oxide levels observed during acute migraine persist after the attacks. BACKGROUND: Involvement of nitric oxide has been suggested in the initiation of acute migraine. Recent studies have shown alteration in the platelet response and platelet nitrite levels during migraine attacks. METHODS: Patients with migraine with aura and patients without aura were included in the study. The study was conducted on 50 patients with migraine and 90 healthy controls. Blood from the patients was collected at least 7 +/- 0.8 days after the last attack of migraine. Nitrite levels in the polymorphonuclear leukocytes, platelets, and plasma were estimated. Platelet aggregation response in some of these patients was also studied. RESULTS: No significant change in the polymorphonuclear leukocyte, platelet, and plasma nitrite levels in patients with migraine compared to controls was observed. Patients with migraine with aura had significantly lower polymorphonuclear leukocyte nitrite levels compared to those without aura (P<.05). In addition, no significant difference in the adenosine diphosphate-induced platelet aggregation was observed in the migraineurs compared to the healthy controls. CONCLUSIONS: Results obtained indicate that the platelet aggregation response and the blood nitrite levels were not altered significantly after an attack in the patients with migraine." [Abstract]

Costa A, Ravaglia S, Sances G, Antonaci F, Pucci E, Nappi G.
Nitric oxide pathway and response to nitroglycerin in cluster headache patients: plasma nitrite and citrulline levels.
Cephalalgia. 2003 Jul;23(6):407-13.
"Nitric oxide (NO) may participate in the mechanisms underlying vascular headaches, such as migraine and cluster headache (CH), by triggering neurogenic inflammation and activation of fibres conveying nociceptive inputs to the trigeminal ganglion. Similarly to migraine, the administration of the NO donor glyceryltrinitrate (GTN) to CH patients is a known model of inducing spontaneous-like attacks. We carried out a GTN test (0.9 mg, sublingually) in 18 patients with episodic CH in active phase and 12 controls. The plasma levels of NO metabolite nitrites (NO2-), after conversion of nitrates to NO2-, were measured spectrophotometrically at baseline, at the maximum intensity of the induced response (or 45 min after GTN in controls), and 120 min after GTN administration. The basal plasma levels of L-citrulline were also assayed in patients and controls using high-performance liquid chromatography. Basal NO2- levels, similar in GTN-responsive patients and controls (48.3 +/- 10.6 and 44.6 +/- 9.5 micromol/l, respectively) were found to be increased significantly at pain peak in patients (76.1 +/- 10.2 micromol/l) and after 45 min in controls (78.2 +/- 9.6 micromol/l) (P < 0.01 vs. respective baseline values), but not after 120 min, without differences between groups. L-citrulline levels in basal conditions showed no differences between groups (patients 64.8 +/- 11.7, controls 67.3 +/- 10.8 micromol/l). These data do not support the presence of a basal hyperactivity of the L-arginine-NO pathway in CH patients. Increased NO production may be of importance in the mechanisms leading to CH attacks, but other factors are likely to render CH patients hyperresponsive to NO, and ultimately to cause the occurrence of pain and associated features." [Abstract]

Ciancarelli I, Tozzi-Ciancarelli MG, Di Massimo C, Marini C, Carolei A.
Urinary nitric oxide metabolites and lipid peroxidation by-products in migraine.
Cephalalgia. 2003 Feb;23(1):39-42.
"Enhanced endothelium nitric oxide (NO) and superoxide anion release may cause migraine through related cerebral blood flow changes. Thirty subjects suffering from migraine with and without aura and 20 healthy controls were investigated. Urine samples collected for 24 h during and after the migraine attack, and during the headache-free period, were assayed for urinary NO stable metabolites (NOx) and thiobarbituric acid reactive substances (TBARS). During the headache-free period urinary NOx and TBARS levels were higher in migraine sufferers than in controls (NOx 0.77 +/- 0.14 vs. 0.28 +/- 0.15 mmol/mmol creatinine, P < 0.05; TBARS 0.40 +/- 0.19 vs. 0.26 +/- 0.13 micro mol/mol creatinine, P < 0.05). Also, NOx excretion was higher during the headache-free period than during or after the migraine attack (P < 0.05). Urinary TBARS were increased during the attack with respect to the headache-free period (P < 0.05). No differences were observed in the same parameters between sufferers of migraine with and without aura. Urinary NOx and TBARS might be promising as markers of their systemic levels to evaluate the increased vulnerability to oxidative stress in migraine sufferers." [Abstract]

D'Andrea G, Cananzi AR, Perini F, Alecci M, Zamberlan F, Hasselmark L, Welch KM.
Decreased collagen-induced platelet aggregation and increased platelet arginine levels in migraine: a possible link with the NO pathway.
Cephalalgia. 1994 Oct;14(5):352-6.
"We studied whole blood platelet aggregation induced by collagen, platelet activating factor (PAF) and measured basal platelet L-arginine (L-arg) levels, as an indirect index of the nitric oxide (NO) pathway in migraine. Migraine, both with and without aura groups, showed a reduced aggregation to collagen, but not to PAF, compared with control subjects. Platelet L-arg levels were significantly increased in migraine with aura sufferers, whereas the plasma levels were in the same range in migraineurs and controls. Platelet hyperesponsiveness to collagen stimulation in migraine may be linked to an increased availability of the amino acid precursor and an abnormal NO synthesis." [Abstract]

Sarchielli P, Tognoloni M, Russo S, Vulcano MR, Feleppa M, Mala M, Sartori M, Gallai V.
Variations in the platelet arginine/nitric oxide pathway during the ovarian cycle in females affected by menstrual migraine.
Cephalalgia. 1996 Nov;16(7):468-75.
"Previous studies have reported the existence of an arginine/nitric oxide (NO) pathway and the involvement of a Ca2+, NADPH-dependent nitric oxide synthase enzyme (NOS) in the generation of NO in human platelets. In the present research, we determined the rate of production of NO and cGMP in the cytosol of platelets stimulated by collagen in 20 females with menstrual migraine (MM), (age range 24-40 years), assessed in the follicular and luteal phases, interictally and ictally in the latter period. The same patients were also assessed at mid-cycle. At the same time, the variations in the collagen response of platelets were evaluated. Moreover, these parameters were determined in the same periods in 20 age-matched control females and in 20 females affected by non-menstrually related migraine (nMM). The collagen-stimulated production of NO in the cytosol of the platelet cytosol was significantly higher in migraine patients with MM than in the control subjects. In MM patients, the increase was greater in the luteal phase of the cycle than during the follicular phase (p < 0.005). A rise in NO production in platelets was also present, although to a lesser extent, in females affected by nMM compared to the healthy females, but this rise was most evident at ovulation (p < 0.001). A slight but significant increase was also observed at mid-cycle in control women, but this increase did not reach the values determined in the migraine groups (p < 0.02). NO production in platelets stimulated by collagen was significantly increased during attacks with respect to the interictal period in both patient groups. Similar variations were observed in the production of cGMP in MM and nMM patients. The increase in NO production was accompanied by a decrease in platelet aggregation in the migraine groups compared with the control group; this decrease was most evident at mid-cycle in nMM patients and in the luteal phase in MM patients. These data suggest an activation of the L-arginine/ NO pathway in MM and nMM patients which could explain the modifications in the platelet response to collagen evidenced in migraine-free periods and during attacks. The activation of this pathway is more accentuated in the luteal phase in MM patients, and this could be the cause of the increased susceptibility to migraine attacks in perimenstrual and menstrual periods in these patients." [Abstract]

Pardutz A, Multon S, Malgrange B, Parducz A, Vecsei L, Schoenen J.
Effect of systemic nitroglycerin on CGRP and 5-HT afferents to rat caudal spinal trigeminal nucleus and its modulation by estrogen.
Eur J Neurosci. 2002 Jun;15(11):1803-9.
"Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraine patients a delayed attack of unknown mechanism. After puberty migraine is more prevalent in women. Attacks can be triggered by abrupt falls in plasma estrogen levels, which accounts in part for sexual dimorphism, but lacks an established neurobiological explanation. We studied the effect of nitroglycerin on the innervated area of calcitonin gene-related peptide (CGRP) and serotonin-immunoreactive afferents to the superficial laminae of the spinal portion of trigeminal nucleus caudalis, and its modulation by estrogen. In male rats, nitroglycerin produced after 4 h a significant decrease of the area innervated by CGRP-immunoreactive afferents and an increase of that covered by serotonin-immunoreactive fibres. These effects were not observed in the superficial laminae of thoracic dorsal horns. The effect of nitroglycerin was similar in ovariectomized females. In estradiol-treated ovariectomized females the area in the spinal portion of trigeminal nucleus caudalis laminae I-II covered by CGRP-immunoreactive fibres was lower and that of serotonin-immunoreactive fibres was higher than in males and for both transmitters not significantly changed after nitroglycerin. The bouton size of CGRP profiles was smaller in estradiol-treated ovariectomized females, whereas after nitroglycerin it decreased significantly but only in males and ovariectomized females. Nitroglycerin, i.e. nitric oxide, is thus able to differentially influence afferent fibres in the superficial laminae of rat spinal trigeminal nucleus caudalis. Estradiol modulates the basal expression of these transmitters and blocks the nitroglycerin effect. These data may contribute to understanding the mechanisms by which estrogens influence migraine severity and the triggering of attacks by nitric oxide." [Abstract]

Geary GG, Krause DN, Duckles SP.
Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries.
Am J Physiol. 1998 Jul;275(1 Pt 2):H292-300.
"Gender differences in the incidence of stroke and migraine appear to be related to circulating levels of estrogen; however, the underlying mechanisms are not yet understood. Using resistance-sized arteries pressurized in vitro, we have found that myogenic tone of rat cerebral arteries differs between males and females. This difference appears to result from estrogen enhancement of endothelial nitric oxide (NO) production. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (Ovx), or ovariectomized with estrogen replacement (Ovx + Est). The maximal passive diameters (0 Ca2+ + 1 mM EDTA) of arteries from all four groups were identical. In response to a series of 10-mmHg step increases in transmural pressure (20-80 mmHg), myogenic tone was greater and vascular distensibility less in arteries from males and Ovx females compared with arteries from either untreated or Ovx + Est females. In the presence of NG-nitro-L-arginine methyl ester (L-NAME; 1 microM), an NO synthase inhibitor, myogenic tone was increased in all arteries, but the differences among arteries from the various groups were abolished. Addition of L-arginine (1 mM) in the presence of L-NAME restored the differences in myogenic tone, suggesting that estrogen works through an NO-dependent mechanism in cerebral arteries. To determine the target of NO-dependent modulation of myogenic tone, we used tetraethylammonium (TEA; 1 mM) to inhibit large-conductance, calcium-activated K+ (BKCa) channels. In the presence of TEA, the myogenic tone of arteries from all groups increased significantly; however, myogenic tone in arteries from males and Ovx females remained significantly greater than in arteries from either untreated or Ovx + Est females. This suggests that activity of BKCa channels influences myogenic tone but does not directly mediate the effects of estrogen. Estrogen appears to alter myogenic tone by increasing cerebrovascular NO production and/or action." [Abstract]

Sarchielli P, Alberti A, Floridi A, Gallai V.
L-Arginine/nitric oxide pathway in chronic tension-type headache: relation with serotonin content and secretion and glutamate content.
J Neurol Sci. 2002 Jun 15;198(1-2):9-15.
"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 Ca(2+) levels. In the present work, we assessed the variations in L-arginine/NO pathway activity and platelet cyclic guanosine 3',5'-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 microg/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 Ca(2+) 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." [Abstract]

Thomsen LL.
Investigations into the role of nitric oxide and the large intracranial arteries in migraine headache.
Cephalalgia. 1997 Dec;17(8):873-95.
"Previous studies suggest that nitric oxide (NO) is involved in headaches induced by i.v. infusion of the vasodilator and NO donor glyceryl trinitrate (GTN) in healthy subjects. Extending these studies to sufferers of migraine without aura, it was found that migraineurs experienced a stronger headache than non-migraineurs. In addition, most migraineurs experienced a delayed migraine attack at variable times (mean 5.5 h) after GTN provocation. This biphasic headache response in migraineurs may be linked to hypersensitivity in the NO-cGMP pathway. Thus, compared to controls, migraineurs were found to be more sensitive to GTN-induced intracranial arterial dilatation, which is known to be mediated via liberation of NO and subsequent synthesis of cGMP Furthermore, histamine infusions in migraineurs induced headache responses and intracranial arterial responses resembling those induced by GTN in migraineurs. Histamine is known to liberate NO from the endothelium via stimulation of the H1 receptor, which is present in the large intracranial arteries in man. Because both immediate histamine-induced headache and intracranial arterial dilatation and delayed histamine-induced migraine are blocked by H1-receptor blockade, a likely common pathway for GTN and histamine-induced headaches/migraines and intracranial arterial responses may be via activation of the NO-cGMP pathway. The delay in the development of these experimental migraines may reflect activation of multiple physiological processes. The intracranial arteries of migraineurs were found supersensitive to the vasodilating effect of GTN (exogenous NO). This relates to clinical findings suggesting dilatation of the large intracranial arteries on the headache side during spontaneous migraine attacks. The function of arterial regulatory mechanisms involving NO in migraine was therefore studied. In peripheral arteries, no endothelial dysfunction of NO was found and cardiovascular and intracranial arterial sympathetic function was normal. A mild parasympathetic dysfunction may be involved and may, via denervation supersensitivity, be responsible for the observed supersensitivity to NO. Another possibility is that NO initiates a perivascular neurogenic inflammation with liberation of vasoactive peptides. NO also mediates a variety of other physiological phenomena. One of these, the pain-modulating effect observed in animals, was evaluated in a human study using GTN infusion and measurements of pain thresholds. No definite effects of GTN were demonstrated. The precise mechanisms involved in NO-triggered migraines and which part of the NO-activated cascade that is involved remain to be determined. The possibilities for pharmacological stimulation and/or inhibition of several steps of the NO-activated cascade increase rapidly and soon may be available for human studies." [Abstract]

Christiansen I, Daugaard D, Lykke Thomsen L, Olesen J.
Glyceryl trinitrate induced headache in migraineurs - relation to attack frequency.
Eur J Neurol. 2000 Jul;7(4):405-11.
"Glyceryl trinitrate, a prodrug of nitric oxide, induces a mild to moderate headache in healthy subjects, whilst migraineurs develop a more severe headache, resembling spontaneous migraine attacks. In order to investigate whether this increased nitric oxide sensitivity depends upon the frequency of spontaneously occurring migraine attacks, intravenous infusion of glyceryl trinitrate (0.5 microg/kg/min) was given to 15 migraine patients with rare attacks (</=4 attacks/year) of migraine without aura. Fourteen age-matched migraine patients with frequent attacks of migraine without aura (>/=12 attacks/year) and 14 healthy subjects served as controls. No significant difference between the migraine groups for any of several parameters was detected, although the trend was always towards more headaches in frequent migraineurs. Both migraineurs with frequent and rare attacks experienced a headache that was significantly more severe, longer lasting, and fulfilled the diagnostic criteria for migraine without aura more often, compared to the healthy subjects (P = 0.0001). Conclusively, supersensitivity to glyceryl trinitrate in migraineurs seems to be related to a basic - probably genetically determined - pathophysiological mechanism involving nitric oxide, and not to the environmental influences, which to a large extent determine the expression of migraine." [Abstract]

M. Ashina, L. Bendtsen, R. Jensen, and J. Olesen
Nitric oxide-induced headache in patients with chronic tension-type headache
Brain 123: 1830-1837. 2000.
"An experimental model of headache offers unique possibilities to study the mechanisms responsible for head pain. Using the glyceryl trinitrate [GTN; nitric oxide (NO) donor] model of experimental headache, we studied the intensity, quality and time profile of headache after infusion of GTN in 16 patients with chronic tension-type headache and in 16 healthy controls. Subjects were randomized to receive intravenous infusion of GTN (0.5 microg/kg per minute for 20 min) or placebo on two headache-free days separated by at least 1 week. Headache intensity was measured on a 10-point verbal rating scale during 2 h of observation and for the next 10 h after discharge from hospital. The primary endpoints were the difference between the area under the curve (AUC-intensities x duration) for headache recorded on the day of GTN treatment and on the day of placebo treatment in patients, and in patients and controls on the days of GTN treatment. In patients, the AUC on a GTN day [2221 (1572-3704); median with quartiles in parentheses], was significantly greater than on a placebo day [730 (60-1678), P: = 0. 008]. On the GTN day, the AUC in patients [2221 (1572-3704)] was significantly higher than in controls [43 (0-972), P: = 0.0001]. In patients, peak pain intensity occurred 8 h after infusion of GTN, whereas in controls it occurred 20 min after the start of infusion. The present study demonstrates that an NO-induced biphasic response with an immediate and a delayed headache is common to chronic tension-type headache and migraine. Furthermore, the NO-induced delayed headache has the characteristics of the primary headache disorder. This suggests that NO contributes to the mechanisms of several types of primary headaches and that NO-related central sensitization may be an important common denominator in the pain mechanisms of primary headaches." [Full Text]

Christiansen I, Thomsen LL, Daugaard D, Ulrich V, Olesen J.
Glyceryl trinitrate induces attacks of migraine without aura in sufferers of migraine with aura.
Cephalalgia. 1999 Sep;19(7):660-7; discussion 626.
"Migraine with aura and migraine without aura have the same pain phase, thus indicating that migraine with aura and migraine without aura share a common pathway of nociception. In recent years, increasing evidence has suggested that the messenger molecule nitric oxide (NO) is involved in pain mechanisms of migraine without aura. In order to clarify whether the same is true for migraine with aura, in the present study we examined the headache response to intravenous infusion of glyceryl trinitrate (GTN) (0.5 microg/kg/min for 20 min) in 12 sufferers of migraine with aura. The specific aim was to elucidate whether an aura and/or an attack of migraine without aura could be induced. Fourteen healthy subjects served as controls. Aura symptoms were not elicited in any subject. Headache was more severe in migraineurs than in the controls during and immediately after GTN infusion (p=0.037) as well as during the following 11 h (p = 0.008). In the controls, the GTN-induced headache gradually disappeared, whereas in migraineurs peak headache intensity occurred at a mean time of 240 min post-infusion. At this time the induced headache in 6 of 12 migraineurs fulfilled the diagnostic criteria for migraine without aura of the International Headache Society. The results therefore suggest that NO is involved in the pain mechanisms of migraine with aura. Since cortical spreading depression has been shown to liberate NO in animals, this finding may help our understanding of the coupling between cortical spreading depression and headache in migraine with aura." [Abstract]

Ashina M, Bendtsen L, Jensen R, Sakai F, Olesen J.
Possible mechanisms of glyceryl-trinitrate-induced immediate headache in patients with chronic tension-type headache.
Cephalalgia. 2000 Dec;20(10):919-24.
"Nitric oxide (NO) plays an important role in the pathophysiology of primary headaches including chronic tension-type headache (CTTH). Thus, a NO synthase inhibitor reduces headache and muscle hardness while the NO donor glyceryl trinitrate (GTN) causes more headache in patients than in healthy controls. Sensitization of myofascial pain pathways is important in CTTH, and the aim of the present study was to investigate if such mechanisms may also explain GTN-induced immediate headache in patients with CTTH. In a randomized, double-blind, crossover study 16 patients with CTTH and 16 healthy subjects received intravenous infusion of GTN (0.5 microg/kg per min for 20 min) or placebo on two headache-free days separated by at least 1 week. Muscle hardness, myofascial tenderness, mechanical and heat pain thresholds were measured at baseline and at 60 min and 120 min after start of infusion. In patients, GTN infusion resulted in a biphasic response with immediate headache and more pronounced delayed headache. A similar but less pronounced response was seen in controls. There was no difference between GTN and placebo regarding muscle hardness, myofascial tenderness or pressure and heat pain thresholds in either patients or controls (P>0.05). The unchanged sensitivity of pericranial myofascial pain pathways indicates that peripheral and central sensitization is not involved in the mechanisms of GTN-induced immediate headache." [Abstract]

Bellantonio P, Micieli G, Buzzi MG, Marcheselli S, Castellano AE, Rossi F, Nappi G.
Haemodynamic correlates of early and delayed responses to sublingual administration of isosorbide dinitrate in migraine patients: a transcranial Doppler study.
Cephalalgia. 1997 May;17(3):183-7.
"In normal subjects or migraine patients, nitrates induce a non-specific early headache caused by vasodilation of intracranial arteries. In migraineurs a delayed headache response to nitrates may have a typical clinical profile of a spontaneous migraine attack. The cerebral vasomotor changes of this delayed response require further study. Isosorbide dinitrate (IDN), an exogenous nitric oxide (NO) donor, was given at a dose of 5 mg sublingually and a bilateral transcranial Doppler device was used to monitor bilateral mean velocity (Vm) changes at the middle cerebral artery (MCA) after IDN administration and until delayed headache occurred. Spontaneous migraine-like headache occurred only in migraine patients during the delayed phase after IDN and was accompanied by a prolonged arterial vasodilation compared to normal subjects. This vasomotor response was more evident on the customary side of the head pain of a spontaneous migraine attack. Our findings suggest a particular vasomotor response to nitrates in migraine patients. This response is associated with the nitrate-induced headache and it is not evident in healthy pain-free controls during the delayed phase after administration of an NO donor. Owing to the short half-life of NO, the neurotransmitter released by IDN, and because of the late onset of headache, we believe the mechanism is unlikely to be vascular in origin, but may have a neurogenic component." [Abstract]

Thomsen LL, Iversen HK, Brinck TA, Olesen J.
Arterial supersensitivity to nitric oxide (nitroglycerin) in migraine sufferers.
Cephalalgia. 1993 Dec;13(6):395-9; discussion 376.
"The sensitivity to nitroglycerin-induced dilatation of large intracranial arteries was studied in 17 patients with migraine without aura, 17 age and sex-matched healthy subjects and 9 patients with episodic tension-type headache. Nitroglycerin in the doses of 0.015, 0.03, 0.25 microgram/kg/min was successively infused for 15 min per dose. Blood velocity (Vmean) in the middle cerebral artery (MCA) was recorded with transcranial Doppler before and at the end of every infusion period, and 30 and 60 min after end of the last infusion. In all three groups Vmean decreased with increasing doses (p < 0.001). The response was more pronounced in migraine patients at the two higher doses (p < 0.05). Since nitroglycerin acts as an exogenous source of nitric oxide (NO), these data support that NO supersensitivity may be an important molecular mechanism of migraine pain." [Abstract]

Olesen J, Iversen HK, Thomsen LL.
Nitric oxide supersensitivity: a possible molecular mechanism of migraine pain.
Neuroreport. 1993 Aug;4(8):1027-30.
"Nitroglycerin, which may be regarded as a prodrug for nitric oxide, induces a mild to moderate headache in healthy subjects. In order to study whether migraine patients are more sensitive to nitric oxide than non-migrainous subjects, four different doses of intravenous nitroglycerin were given in a double blind design to 17 migraine patients, 17 age and sex matched healthy controls and 9 subjects with tension-type headache. The nitroglycerin-induced headache was significantly more severe in migraine sufferers, lasted longer and fulfilled diagnostic criteria for migraine more often. We have previously shown a similar supersensitivity to histamine which in human cerebral arteries activates endothelial H1 receptors and causes endothelial production of nitric oxide. Migraine patients are thus supersensitive to exogenous nitric oxide from nitroglycerin as well as to endothelially produced nitric oxide. It is suggested that nitric oxide may be partially or completely responsible for migraine pain." [Abstract]

Martelletti P, D'Alo S, Stirparo G, Rinaldi C, Cifone MG, Giacovazzo M.
Modulation of nitric oxide synthase by nitric oxide donor compounds in migraine.
Int J Clin Lab Res. 1998;28(2):135-9.
"A controlled study was performed to assess the involvement of the nitric oxide pathway in migraine pathophysiology. Thirteen patients with migraine without aura and seven clinically healthy subjects (C) were selected. All of the migraine patients were studied both before, during an asymptomatic phase (t0), and 1 h after the administration of 5 mg isosorbide dinitrate, a nitric oxide donor able to induce an experimental migraine attack (t1). The nitric oxide levels were analyzed as nitrite accumulation in serum samples, in peripheral blood mononuclear cell extracts, and culture supernatants. Basal nitrite levels in serum samples and peripheral blood mononuclear cell culture supernatants or migraine patients and healthy subjects indicated that migraine patients possess an activated nitric oxide synthesis pathway (t0 vs. CF = 8.16, P < 0.01 and F = 16.2, P < 0.01, respectively). As expected, in the migraine patients treated with the nitric oxide donor, a marked increase of nitrite levels was observed in sera (t1 vs. t0 P < 0.05, t = 3.05). In contrast, during the nitric oxide donor-induced migraine attacks a statistically significant decrease of nitrite levels in peripheral blood mononuclear cell culture supernatants was observed (t1 vs. t0 P < 0.01, t = -4.03), whereas a significant increase of nitrite in total cell extracts was detected (t1 vs. t0 P < 0.001, t = -6.89). These preliminary data suggest that nitric oxide could be involved in the neurovascular modifications leading to a migraine attack." [Abstract]

Kruuse C, Thomsen LL, Birk S, Olesen J.
Migraine can be induced by sildenafil without changes in middle cerebral artery diameter.
Brain. 2003 Jan;126(Pt 1):241-7.
"Migraine is considered a neurovascular disease involving dilatation of cerebral arteries. Nitric oxide (NO) donors induce dilatation of cerebral and extracranial arteries and migraine, but NO has several mechanisms of action in addition to its cyclic guanosine monophosphate (cGMP)-mediated vasodilatation. We examined whether sildenafil (Viagra), a selective inhibitor of cGMP-hydrolysing phosphodiesterase 5 (PDE5), which acts exclusively by increasing cGMP, can induce migraine and dilatation of cerebral arteries. We included 12 patients with migraine without aura in this double-blind, placebo-controlled crossover study, in which placebo or sildenafil 100 mg was administered orally on two separate days. Blood flow velocity in the middle cerebral artery (V(mca)) was recorded by transcranial Doppler ultrasonography and regional cerebral blood flow in the territory of the middle cerebral artery (rCBF(mca)) was measured using SPECT (single photon emission computed tomography) and xenon 133 inhalation. Radial and temporal artery diameters were studied using high-frequency ultrasonography. Headache response, tenderness of pericranial muscles, blood pressure and heart rate were measured repeatedly. We found that migraine attack was induced by sildenafil in 10 of 12 migraine patients and by placebo in two of 12 patients (P = 0.01). V(mca) (P = 0.1) and rCBF(mca) (P = 0.93) remained unchanged after sildenafil. Temporal (P = 0.47) and radial (P = 0.87) artery diameter and pericranial tenderness (P = 0.16) were unaffected by sildenafil. Systolic and diastolic blood pressures were unchanged but heart rate increased from a mean of 62 +/- 2 to 74 +/- 3 beats/min (P = 0.01) after sildenafil. Our results demonstrate that migraine may be induced via a cGMP-dependent mechanism, and we show for the first time that this occurs without initial dilatation of the middle cerebral artery. We propose that triggering mechanisms may reside within the perivascular sensory nerve terminals or the brainstem. However, other sites of action may also be possible and future studies are needed to elucidate this. In the clinical use of sildenafil, patients who have migraine should be informed about the risk of migraine attacks." [Abstract]

Thomsen LL, Brennum J, Iversen HK, Olesen J.
Effect of a nitric oxide donor (glyceryl trinitrate) on nociceptive thresholds in man.
Cephalalgia. 1996 May;16(3):169-74.
"Several animal studies suggest that nitric oxide (NO) plays a role in central and peripheral modulation of nociception. Glyceryl trinitrate (GTN) exerts its physiological actions via donation of NO. The purpose of the present study was to examine the effect of this NO donor on nociceptive thresholds in man. On two different study days separated by at least a week 12 healthy subjects received a staircase infusion of GTN (0.015, 0.25, 1.0, 2.0 micrograms/kg/min, 20 min each dose) or placebo in a randomized double-blind crossover design. Before the infusion and after 15 min of infusion on each dose, pressure pain detection and tolerance thresholds were determined by pressure algometry (Somomedic AB, Sweden) in three different anatomic regions (finger, a temporal region with interposed myofascial tissue and a temporal region without interposed myofascial tissue). Relative to placebo, the three higher GTN doses induced a decrease in both detection and tolerance thresholds in the temporal region with interposed myofascial tissue (p = 0.003 detection and p = 0.002 tolerance thresholds, Friedman). No such changes were observed in the other two stimulated regions. These results could reflect central facilitation of nociception by NO. However, we regard convergence of nociceptive input from pericranial myofascial tissue and from cephalic blood vessels dilated by NO as a more likely explanation of our findings." [Abstract]

Sarchielli P, Alberti A, Codini M, Floridi A, Gallai V.
Nitric oxide metabolites, prostaglandins and trigeminal vasoactive peptides in internal jugular vein blood during spontaneous migraine attacks.
Cephalalgia. 2000 Dec;20(10):907-18.
"Despite evidence emerging from the experimental model of nitroglycerin-induced headache, the endogenous increase in nitric oxide (NO) production during migraine attacks is only speculative. It has been hypothesized that there is a close relationship between activation of the L-arginine/NO pathway and production of certain vasoactive and algogenic prostaglandins during spontaneous migraine attacks, but this suggestion also needs to be confirmed. In the present study the levels of nitrites, the stable metabolites of NO, were determined with high performance liquid chromatography (HPLC) in the internal jugular venous blood of five patients affected by migraine without aura examined ictally. These samples were taken within 30 min, 1, 2, and 4 h from the onset of the attack and at the end of the ictal period. At the same time, the plasma levels of calcitonin gene-related peptide (CGRP), neurokinin A (NKA), prostaglandin E2 (PGE2) and 6 keto PGF1alpha, the stable product of PGI2, were assessed with radioimmunoassay (RIA) kits in the same samples. The levels of the intracellular messengers, cGMP and cAMP, were also measured with the RIA method. Nitrite, cGMP, CGRP and NKA levels reached their highest values at the first hour, then they tended to decrease progressively and returned, after the end of attacks, to values similar or below those detected at the time of catheter insertion (ANOVA, statistical significance: P<0.001; P<<0.002; P<0.002; P<0.003, respectively). PGE2 and 6 keto PGF1alpha, as well as cAMP levels also significantly increased at the first hour but reached a peak at the 2nd hour and remained in the same range until the 4th and 6th hours. Then their values tended to decrease after the end of attacks, becoming lower than those measured immediately after catheter positioning for internal jugular venous blood drawing (ANOVA: P<0.002, P<0.004, P<0.001, respectively). Our results support early activation of the L-arginine/NO pathway which accompanies the release of vasoactive peptides from trigeminal endings and a late rise in the synthesis of prostanoids with algogenic and vasoactive properties which may intervene in maintaining the headache phase." [Abstract]

Stirparo G, Zicari A, Favilla M, Lipari M, Martelletti P.
Linked activation of nitric oxide synthase and cyclooxygenase in peripheral monocytes of asymptomatic migraine without aura patients.
Cephalalgia. 2000 Mar;20(2):100-6.
"Many reports indicate that nitric oxide (NO) could be involved in migraine without aura (MWA), an extremely diffuse clinical event. Since monocyte may be a relevant source of NO, we analysed monocyte activation in MWA patients, in a period in which they were free of symptoms. NO basal production by MWA peripheral monocytes was significantly higher than in healthy subjects (91.25+/-8.6 microM/10(6) cells vs. 22.6+/-3.2 microM/106 cells). Interestingly, even the release of prostaglandin E2 (PGE2), was higher in MWA patients than in healthy subjects (3137+/-320 pg/10(6) cells vs. 1531+/-220 pg/10(6) cells). The incubation of monocytes from healthy subjects and MWA patients with N-nitro-L-arginine methyl ester caused a marked decrease of both NO and PGE2 release. We hypothesise that NOS and cyclooxygenase pathways in monocytes are linked and are, in MWA patients, up-regulated, even in a symptoms-free period. NO and PGE2 hyperproduction could therefore be involved in the neurovascular modifications leading to migraine attacks." [Abstract]

Castellano AE, Micieli G, Bellantonio P, Buzzi MG, Marcheselli S, Pompeo F, Rossi F, Nappi G.
Indomethacin increases the effect of isosorbide dinitrate on cerebral hemodynamic in migraine patients: pathogenetic and therapeutic implications.
Cephalalgia. 1998 Nov;18(9):622-30.
"Intracerebral vascular reactivity induced by the nitric oxide (NO) donor isosorbide dinitrate (IDN, 5 mg sublingually) is more major and longer-lasting in migraine patients who develop delayed headache in response to the drug. The headache is purportedly due to neuronally-mediated vascular mechanisms. Indomethacin inhibits prostaglandin synthesis, which is involved in NO generation. Indomethacin also decreases cerebral blood flow by constricting precapillary resistance vessels. In the present study, the hemodynamic effects of indomethacin were evaluated in migraine patients and healthy controls by means of transcranial Doppler monitoring. Indomethacin caused a significant decrease in mean flow velocity in the middle cerebral artery. This was an additional effect to the mean velocity decrease induced by IDN. The interactions between the two drugs suggest that their effects on cerebral hemodynamics (and pain) may be of relevance both in understanding the role of NO in migraine pathogenesis and in evaluating symptomatic treatments for migraine attacks." [Abstract]

Reuter U, Bolay H, Jansen-Olesen I, Chiarugi A, Sanchez del Rio M, Letourneau R, Theoharides TC, Waeber C, Moskowitz MA.
Delayed inflammation in rat meninges: implications for migraine pathophysiology.
Brain. 2001 Dec;124(Pt 12):2490-502.
"Nitric oxide (NO) has been implicated in migraine pathogenesis based on the delayed development of typical migraine headache 4-6 h after infusing the NO donor nitroglycerin [glyceryl trinitrate (GTN)] to migraineurs. Furthermore, inhibiting the synthesis of NO by treatment with a NO synthase (NOS) inhibitor attenuates spontaneous migraine headaches in 67% of subjects. Because NO has been linked to inflammation and cytokine expression, we investigated the delayed consequences of brief GTN infusion (30 min) on the development of meningeal inflammation in a rat model using doses relevant to the human model. We found dose-dependent Type II NOS [inducible NOS (iNOS)] mRNA upregulation in dura mater beginning at 2 h and an increase in the corresponding protein expression at 4, 6 and 10 h after infusion. Type II NOS immunoreactivity was expressed chiefly within resident meningeal macrophages. Consistent with development of a delayed inflammatory response, we detected induction of interleukin 1beta in dura mater at 2 and 6 h and increased interleukin 6 in dural macrophages and in rat cerebrospinal fluid at 6 h after GTN infusion. Myeloperoxidase-positive cells were rarely found. Leakage of plasma proteins from dural blood vessels was first detected 4 h after GTN infusion, and this was suppressed by administering a specific Type II NOS inhibitor [L-N(6)-(1-iminoethyl)-lysine (L-NIL)]. In addition to cytokine induction, macrophage iNOS upregulation and oedema formation after GTN infusion, dural mast cells exhibited granular changes consistent with secretion at 4 and 6 h. Because iNOS was expressed in dural macrophages following topical GTN, and in the spleen after intravenous injection, the data suggest that the inflammatory response is mediated by direct actions on the dura and does not develop secondary to events within the brain. Our findings point to the importance of new gene expression and cytokine expression as fundamental to the delayed response following GTN infusion, and support the hypothesis that a similar response develops in human meninges after GTN challenge." [Abstract]

Martelletti P, Stirparo G, Morrone S, Rinaldi C, Giacovazzo M.
Inhibition of intercellular adhesion molecule-1 (ICAM-1), soluble ICAM-1 and interleukin-4 by nitric oxide expression in migraine patients.
J Mol Med. 1997 Jun;75(6):448-53.
"The mechanisms of the postulated "sterile" inflammation in migraine were studied utilizing flow cytometry (intercellular adhesion molecule 1, ICAM-1; interleukin-1 receptor, IL-1R) and enzyme-linked immunosorbent assay (soluble intercellular adhesion molecule 1, sICAM-1; interleukin-4, IL-4). Twenty patients suffering from migraine without aura, 20 healthy subjects, and 10 patients suffering from episodic tension headache were selected. All of the migraine patients were studied during a migraine crisis experimentally induced by the administration of isosorbide dinitrate (a nitric oxide donor), and 10 out the 20 were also studied during a spontaneous migraine attack. A sharp decrease in the expression of ICAM-1 (F=5.09, p<0.001 and F=2.46, p<0.05, respectively), sICAM-1 1 (F=6.21, p<0.0001 and F=3.99, p<0.007, respectively) and serum IL-4 (F=6.23, p<0.001 and F=3.64, p<0.01, respectively) were observed in experimentally induced and spontaneous migraine attacks. There was no change with respect to IL-IR 1 receptor expression values. The two control groups, tested with the same experimental procedure, showed no changes in ICAM-1 and IL-1R or in in sICAM-1 and IL-4. Our data suggest that migraine patients are more sensitive to exogenous NO than controls. In addition, our results indicate that experimental migraine crisis, induced by an NO donor, is mediated by the inhibition of IL-4 and subsequently of ICAM-1. It is likely that the described ICAM-1 downregulation inhibits during a migraine attack the critical step of transendothelial migration into the cerebral tissues of activated leukocytes, as proposed in the "sterile inflammation" hypothesis." [Abstract]

Srikiatkhachorn A, Anuntasethakul T, Maneesri S, Phansuwan-Pujito P, Patumraj S, Kasantikul V.
Hyposerotonin-induced nitric oxide supersensitivity in the cerebral microcirculation.
Headache. 2000 Apr;40(4):267-75.
"OBJECTIVE: To investigate the relationship between hyposerotonin and cranial microvascular responses to nitric oxide (NO). BACKGROUND: Although the mechanism underlying NO supersensitivity in migraine is still unclear, an alteration of the serotonin system is a possible explanation. METHODS: Wistar rats were divided into control and hyposerotonin groups. Serotonin was depleted by intraperitoneal injection with 300 mg/kg of para-chlorophenylalanine (PCPA), a tryptophan hydroxylase inhibitor. Three days after PCPA pretreatment, the animals were prepared for assessment of their NO-induced vasomotor response using glyceryl trinitrate (GTN: 8 to 10 mg/kg, intravenously) as an NO donor. Pial circulation was visualized by the intravital fluorescein videomicroscopic technique. Images of vessels at 0, 5, 15, 30, and 60 minutes post GTN infusion were digitized and measured. At the end of monitoring, the rat brains were removed for ultrastructural study of the brain microvessels. RESULTS: Infusion of GTN produced dose-dependent pial arteriolar dilatation. This vasodilator effect was significantly increased in the PCPA-treated groups, especially at 30 and 60 minutes. The percentage change from baseline diameter at 30 minutes after the 8-mg/kg GTN infusion was 42.6 +/- 3.1 for the hyposerotonin group and 16.8 +/- 2.9 for the control group (P<.001). Electron microscopic study revealed that exposure to the NO donor produced considerable changes in cerebral microvessels, characterized by focal ballooning of endothelial cells, increased microvillous formation, and increased endothelial pinocytosis. These anatomical changes were significantly more prominent in the hyposerotonin group. CONCLUSIONS: A hyposerotoninergic condition can facilitate the NO-induced physiological and pathological responses in meningeal and cerebral microvessels and, therefore, is a possible explanation for the supersensitivity to NO observed in patients with migraine." [Abstract]

Sarchielli P, Alberti A, Russo S, Codini M, Panico R, Floridi A, Gallai V.
Nitric oxide pathway, Ca2+, and serotonin content in platelets from patients suffering from chronic daily headache.
Cephalalgia. 1999 Nov;19(9):810-6.
"An alteration in serotonin concentration has been found in patients with chronic headache caused by abuse of analgesic substances as well as an up-regulation of 5HT2 platelet receptors, which has been correlated with chronicization of the headache. In a previous study we demonstrated an increase in L-arginine/nitric oxide (NO) pathway activity in platelets from patients affected by migraine with or without aura, particularly during attacks. In the present research we assessed the variations in platelet L-arginine/NO pathway and cyclic guanosine monophosphate (cGMP) levels in 32 patients affected by chronic daily headache (CDH) (8 M, 24 F, age range 34-50 years) both during and between attacks. In these same patients, the platelet aggregation to different collagen concentrations (0.3, 1, 3 micrograms/ml) was determined as well as the intracellular platelet calcium concentration using fluorescence polarization spectrometry. These parameters were compared with those of an age- and sex-matched control group (n = 25; n = 10, n = 15, age range 35-51 years). A reduction found in platelet aggregation response to each collagen concentration used (p < 0.001) was coupled with an increased NO and cGMP production (NO: p < 0.0001; cGMP: p < 0.001). This was accompanied by a significant increase in intracytosolic Ca2+ (p < 0.0001) concentration and a reduced platelet serotonin content compared to those in control individuals (p < 0.0002). Changes in the above platelet parameters were accentuated more in patients with analgesic abuse than in CDH patients with no drug abuse. These findings suggest the occurrence of an activation of cGMP-Ca2+ mediated events in CDH patients with analgesic abuse. This physiologic compensatory mechanism, which intervenes in overcoming the increase in cytosolic Ca2+ levels, is not as efficient at limiting serotonin depletion by platelet dense bodies. A similar depletion in the central serotoninergic pathway can be assumed in the same patients." [Abstract]

Tassorelli C, Blandini F, Costa A, Preza E, Nappi G.
Nitroglycerin-induced activation of monoaminergic transmission in the rat.
Cephalalgia. 2002 Apr;22(3):226-32.
"When administered to migraine patients, nitroglycerin induces a spontaneous-like migraine attack, with a latency of several hours. Nitroglycerin acts directly and/or indirectly on the central nervous system, through the release of nitric oxide (NO). Systemic administration of the drug to the rat causes neuronal activation in selected subcortical areas, particularly in monoaminergic nuclei of the brainstem. In this study, we sought to investigate whether this activation correlates with changes in monoaminergic neurotransmission. For this purpose, we evaluated the tissue levels of catecholamines and serotonin in the hypothalamus, mesencephalon, pons and medulla of rats treated with systemic nitroglycerin or vehicle, at different time points (1, 2 and 4 h). We also evaluated the peripheral sympathetic response to the drug by measuring the concentrations of plasma catecholamines. Nitroglycerin caused an early (1 h) increase in cerebral (pons) and plasma levels of norepinephrine, followed by a delayed (4 h) decrease in medullary and pontine levels of serotonin. The initial noradrenergic activation may reflect the autonomic response to the rapid cardiovascular effects of the drug, while the delayed response may result from the interaction of nitroglycerin-released NO and 5-HT in central areas devoted to the modulation of nociception. These data might therefore help to clarify the mechanisms underlying the delayed migraine attack observed in migraine sufferers after systemic administration of nitroglycerin." [Abstract]

Srikiatkhachorn A, Suwattanasophon C, Ruangpattanatawee U, Phansuwan-Pujito P.
2002 Wolff Award. 5 -HT2A receptor activation and nitric oxide synthesis: a possible mechanism determining migraine attacks.
Headache. 2002 Jul-Aug;42(7):566-74.
"OBJECTIVE: To determine the effect of the 5-HT2A receptor in control of spinal nociception, cerebral circulation, and nitric oxide synthase (nNOS) expression in trigeminovascular neurons. BACKGROUND: The plasticity of the 5-HT2A receptor is a possible factor determining the course of migraine. Up-regulation of this receptor has been demonstrated to correlate with the increasing frequency of migraine attacks and may underlie the development of chronic daily headache. METHODS: Adult male Wistar rats were divided into groups receiving the 5-HT2A agonist, 1,2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), nitroglycerin, or normal saline. The tail flick test and chemical nociception-evoked Fos-expression in dorsal horn neurons were used as indicators of nociception. Regional cerebral blood flow was monitored using laser Doppler flowmetry. Expression of Fos and nNOS was studied using immunohistochemical method. RESULTS: Administration of DOI led to the shortening of tail flick latency (1.3 +/- 0.2 and 7.2 +/- 0.6 seconds for DOI-treated and control groups, respectively). The number of Fos-immunoreactive neurons was also greater in the DOI-treated group compared with the control group. DOI also produced long-lasting cerebral hyperemia (123% of baseline value) associated with the enlargement of perivascular nNOS-immunoreactive nerve fibers and increased nNOS-immunoreactive neurons in trigeminal ganglia and trigeminal nucleus caudalis. These findings resembled those observed in the rats exposed to nitroglycerin. CONCLUSION: Our results suggest that activation of the 5-HT2A receptor leads to an enhancement of NO production in trigeminovascular pathway. NO may trigger migraine attacks by inducing cerebral vasodilation and sensitizing the perivascular nociceptors and central nociceptive neurons in trigeminovascular system. Up-regulation of this pronociceptive receptor can increase headache attacks and contributes to the development of chronic daily headache." [Abstract]

Johnson KW, Nelson DL, Dieckman DK, Wainscott DB, Lucaites VL, Audia JE, Owton WM, Phebus LA.
Neurogenic dural protein extravasation induced by meta-chlorophenylpiperazine (mCPP) involves nitric oxide and 5-HT2B receptor activation.
Cephalalgia. 2003 Mar;23(2):117-23.
"The compound m-chlorophenylpiperazine (mCPP), which is known to trigger migraine-like head pain in some subjects, was evaluated for its ability to induce dural plasma protein extravasation (PPE) in guinea pigs. Intravenous mCPP dose-dependently increased PPE. This effect was inhibited by non-selective 5-HT2 receptor antagonists (methysergide, LY53857, LY215840), by a peripherally restricted 5-HT2 receptor antagonist (xylamidine) and by a 5-HT2B selective receptor antagonist (LY202146). These data suggests that peripheral 5-HT2B receptors mediate mCPP-induced PPE. The nitric oxide synthase inhibitor L-NAME and 5-HT1 agonist sumatriptan also blocked mCPP-induced PPE, suggesting a role for nitric oxide (NO) and the trigeminal system, respectively. NO release has been linked to activation of the 5-HT2B receptor on the vascular endothelium. However, LY202146 did not block PPE induced by electrical stimulation of the trigeminal ganglion. These data are consistent with activation of peripheral 5-HT2B receptors initiating PPE and the theory that selective 5-HT2B antagonists might be effective prophylactic therapies for migraine." [Abstract]

Schmetterer L, Wolzt M, Graselli U, Findl O, Strenn K, Simak S, Kastner J, Eichler HG, Singer EA.
Nitric oxide synthase inhibition in the histamine headache model.
Cephalalgia. 1997 May;17(3):175-82.
"Histamine has been widely used experimentally to induce headache in healthy subjects and migraine in migraineurs. There is evidence that the vascular effects of histamine are at least partially mediated by nitric oxide (NO). Hence we hypothesized that subjective symptoms and hemodynamic effects of histamine could be reduced by systemic NO-synthase inhibition. We therefore studied the effect of pretreatment with N-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO-synthase, or placebo on headache, flush and discomfort scores during histamine infusion. Additionally, blood flow velocities in the middle cerebral and the ophthalmic artery and ocular fundus pulsations were measured. Whereas L-NMMA blunted the effect of histamine in the ophthalmic artery and the ocular circulation, NO-synthase inhibition did not mitigate subjective symptoms. Histamine did not affect mean blood flow velocities in the middle cerebral artery. Hence, we conclude that NO-synthase inhibition reduces the histamine-induced vascular effects in the ocular circulation, but is not sufficient to attenuate or abort the subjective symptoms provoked by histamine infusion." [Abstract]

Akerman S, Williamson DJ, Kaube H, Goadsby PJ.
The role of histamine in dural vessel dilation.
Brain Res. 2002 Nov 22;956(1):96-102.
"The pain of migraine is often throbbing suggesting an important role for the cranial blood vessels and their innervation by the trigeminal nerve. It is proposed that clinically effective anti-migraine compounds, such as 5-HT(1B/1D) agonists, have actions that include inhibiting calcitonin gene-related peptide (CGRP) release from trigeminal nerves. Human studies suggest that histamine can induce migraine possibly by activating nitric oxide (NO) synthase to promote endogenous NO production. The present studies investigated the effect of histamine and its antagonists on the cranial blood vessels using intravital microscopy to assess directly the diameter of dural arteries in sodium pentobarbitone anaesthetised rats. Electrical stimulation of a closed cranial window produces, by local depolarisation of nerves, dural vessel dilation that is monitored continuously on-line using video-microscopy and a video dimension analyser. Histamine infusion caused immediate and reproducible dilation of meningeal blood vessels (103.5+/-6%; n=40) that could be blocked by H(1)- (mepyramine) and H(2) (famotidine)-receptor antagonists (P<0.05), as well as a nitric oxide synthase inhibitor (N(G)-nitro-L-arginine methylester; P<0.05). Neurogenic dural vasodilation was not inhibited by H(2)-receptor antagonists, but was significantly inhibited by a H(1)-receptor antagonist at the high dose of 10 mg/kg. The present studies demonstrate that histamine is likely to activate NO synthase to promote NO production. There is also evidence that H(1)-receptors may be present on trigeminal neurones as the H(1)-receptor antagonist inhibited neurogenic vasodilation, albeit at a large dose." [Abstract]

Lassen LH, Thomsen LL, Kruuse C, Iversen HK, Olesen J.
Histamine-1 receptor blockade does not prevent nitroglycerin induced migraine. Support for the NO-hypothesis of migraine.
Eur J Clin Pharmacol. 1996;49(5):335-9.
"It has previously been shown that in migraine sufferers infusion of glyceryl trinitrate (GTN) and histamine causes an immediate headache during the infusion and a genuine migraine attack one to several hours after the infusion. This identical time profile indicates a common mechanism of action. To evaluate whether GTN causes headache via liberation of histamine, we studied the effect of GTN 0.5 micrograms.kg-1.min-1 for 20 min in seven migraine sufferers, once after pretreatment with the histamine-1 (H1)-receptor blocker mepyramine (0.5 mg.kg-1) and once without pretreatment. This mepyramine dose is known to completely abolish histamine-induced headache. After pretreatment with mepyramine five patients experienced migraine, and without pretreatment six patients did so. The median peak headache score was 7 on a 0-10 scale with and without mepyramine pretreatment. The arterial responses, evaluated with transcranial Doppler, were also unaffected by the mepyramine pretreatment. Our results demonstrate that neither headache nor arterial dilatation due to GTN infusion is caused by histamine release. In all likelihood the common mediator of migraine induction by GTN and histamine is nitric oxide." [Abstract]

Lassen LH, Thomsen LL, Olesen J.
Histamine induces migraine via the H1-receptor. Support for the NO hypothesis of migraine.
Neuroreport. 1995 Jul 31;6(11):1475-9.
"In primates, histamine activates cerebral endothelial H1-receptors leading to formation of nitric oxide (NO). Twenty migraine patients received pretreatment with placebo or the histamine-H1-receptor antagonist, mepyramine, in a randomized, double blind fashion, followed in both groups by i.v. histamine (0.5 microgram kg-1 min-1 for 20 min). Headache characteristics were subsequently observed for 12 h. In patients given placebo histamine caused immediate headache during the infusion followed by a delayed migraine attack fulfilling IHS criteria for migraine without aura. The temporal profile of induced headache was exactly the same as after glyceryl trinitrate. Mepyramine pretreatment abolished both immediate headache and delayed migraine attacks. Our results suggest that a migraine attack can be caused by NO formation in the endothelium of cerebral arteries." [Abstract]