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]

Juhasz G, Zsombok T, Modos EA, Olajos S, Jakab B, Nemeth J, Szolcsanyi J, Vitrai J, Bagdy G.
NO-induced migraine attack: strong increase in plasma calcitonin gene-related peptide (CGRP) concentration and negative correlation with platelet serotonin release.
Pain. 2003 Dec;106(3):461-70.
"The aim of the present study was to investigate changes in the plasma calcitonin gene-related peptide (CGRP) concentration and platelet serotonin (5-hydroxytriptamine, 5-HT) content during the immediate headache and the delayed genuine migraine attack provoked by nitroglycerin. Fifteen female migraineurs (without aura) and eight controls participated in the study. Sublingual nitroglycerin (0.5 mg) was administered. Blood was collected from the antecubital vein four times: 60 min before and after the nitroglycerin application, and 60 and 120 min after the beginning of the migraine attack (mean 344 and 404 min; 12 subjects). In those subjects who had no migraine attack (11 subjects) a similar time schedule was used. Plasma CGRP concentration increased significantly (P<0.01) during the migraine attack and returned to baseline after the cessation of the migraine. In addition, both change and peak, showed significant positive correlations with migraine headache intensity (P<0.001). However, plasma CGRP concentrations failed to change during immediate headache and in the subjects with no migraine attack. Basal CGRP concentration was significantly higher and platelet 5-HT content tended to be lower in subjects who experienced a migraine attack. Platelet serotonin content decreased significantly (P<0.01) after nitroglycerin in subjects with no migraine attack but no consistent change was observed in patients with migraine attack. In conclusion, the fact that plasma CGRP concentration correlates with the timing and severity of a migraine headache suggests a direct relationship between CGRP and migraine. In contrast, serotonin release from platelets does not provoke migraine, it may even counteract the headache and the concomitant CGRP release in this model." [Abstract]

Fanciullacci M, Alessandri M, Figini M, Geppetti P, Michelacci S.
Increase in plasma calcitonin gene-related peptide from the extracerebral circulation during nitroglycerin-induced cluster headache attack.
Pain. 1995 Feb;60(2):119-23.
"In this study, changes in plasma levels of calcitonin gene-related peptide (CGRP) and substance P (SP) during a spontaneous-like cluster headache attack provoked by nitroglycerin were evaluated. Peptide variations after spontaneous or sumatriptan-induced remission were also assessed. Blood was collected from the external jugular vein homolateral to the pain side of 30 male cluster headache patients; 18 men were in an active and 12 in a remission one. Plasma levels of CGRP and SP were determined using sensitive radioimmunoassays for each peptide. CGRP-like immunoreactivity (CGRP-LI) was found to be augmented in patients in an active period and became elevated further at the peak of the provoked attack. A complete reversal occurred both after spontaneous and sumatriptan-induced remission. On the contrary, nitroglycerin neither provoked a cluster headache attack nor altered CGRP-LI in the patients in a remission period. The augmented levels of CGRP-LI measured before and after nitroglycerin administration, when the provoked attack reached the maximum intensity, suggest an activation of the trigeminovascular system during the active period of cluster headache. Moreover, the clinical and biochemical actions showed by sumatriptan stress the involvement of serotonin in cluster headache mechanisms." [Abstract]

M Fanciullacci, M Alessandri, R Sicuteri, and S Marabini
Responsiveness of the trigeminovascular system to nitroglycerine in cluster headache patients
Brain 120: 283-288. 1997.
"Nitroglycerine is known to induce a headache attack in cluster headache patients, which is indistinguishable from a spontaneous attack. It has recently been suggested that a release of calcitonin gene-related peptide (CGRP) from peripheral terminals of trigeminal nociceptive neurons, which supply cephalic blood vessels, underlies symptoms of cluster headache. The aim of this study was to investigate whether the provocative action of nitroglycerine in cluster headache is due, at least in part, to activation of the trigeminovascular system. Nineteen subjects suffering from episodic cluster headache participated in the study. Eleven of them were in an active period, whilst the others were in remission at the time of the study. CGRP-like immunoreactivity (CGRP-LI) was measured in blood samples from the extracerebral circulation before and after the sublingual administration of nitroglycerine. Baseline CGRP-LI plasma levels were higher (P < 0.05) in the patients who were in an active period. Only in these patients did nitroglycerine induce an attack, which was preceded by a latent period with a mean duration of 27 +/- 3 min. When compared with the baseline, a significant (P < 0.01) increase in plasma CGRP-LI was detected at the peak of the provoked attack; no such increase was detected during the latent period, or at the onset of the attack. The results of this study suggests that the provocative action of nitroglycerine in cluster headache is due, at least in part, to activation of the trigeminovascular system. This mechanism seems to be slow and unrelated to the well-known rapidly occurring vasodilator effects of the drug. Finally, activation of the trigeminovascular system only occurs in those patients already in an active cluster headache period who also have high basal CGRP-LI plasma levels. This suggests that a hyperactivity of trigeminal nociceptive fibres could make the trigeminovascular system of these patients sensitive to the triggering action of nitroglycerine." [Abstract/Full Text]

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]

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, 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]

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]

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]

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]

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]

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]

Lassen LH, Haderslev PA, Jacobsen VB, Iversen HK, Sperling B, Olesen J.
CGRP may play a causative role in migraine.
Cephalalgia. 2002 Feb;22(1):54-61.
"Calcitonin gene-related peptide (CGRP) has been detected in increased amounts in external jugular venous blood during migraine attacks. However, it is unknown whether this is secondary to migraine or whether CGRP may cause headache. In a double-blind crossover study, the effect of human alphaCGRP (2 microg/min) or placebo infused intravenously for 20 min was studied in 12 patients suffering from migraine without aura. Headache intensity was scored on a scale from 0 to 10. Two patients were excluded due to severe hypotension and one because she had an infection. In the first hour median peak headache score was 1.0 in the halphaCGRP group vs. 0 in the placebo group (P < 0.01). During the following 11 h all patients experienced headaches after halphaCGRP vs. one patient after placebo (P = 0.0004). The median maximal headache score was 4 after CGRP and 0 after placebo (P = 0.006). In three patients after halphaCGRP, but in no patients after placebo, the delayed headache fulfilled the IHS criteria for migraine without aura. As intravenous administration of halphaCGRP causes headache and migraine in migraineurs, our study suggests that the increase in CGRP observed during spontaneous migraine attacks may play a causative role." [Abstract]

Goadsby PJ, Edvinsson L, Ekman R.
Vasoactive peptide release in the extracerebral circulation of humans during migraine headache.
Ann Neurol. 1990 Aug;28(2):183-7.
"The innervation of the cranial vessels by the trigeminal nerve, the trigeminovascular system, has recently been the subject of study in view of its possible role in the mediation of some aspects of migraine. Since stimulation of the trigeminal ganglion in humans leads to facial pain and flushing and associated release of powerful neuropeptide vasodilator substances, their local release into the extracerebral circulation of humans was determined in patients who had either common or classic migraine. Venous blood was sampled from both the external jugular and cubital fossa ipsilateral to the side of headache. Plasma levels of neuropeptide Y, vasoactive intestinal polypeptide, substance P, and calcitonin gene-related peptide were determined using sensitive radioimmunoassays for each peptide, and values for the cubital fossa and external jugular and a control population were compared. A substantial elevation of the calcitonin gene-related peptide level in the external jugular but not the cubital fossa blood was seen in both classic and common migraine. The increase seen in classic migraine was greater than that seen with common migraine. The other peptides measured were unaltered. This finding may have importance in the pathophysiology of migraine." [Abstract]

Gallai V, Sarchielli P, Floridi A, Franceschini M, Codini M, Glioti G, Trequattrini A, Palumbo R.
Vasoactive peptide levels in the plasma of young migraine patients with and without aura assessed both interictally and ictally.
Cephalalgia. 1995 Oct;15(5):384-90.
"We measured, by RIA methods, ictal and interictal levels of substance P (SP), calcitonin-gene related peptide (CGRP) and neurokinin A (NKA) in the plasma of 30 young migraine patients with aura (MPA) and 45 migraine patients without aura (MWA), and compared the results with those of 30 age-matched controls. There were no significant differences between the levels of these vasoactive peptides in the control group and the levels in both migraine groups studied in headache-free periods. An elevation of CGRP levels in plasma was found during attacks in MPA and, to a lesser extent, in MWA (p < 0.03 and p < 0.05, respectively). A significant increase in NKA levels was also demonstrated in the MPA and MWA groups (p < 0.02 and p < 0.04, respectively). These data suggest, although indirectly, that CGRP and NKA could be involved in the pathogenesis of migraine attacks in juvenile migraine patients." [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.
"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." [Abstract]

Sarchielli P, Alberti A, Floridi A, Gallai V.
Levels of nerve growth factor in cerebrospinal fluid of chronic daily headache patients.
Neurology. 2001 Jul 10;57(1):132-4.
"Nerve growth factor (NGF) levels were determined in the CSF of patients with chronic daily headache (CDH) and correlated with levels of sensory neuropeptides. Patients with CDH showed higher NGF levels in the CSF compared with control subjects (p < 0.0001). Higher CSF levels of substance P (SP) (p < 0.002) and calcitonin-gene-related peptide (CGRP) (p < 0.0001) were also found. There was a significant positive correlation between NGF and both SP and CGRP values. These findings suggest that NGF is involved in the long-lasting sensitization and sustained activation of the trigeminal system in CDH." [Abstract]

Fischer HP, Eich W, Russell IJ.
A possible role for saliva as a diagnostic fluid in patients with chronic pain.
Semin Arthritis Rheum. 1998 Jun;27(6):348-59.
"OBJECTIVES: The focus of this review was on proteins and peptides found in saliva. Of greatest interest were those neuropeptides relevant to nociception and to the pathogenesis of chronic pain syndromes. An additional goal was to develop a standardized protocol to collect saliva for laboratory assessment. METHODS: Data were obtained through discussion with experts at the medical schools in San Antonio and Heidelberg and a Medline literature search involving all relevant studies from 1966 to 1997. The literature search was based on the following key terms: saliva, serotonin, neuropeptide, substance P (SP), calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF). RESULTS: The mean concentration of SP in the saliva of healthy normal controls ranged from 9.6 to 220 pg/mL. Generally, the concentration of SP was approximately three times higher in saliva than in plasma. In a number of painful conditions, particularly tension headache, substantial elevations of salivary SP were found. Mean values for salivary CGRP in healthy controls were approximately 22 pmol/L and were significantly elevated in patients with migraine attacks or cluster headache. There were no data to indicate prior quantitative determination of NGF in human saliva. CONCLUSIONS: After sampling and processing techniques have been standardized, measurement of neuropeptides in human saliva could provide a valuable tool for study of patients with chronic painful disorders such as rheumatoid arthritis, osteoarthritis, and even fibromyalgia syndrome." [Abstract]

Goadsby PJ, Edvinsson L.
Human in vivo evidence for trigeminovascular activation in cluster headache. Neuropeptide changes and effects of acute attacks therapies.
Brain. 1994 Jun;117 ( Pt 3):427-34.
"Cluster headache is a rare very severe disorder that is clinically well characterized with a relatively poorly understood pathophysiology. In this study patients with episodic cluster headache fulfilling the criteria of the International Headache Society were examined during an acute spontaneous attack of headache to determine the local cranial release of neuropeptides. Blood was sampled from the external jugular vein ipsilateral to the pain before and after treatment of the attack. Samples were assayed for calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), substance P and neuropeptide Y. Attacks were treated with either oxygen inhalation, sumatriptan or an opiate. Thirteen patients were studied of whom 10 were male and three female. All had well-established typical attacks of cluster headache when blood was sampled. During the attacks external jugular vein blood levels of CGRP and VIP were raised while there was no change in neuropeptide Y or substance P. Calcitonin gene-related peptide levels rose to 110 +/- 7 pmol/l (normal: < 40) while VIP levels rose to 20 +/- 3 pmol/l (normal: < 7). Treatment with both oxygen and subcutaneous sumatriptan reduced the CGRP level to normal, while opiate administration did not alter the peptide levels. These data demonstrate for the first time in vivo human evidence for activation of the trigeminovascular system and the cranial parasympathetic nervous system in an acute attack of cluster headache. Furthermore, it is shown that both oxygen and sumatriptan abort the attacks and terminate activity in the trigeminovascular system." [Abstract]

Ashina M, Bendtsen L, Jensen R, Schifter S, Jansen-Olesen I, Olesen J.
Plasma levels of calcitonin gene-related peptide in chronic tension-type headache.
Neurology. 2000 Nov 14;55(9):1335-40.
"BACKGROUND: Calcitonin gene-related peptide (CGRP) is involved in the pathophysiology of migraine and cluster headache. Whether CGRP has any role in chronic tension-type headache is unknown. OBJECTIVES: To compare interictal plasma levels of CGRP between patients with chronic tension-type headache and healthy control subjects, to investigate plasma CGRP in relation to headache state, and to compare plasma CGRP between the peripheral and the cranial circulation. METHODS: Blood from the antecubital vein was drawn from 30 patients with chronic tension-type headache and 34 healthy control subjects. In addition, blood samples from the consecutive first 15 patients and from the consecutive first 20 healthy control subjects were also collected from the external jugular vein. RESULTS: CGRP levels measured in the peripheral circulation in patients on days without headache, 63+/-5 pmol/L, tended to be higher than CGRP levels in control subjects, 53+/-3 pmol/L (p = 0.06). In patients, no differences were found between CGRP levels assessed ictally and interictally in either the cranial (p = 0.91) or the peripheral (p = 0.62) circulation. Plasma CGRP level was higher in the external jugular vein than in the antecubital vein on days without headache (p = 0.03) but not on days with headache (p = 0.82). In control subjects, CGRP levels in the cranial circulation did not differ from CGRP levels in the peripheral circulation (p = 0.92). Exploratory analyses showed that 8 patients whose usual headache quality was throbbing had a higher interictal plasma CGRP level than control subjects (p = 0.002), whereas plasma CGRP level was normal in 22 patients with pressing headaches (p = 0.36). CONCLUSIONS: Plasma levels of CGRP are normal in patients with chronic tension-type headache and are unrelated to headache state. Interictal plasma CGRP was increased in patients with a pulsating pain quality. Because the authors have previously shown a similar increase of interictal CGRP levels in migraine, this study suggests that headaches with symptoms that fulfill International Headache Society criteria for tension-type headache may be pathophysiologically related to migraine, if the headache has a pulsating quality." [Abstract]

Ashina M.
Calcitonin gene-related Peptide in tension-type headache.
ScientificWorldJournal. 2002 Jun 7;2(6):1527-31.
"In the last 10 years there has been increasing interest in the role of calcitonin gene-related peptide (CGRP) in primary headaches. Tension-type headache is one of the most common and important types of primary headaches, and ongoing nociception from myofascial tissues may play an important role in the pathophysiology of this disorder. CGRP sensory fibers are preferentially located in the walls of arteries, and nerve fibers containing CGRP accompany small blood vessels in human cranial muscles. It is well established that nociception may lead to release of CGRP from sensory nerve endings and from central terminals of sensory afferents into the spinal cord. It has also been shown that density of CGRP fibers around arteries is increased in persistently inflamed muscle. These findings indicate that ongoing activity in sensory neurons in the cranial muscles may be reflected in changes of plasma levels of neuropeptides in patients with chronic tension-type headache. To explore the possible role of CGRP in tension-type headache, plasma levels of CGRP were measured in patients with chronic tension-type headache. This study showed that plasma levels of CGRP are normal in patients and unrelated to headache state. However, the findings of normal plasma levels of CGRP do not exclude the possibility that abnormalities of this neuropeptide at the neuronal or peripheral (pericranial muscles) levels play a role in the pathophysiology of tension-type headache. Investigation of CGRP in other compartments with new sensitive methods of analysis is necessary to clarify its role in tension-type headache." [Abstract]

Ashina M, Bendtsen L, Jensen R, Schifter S, Olesen J.
Evidence for increased plasma levels of calcitonin gene-related peptide in migraine outside of attacks.
Pain. 2000 May;86(1-2):133-8.
"Although calcitonin gene-related peptide (CGRP) has been shown to be elevated in jugular venous blood of adult migraineurs during acute migraine attacks, it remains unknown whether CGRP is increased outside of attacks in jugular or cubital venous blood. The aim of the present study was to compare interictal plasma levels of CGRP in adult migraine patients and in healthy controls. Twenty patients with a diagnosis of migraine with or without aura and 20 healthy controls were included. In blood from the cubital vein, CGRP levels were significantly higher in patients (75+/-8 pmol/l (mean+/-SEM)) than in controls (49+/-3 pmol/l) (P=0.005). The subgroup of patients suffering exclusively from migraine without aura (n=14) also had significantly higher levels of CGRP (82+/-10 pmol/l) than controls (n=20; 49+/-3 pmol/l) (P=0.001). The findings could not be explained by confounding factors such as age, sex or use of contraceptive pills. We therefore conclude that CGRP is increased in cubital venous blood of migraineurs outside of attack. It is hypothesized that this finding may reflect a long-lasting or permanent abnormal neurogenic vascular control in patients with migraine." [Abstract]

Edvinsson L.
Sensory nerves in man and their role in primary headaches.
Cephalalgia. 2001 Sep;21(7):761-4.
"The sensory innervation of intracranial vessels originate in the trigeminal ganglion and comprise the following signal substances; calcitonin gene-related peptide (CGRP), substance P, neurokinin A, pituitary adenylate cyclase activating peptide (PACAP) and nitric oxide (NO). Studies in patients have revealed a clear association between head pain and the release of CGRP. In cluster headache and in a case of chronic paroxysmal headache there is in addition release of vasoactive intestinal peptide (VIP), which was associated with the facial symptoms (nasal congestion, rhinorrhea). In parallel with triptan administration, acting via 5-HT(1B/1D) receptors, head pain subside and neuropeptide release normalise. These data show the involvement of sensory and parasympathetic mechanisms in the pathophysiology of primary headaches." [Abstract]

Friberg L, Olesen J, Olsen TS, Karle A, Ekman R, Fahrenkrug J.
Absence of vasoactive peptide release from brain to cerebral circulation during onset of migraine with aura.
Cephalalgia. 1994 Feb;14(1):47-54.
"In eight patients carotid angiography was required for evaluation of transient neurological attacks. Cerebral blood flow results, angiography and clinical observations subsequently suggested the diagnosis of migraine. We measured plasma concentrations of substance P(SP), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) in repeated blood samples obtained from the carotid artery and the internal jugular vein in conjunction with cerebral angiography followed by 4 to 6 repeated recordings of regional cerebral blood flow (rCBF) with the intracarotid Xenon-133 injection technique. This technique is known to induce attacks of migraine with aura in many sufferers. Four patients developed aura symptoms. In three this was succeeded by throbbing headache. Typical, migraine-related, focal hypoperfusion occurred in conjunction with the aura symptoms. The remaining four patients had no symptoms or rCBF changes. There were no systematic or statistically significant changes over time in arterial-venous plasma concentrations or in the release rates of any of the peptides. All migraineurs had an overall elevated mean CGRP value compared to control values from the literature. The overall plasma levels of the potent vasoconstrictor NPY were higher (p < 0.10) in the group that developed symptoms and rCBF changes (136 pmol/l) than in the non-symptomatic group (97 pmol/l). The difference in NPY levels could perhaps be associated with the focal rCBF decrease seen in the attack group." [Abstract]

Asakura K, Kanemasa T, Minagawa K, Kagawa K, Yagami T, Nakajima M, Ninomiya M.
alpha-eudesmol, a P/Q-type Ca(2+) channel blocker, inhibits neurogenic vasodilation and extravasation following electrical stimulation of trigeminal ganglion.
Brain Res. 2000 Aug 4;873(1):94-101.
"In this study, we investigated the effect of alpha-eudesmol, which potently inhibits the presynaptic omega-agatoxin IVA-sensitive (P/Q-type) Ca(2+) channel, on neurogenic inflammation following electrical stimulation of rat trigeminal ganglion. Treatment with alpha-eudesmol (0.1-1 mg/kg. i.v.) dose-dependently attenuated neurogenic vasodilation in facial skin monitored by a laser Doppler flowmetry. In addition, alpha-eudesmol (1 mg/kg. i.v.) significantly decreased dural plasma extravasation in analysis using Evans blue as a plasma marker. On the other hand, alpha-eudesmol (1 mg/kg, i.v.) did not affect mean arterial blood pressure in rats. The calcitonin gene-related peptide (CGRP) and substance P (SP) released from activated sensory nerves have recently been suggested to be associated with the neurogenic inflammation. In this study, we also showed that alpha-eudesmol (0.45-45 microM) concentration-dependently inhibits the depolarization-evoked CGRP and SP release from sensory nerve terminals in spinal cord slices. These results indicate that the anti-neurogenic inflammation action of alpha-eudesmol, which does not affect the cardiovascular system, may be due to its presynaptic inhibition of the neuropeptide release from perivascular trigeminal terminals. We also suggest that the omega-agatoxin IVA-sensitive Ca(2+) channel blocker, alpha-eudesmol, may become useful for the treatment of the neurogenic inflammation in the trigemino-vascular system such as migraine." [Abstract]

Piper RD, Edvinsson L, Ekman R, Lambert GA.
Cortical spreading depression does not result in the release of calcitonin gene-related peptide into the external jugular vein of the cat: relevance to human migraine.
Cephalalgia. 1993 Jun;13(3):180-3; discussion 149.
"There is circumstantial evidence that cortical spreading depression (SD) may account for the scotoma and the "spreading cortical oligemia" seen during migraine with aura. It has been shown that calcitonin gene-related peptide (CGRP) is increased in blood taken from the external jugular vein (EJV) in humans during migraine and after stimulation of the trigeminal ganglion. To test the hypothesis that cortical SD may elevate the concentration of this vasoactive peptide in the EJV during migraine, we have measured its concentration in the external jugular vein of cats during cortical SD. This study demonstrates that SD has no effect on the concentration of CGRP either during the passage of a wave of spreading depression across the cortex or, 60 min later, during the period of post-SD cortical oligemia." [Abstract]

Williamson DJ, Shepheard SL, Cook DA, Hargreaves RJ, Hill RG, Cumberbatch MJ.
Role of opioid receptors in neurogenic dural vasodilation and sensitization of trigeminal neurones in anaesthetized rats.
Br J Pharmacol. 2001 Jul;133(6):807-14.
"Migraine headache is thought to be caused by a distension of meningeal blood vessels, the activation of trigeminal sensory neurones and the the development of a central sensitization within the trigeminal nucleus caudalis (TNC). It has been proposed that clinically effective 5-HT(1B/1D) agonists act peripherally to inhibit the release of calcitonin gene-related peptide (CGRP) and neurogenic dural vasodilation, and to attenuate nociceptive neurotransmission within the TNC. Since opioids are also effective anti-migraine agents the present studies investigated the role of opioids within the trigemino-vascular system in anaesthetised rats. Electrical stimulation of the dura mater evoked neurogenic dural vasodilation which was significantly inhibited by morphine (1 mg kg(-1)) the selective mu-opioid agonist DAGO (10 microg kg(-1)) and the mixed agonist/antagonist butorphanol (1 mg kg(-1)) but not by the kappa- and delta-opioid agonists (+/-) U50488H (100 microg kg(-1)) and DPDPE (1 mg kg(-1)). Morphine had no effect on CGRP-evoked dural vasodilation. In electrophysiological studies morphine (1 - 10 mg kg(-1)) significantly attenuated brainstem neuronal activity in response to electrical stimulation of the dura by 65% at 10 mg kg(-1). Morphine (3 mg kg(-1)) also inhibited the TNC neuronal sensitization following CGRP-evoked dilation. The present studies have demonstrated that opioids block the nociceptive neurotransmission within the trigeminal nucleus caudalis and in addition inhibit neurogenic dural vasodilation via an action on mu-opioid receptors located on trigeminal sensory fibres innervating dural blood vessels. These peripheral and central actions are similar to those of the 'triptan' 5-HT(1B/1D) agonists and could account for the anti-migraine actions of opioids." [Abstract]

Akerman, S., Kaube, H., Goadsby, P. J.
Anandamide Is Able to Inhibit Trigeminal Neurons Using an in Vivo Model of Trigeminovascular-Mediated Nociception
J Pharmacol Exp Ther 2004 309: 56-63
"Arachidonylethanolamide (anandamide, AEA) is believed to be the endogenous ligand of the cannabinoid CB(1) and CB(2) receptors. CB(1) receptors have been found localized on fibers in the spinal trigeminal tract and spinal trigeminal nucleus caudalis. Known behavioral effects of anandamide are antinociception, catalepsy, hypothermia, and depression of motor activity, similar to Delta(9)-tetrahydocannanbinol, the psychoactive constituent of cannabis. It may be a possible therapeutic target for migraine. In this study, we looked at the possible role of the CB(1) receptor in the trigeminovascular system, using intravital microscopy to study the effects of anandamide against various vasodilator agents. Anandamide was able to inhibit dural blood vessel dilation brought about by electrical stimulation by 50%, calcitonin gene-related peptide (CGRP) by 30%, capsaicin by 45%, and nitric oxide by 40%. CGRP(8-37) was also able to attenuate nitric oxide (NO)-induced dilation by 50%. The anandamide inhibition was reversed by the CB(1) receptor antagonist AM251. Anandamide also reduced the blood pressure changes caused by CGRP injection, this effect was not reversed by AM251. It would seem that anandamide acts both presynaptically, to prevent CGRP release from trigeminal sensory fibers, and postsynaptically to inhibit the CGRP-induced NO release in the smooth muscle of dural arteries. CB(1) receptors seem to be involved in the NO/CGRP relationship that exists in causing headache and dural blood vessel dilation. It also seems that some of the blood pressure changes caused by anandamide are mediated by a noncannabinoid receptor, as AM251 was unable to reverse these effects. It can be suggested that anandamide is tonically released to play some form of modulatory role in the trigeminovascular system." [Abstract]

Edvinsson L.
New therapeutic target in primary headaches - blocking theCGRP receptor.
Expert Opin Ther Targets. 2003 Jun;7(3):377-83.
"The primary headaches are among the most prevalent neurological disorders, afflicting up to 16% of the adult population. The associated pain originates from intracranial blood vessels that are innervated by sensory nerves storing several neurotransmitters. In primary headaches, there is a clear association between the headache and the release of calcitonin gene-related peptide (CGRP), but not other neuronal messengers. The specific purpose of this review is to describe CGRP in the human cranial circulation and to elucidate a possible role for a specific antagonist in the treatment of primary headaches. Acute treatment with administration of a 5-HT(1B/1D) agonist (triptan) results in alleviation of the headache and normalisation of the CGRP level. The mechanism of action of triptans involves vasoconstriction of intracranial vessels and a presynaptic inhibitory effect of sensory nerves. The central role of CGRP in migraine and cluster headache pathophysiology has led to the search for small-molecule CGRP antagonists, which are predicted to have fewer cardiovascular side effects in comparison to the triptans. The initial pharmacological profile of such a group of compounds has recently been disclosed. These compounds have high selectivity for human CGRP receptors and are reportedly efficacious in the relief of acute attacks of migraine." [Abstract]

Olesen J, Diener HC, Husstedt IW, Goadsby PJ, Hall D, Meier U, Pollentier S, Lesko LM; BIBN 4096 BS Clinical Proof of Concept Study Group.
Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine.
N Engl J Med. 2004 Mar 11;350(11):1104-10.
"BACKGROUND: Calcitonin gene-related peptide (CGRP) may have a causative role in migraine. We therefore hypothesized that a CGRP-receptor antagonist might be effective in the treatment of migraine attacks. METHODS: In an international, multicenter, double-blind, randomized clinical trial of BIBN 4096 BS, a highly specific and potent nonpeptide CGRP-receptor antagonist, 126 patients with migraine received one of the following: placebo or 0.25, 0.5, 1, 2.5, 5, or 10 mg of BIBN 4096 BS intravenously over a period of 10 minutes. A group-sequential adaptive treatment-assignment design was used to minimize the number of patients exposed. RESULTS: The 2.5-mg dose was selected, with a response rate of 66 percent, as compared with 27 percent for placebo (P=0.001). The BIBN 4096 BS group as a whole had a response rate of 60 percent. Significant superiority over placebo was also observed with respect to most secondary end points: the pain-free rate at 2 hours; the rate of sustained response over a period of 24 hours; the rate of recurrence of headache; improvement in nausea, photophobia, phonophobia, and functional capacity; and the time to meaningful relief. An effect was apparent after 30 minutes and increased over the next few hours. The overall rate of adverse events was 25 percent after the 2.5-mg dose of the drug and 20 percent for the BIBN 4096 BS group as a whole, as compared with 12 percent for placebo. The most frequent side effect was paresthesia. There were no serious adverse events. CONCLUSIONS: The CGRP antagonist BIBN 4096 BS was effective in treating acute attacks of migraine." [Abstract]

Durham P, Russo A.
New insights into the molecular actions of serotonergic antimigraine drugs.
Pharmacol Ther. 2002 Apr-May;94(1-2):77-92.
"Migraine is a painful and debilitating neurological disorder that affects approximately 10% of the adult population in Western countries. Sensitization and activation of the trigeminal ganglia nerves that innervate the meningeal blood vessels is believed to play an important role in the initiation and maintenance of migraine pain. In this capacity, release of the neuropeptide calcitonin gene-related peptide (CGRP) and the resultant neurogenic inflammation is thought to underlie the pathophysiology of migraine. Largely due to the success of the serotonin Type 1 migraine drugs such as sumatriptan, migraine pathology and therapy has become a focus of intensive clinical and physiological research during the past decade. The effectiveness of these drugs is thought to be due to their ability to block the stimulated secretion of neuropeptides from trigeminal nerves to break the vicious nociceptive cycle of migraine. A component of this nociceptive cycle involves activation of mitogen-activated protein kinase signaling pathways. Indeed, activation of mitogen-activated protein kinase pathways can increase CGRP neuropeptide synthesis and secretion. Recently, the serotonin Type 1 agonists have been shown to cause a prolonged increase in intracellular Ca(2+) in trigeminal ganglia neurons and an increased phosphatase activity that can repress stimulated CGRP secretion and transcription. Identification of molecular signaling events in migraine pathology and therapy has provided new insight into the pharmacology and signaling mechanisms of sumatriptan and related drugs, and may provide the foundation for development of novel treatments for migraine." [Abstract]

Doods H.
Development of CGRP antagonists for the treatment of migraine.
Curr Opin Investig Drugs. 2001 Sep;2(9):1261-8.
"Migraine is one of the most common neurological disorders, involving periodical attacks of headache and nausea as well as a plethora of other symptoms. Although considerable progress has been made, the pathophysiology of migraine is still not understood. However, several observations point to an involvement of calcitonin gene-related peptide (CGRP). Migraine headache involves the activation of the trigeminal system and dilatation of cranial vessels. CGRP is localized to neurons in the trigeminal ganglia and CGRP levels are increased during a migraine attack, presumably causing the vasodilation observed. Accordingly, it is conceivable that inhibition of CGRP-evoked dilatation of the cranial vessels may provide a novel treatment for migraine headache. The non-peptidic CGRP antagonist BIBN-4096BS (Boehringer Ingelheim) is presently under clinical investigation to assess the importance of CGRP in migraine headache and to answer the question of whether the concept of CGRP antagonists may offer advantages, e.g., higher efficacy, lower recurrence rate or improved side-effect profile, compared to the currently used antimigraine drugs." [Abstract]

Verheggen R, Bumann K, Kaumann AJ.
BIBN4096BS is a potent competitive antagonist of the relaxant effects of alpha-CGRP on human temporal artery: comparison with CGRP(8-37).
Br J Pharmacol. 2002 May;136(1):120-6.
"Release of CGRP during migraine may produce harmful dilatation of cranial arteries, thereby possibly causing pain. We have compared the antagonism by BIBN4096BS and CGRP(8-37) of the relaxant effects of alpha-CGRP on rings of human temporal artery. alpha-CGRP relaxed the arteries precontracted with 9 - 24 mM KCl (-logEC50=9.4) nearly as efficaciously as sodium nitroprusside (10 microM). BIBN4096BS (0.1 - 100 nM) antagonized the effects of alpha-CGRP in surmountable manner with slopes of Schild-plots not different from unity. -LogKB values of 10.1 and 10.4 were estimated for BIBN4096BS when administered before or during the KCl-contracture respectively. BIBN4096BS (1 microM) did not modify the relaxant effects of papaverine and sodium nitroprusside. CGRP(8-37) (1 - 10 microM) antagonized the effects of alpha-CGRP in a surmountable manner with slopes of Schild-plots not different from unity. -LogKB values of 6.6 and 6.7 were estimated for CGRP(8-37) administered before or during the KCl-contracture respectively. The high affinity of BIBN4096BS for CGRP receptors of human temporal artery makes it an excellent tool to explore the hypothesis of CGRP-evoked cerebral vasodilation in migraine." [Abstract]

Moreno MJ, Abounader R, Hebert E, Doods H, Hamel E.
Efficacy of the non-peptide CGRP receptor antagonist BIBN4096BS in blocking CGRP-induced dilations in human and bovine cerebral arteries: potential implications in acute migraine treatment.
Neuropharmacology. 2002 Mar;42(4):568-76.
"The present data demonstrate that BIBN4096BS is a very potent antagonist that could, depending on its bioavailability and in vivo affinity, be of potential benefit in the acute treatment of migraine headache by blocking and/or reversing the CGRP-mediated dilation of intracranial vessels induced by activation of trigeminovascular afferents." [Abstract]

Aiyar, Nambi, Daines, Robert A., Disa, Jyoti, Chambers, Pamela A., Sauermelch, Charles F., Quiniou, Marie-J., Khandoudi, Nassirah, Gout, Bernard, Douglas, Stephen A., Willette, Robert N.
Pharmacology of SB-273779, a Nonpeptide Calcitonin Gene-Related Peptide 1 Receptor Antagonist
J Pharmacol Exp Ther 2001 296: 768-775 [Full Text]

Durham, Paul L., Russo, Andrew F.
Stimulation of the Calcitonin Gene-Related Peptide Enhancer by Mitogen-Activated Protein Kinases and Repression by an Antimigraine Drug in Trigeminal Ganglia Neurons
J. Neurosci. 2003 23: 807-815
"Calcitonin gene-related peptide (CGRP) is involved in the underlying pathophysiology of all vascular headaches, including migraines. Elevated levels of CGRP during migraine are restored to normal coincident with headache relief after treatment with the antimigraine drug sumatriptan. We have used primary cultures of trigeminal neurons under conditions simulating migraine pathology and therapy to study the mechanisms controlling the CGRP promoter. Using reporter genes in transient transfection assays, we demonstrate that an 18 bp enhancer containing a helix-loop-helix element is both necessary and sufficient for full promoter activity. NGF treatment and cotransfection with an upstream activator of the extracellular signal-regulated MAP kinases (MAPKs) activated the enhancer. Treatment with sumatriptan repressed NGF- and MAPK-stimulated CGRP promoter activity. Repression was also observed using a synthetic MAPK-responsive reporter gene. Sumatriptan regulation of CGRP gene expression did not couple to a G(i)/G(o) pathway, but rather caused a prolonged increase in intracellular calcium. The importance of the prolonged calcium signal in repression of MAPK activity was demonstrated by using the ionophore ionomycin to mimic sumatriptan action. We propose that activation of MAPK pathways may increase CGRP gene expression during migraine, and that sumatriptan can diametrically oppose that activation via a prolonged elevation of intracellular calcium." [Full Text]

Durham, Paul L., Russo, Andrew F.
Regulation of Calcitonin Gene-Related Peptide Secretion by a Serotonergic Antimigraine Drug
J. Neurosci. 1999 19: 3423-3429
"We have investigated the regulation of calcitonin gene-related peptide (CGRP) release from trigeminal neurons by the serotonergic antimigraine drug sumatriptan. Serum levels of the neuropeptide CGRP are elevated during migraine. Treatment with the drug sumatriptan returns CGRP levels to normal coincident with the alleviation of headache. However, despite this clinical efficacy, the cellular target and mechanism of sumatriptan action are not well understood beyond the pharmacology of its recognition of the 5-HT1 class of serotonin receptors. We have used cultured trigeminal neurons to demonstrate that sumatriptan can directly repress CGRP secretion from sensory neurons. The stimulated secretion in response to depolarization or inflammatory agents was inhibited, but not the basal secretion rate. Unexpectedly, sumatriptan did not lower cAMP levels, in contrast to the classical role ascribed to the 5-HT1 receptors. Instead, activation of 5-HT1 receptors caused a slow and remarkably prolonged increase in intracellular calcium. The inhibition of CGRP secretion is attenuated by the phosphatase inhibitor okadaic acid, suggesting that sumatriptan action is mediated by calcium-recruited phosphatases. These results suggest that 5-HT1 agonists may block a deleterious feedback loop in migraine at the trigeminal neurons and provide a general mechanism by which this class of drugs can attenuate stimulated neuropeptide release." [Full Text]

Knight YE, Edvinsson L, Goadsby PJ.
4991W93 inhibits release of calcitonin gene-related peptide in the cat but only at doses with 5HT(1B/1D) receptor agonist activity?
Neuropharmacology. 2001 Mar;40(4):520-5.
"Calcitonin gene-related peptide (CGRP) is a marker for trigeminovascular activation and is released during the headache phase of migraine and cluster headache. CGRP may have a role in migraine through its potent cranial vasodilator effects, or by an action on trigeminal nerve activity, both of which are targeted by 5HT(1B/1D) agonist drugs. CP122,288, a conformationally restricted analogue of sumatriptan that is a potent inhibitor of neurogenic plasma protein extravasation (PPE), was ineffective at inhibiting CGRP release at a single low dose; and is also ineffective as an acute anti-migraine compound. However, it remained unclear as to whether, as a class, the conformationally-restricted triptan analogues could have inhibitory effects on CGRP in higher doses. 4991W93, a conformationally restricted analogue of zolmitriptan, is also a potent inhibitor of PPE at doses without 5HT(1B/1D)-mediated effects, that was developed as an anti-migraine drug, and thus was suitable to test whether higher doses of such conformationally restricted triptan analogues could inhibit trigeminal-evoked CGRP release. The superior sagittal sinus (SSS) was stimulated in 14 anaesthetised cats and external jugular vein blood samples were analysed by radioimmunoassay for CGRP levels before, 1 min after SSS stimulation, and 1 min after SSS stimulation in the presence of 4991W93. Stimulation of the SSS resulted in release of CGRP from the external jugular vein. 4991W93 at a dose of 0.1 and 10 microg/kg, selected for maximal PPE blocking effects in rodents, was ineffective at inhibiting CGRP release, with an SSS stimulation level of 78+/-4 pmol/l compared to a post-4991W93 level of 79+/-3 pmol/l (n=4). In comparison CGRP release was inhibited after a dose of 100 microg/kg 4991W93 from 64+/-6 to 36+/-3 pmol/l (n=5). Given that 4991W93 is inactive clinically at non-vascular doses, it seems clear that the 5HT(1B/1D) agonist effects of the compound are necessary for blockade of CGRP release and thus any anti-migraine action. Taken with the clinical results, these data emphasise the importance of CGRP release in migraine, and suggest that other non-5HT-based pharmacological targets may account for PPE blockade in animal studies." [Abstract]

Williamson DJ, Hargreaves RJ, Hill RG, Shepheard SL.
Sumatriptan inhibits neurogenic vasodilation of dural blood vessels in the anaesthetized rat--intravital microscope studies.
Cephalalgia. 1997 Jun;17(4):525-31.
"This study used intravital microscopy to measure the diameter of dural arteries in anaesthetized rats. Electrical stimulation of the surface of a closed cranial window produced increases in dural vessel diameter which were blocked by the CGRP receptor antagonist human-alpha CGRP(8-37) but unaffected by the NK1 receptor antagonist RP67580. Sumatriptan (3 and 10 mg kg-1, i.v.) significantly reduced the response to electrical stimulation. In contrast, sumatriptan (3 mg kg-1) had no effects on the response to exogenously administered CGRP. These results indicate that neurokinins play no role in neurogenic vasodilation in this preparation and that neurogenic vasodilation in rat dural vessels is mediated predominantly by CGRP. Furthermore, the data indicate that sumatriptan attenuates neurogenic vasodilation, probably by inhibiting the release of CGRP from perivascular trigeminal nerve endings innervating the dura. These experimental data parallel the clinical findings that CGRP levels are elevated in migraine and normalized, concomitantly with headache relief, by sumatriptan." [Abstract]

Williamson DJ, Hill RG, Shepheard SL, Hargreaves RJ.
The anti-migraine 5-HT(1B/1D) agonist rizatriptan inhibits neurogenic dural vasodilation in anaesthetized guinea-pigs.
Br J Pharmacol. 2001 Aug;133(7):1029-34.
"These studies investigated the pharmacology of neurogenic dural vasodilation in anaesthetized guinea-pigs. Following introduction of a closed cranial window the meningeal (dural) blood vessels were visualized using intravital microscopy and the diameter constantly measured using a video dimension analyser. Dural blood vessels were constricted with endothelin-1 (3 microg kg(-1), i.v.) prior to dilation of the dural blood vessels with calcitonin gene-related peptide (CGRP; 1 microg kg(-1), i.v.) or local electrical stimulation (up to 300 microA) of the dura mater. In guinea-pigs pre-treated with the CGRP receptor antagonist CGRP((8-37)) (0.3 mg kg(-1), i.v.) the dilator response to electrical stimulation was inhibited by 85% indicating an important role of CGRP in neurogenic dural vasodilation in this species. Neurogenic dural vasodilation was also blocked by the 5-HT(1B/1D) agonist rizatriptan (100 microg kg(-1)) with estimated plasma levels commensurate with concentrations required for anti-migraine efficacy in patients. Rizatriptan did not reverse the dural dilation evoked by CGRP indicating an action on presynaptic receptors located on trigeminal sensory fibres innervating dural blood vessels. In addition, neurogenic dural vasodilation was also blocked by the selective 5-HT(1D) agonist PNU-142633 (100 microg kg(-1)) but not by the 5-HT(1F) agonist LY334370 (3 mg kg(-1)) suggesting that rizatriptan blocks neurogenic vasodilation via an action on 5-HT(1D) receptors located on perivascular trigeminal nerves to inhibit CGRP release. This mechanism may underlie one of the anti-migraine actions of the triptan class exemplified by rizatriptan and suggests that the guinea-pig is an appropriate species in which to investigate the pharmacology of neurogenic dural vasodilation." [Abstract]

Samsam M, Covenas R, Csillik B, Ahangari R, Yajeya J, Riquelme R, Narvaez JA, Tramu G.
Depletion of substance P, neurokinin A and calcitonin gene-related peptide from the contralateral and ipsilateral caudal trigeminal nucleus following unilateral electrical stimulation of the trigeminal ganglion; a possible neurophysiological and neuroanatomical link to generalized head pain.
J Chem Neuroanat. 2001 Mar;21(2):161-9.
"Primary trigeminal neurons of the trigeminal ganglion (TG) innervate major parts of the face and head, including the dura. Electrical stimulation of the TG at specific parameters, can activate its nociceptive neurons and may serve as an experimental pain model. Markowitz [J. Neurosci. 7 (1987) 4129] reported that electrical stimulation of the trigeminal ganglion (TG) causes extravasation of plasma proteins from venules of the trigeminally innervated domain possibly due to the release of vasoactive substances. Neurogenic inflammation (vasodilatation, plasma protein extravasation, release of vasoactive peptides) in dura may serve as one of the possible pathomechanisms underlying vascular head pain [Moskowitz, Ann. Neurol. 16 (1984) 157]. We performed a unilateral electrical stimulation (7.5 Hz, 5 ms, 0.8-1.4 mA for 5 min) of the TG in rat, to induce a neurogenic inflammation in the peripheral trigeminal domain including the dura, looking for calcitonin gene related peptide (CGRP), substance P (SP) and neurokinin A (NKA) immunoreactivity (IR) in the caudal trigeminal nucleus (CTN) into which massive central trigeminal processes terminate. Here, we show patchy depletion(s) of CGRP-, SP- and NKA-IRs in the contralateral CTN of the rat in addition to their ipsilateral depletion. Such depletion is due to the release of these neuropeptides in the CTN leading to the activation of bilateral trigeminal nociceptive pathway. These data afford the possibility that under specific frequencies (which may roughly correlate to the intensity of the painful stimulus) and/or specific intensities (may correlate to specific areas of the peripheral trigeminal domain) of stimulation, activation of one side of the TG may activate bilateral trigeminal nociceptive pathway leading to the perception of an ill localized/generalized pain or headache rather than a unilateral one." [Abstract]

Samsam M, Covenas R, Ahangari R, Yajeya J, Narvaez JA, Tramu G.
Simultaneous depletion of neurokinin A, substance P and calcitonin gene-related peptide from the caudal trigeminal nucleus of the rat during electrical stimulation of the trigeminal ganglion.
Pain. 2000 Feb;84(2-3):389-95. [Abstract]

Williamson DJ, Hargreaves RJ.
Neurogenic inflammation in the context of migraine.
Microsc Res Tech. 2001 May 1;53(3):167-78.
"Despite considerable research into the pathogenesis of idiopathic headaches, such as migraine, the pathophysiological mechanisms underlying them remain poorly understood. Although it is well established that the trigeminal nerve becomes activated during migraine, the consequences of this activation remain controversial. One theory, based on preclinical observations, is that activation of trigeminal sensory fibers leads to a painful neurogenic inflammation within the meningeal (dural) vasculature mediated by neuropeptide release from trigeminal sensory fibres and characterized by plasma protein extravasation, vasodilation, and mast cell degranulation. Effective antimigraine agents such as ergots, triptans, opioids, and valproate inhibit preclinical neurogenic dural extravasation, suggesting that this activity may be a predictor of potential clinical efficacy of novel agents. However, several clinical trials with other agents that inhibit this process preclinically have failed to show efficacy in the acute treatment of migraine in man. Alternatively, it has been proposed that painful neurogenic vasodilation of meningeal blood vessels could be a key component of the inflammatory process during migraine headache. This view is supported by the observation that jugular plasma levels of the potent vasodilator, calcitonin gene-related peptide (CGRP) are elevated during the headache and normalized by successful sumatriptan treatment. Preclinically, activation of trigeminal sensory fibers evokes a CGRP-mediated neurogenic dural vasodilation, which is blocked by dihydroergotamine, triptans, and opioids but unaffected by NK1 receptor antagonists that failed in clinical trials. These observations suggest that CGRP release with associated neurogenic dural vasodilation may be important in the generation of migraine pain, a theory that would ultimately be tested by the clinical testing of a CGRP receptor antagonist." [Abstract]

Ebersberger A, Averbeck B, Messlinger K, Reeh PW.
Release of substance P, calcitonin gene-related peptide and prostaglandin E2 from rat dura mater encephali following electrical and chemical stimulation in vitro.
Neuroscience. 1999 Mar;89(3):901-7.
"Neurogenic inflammation of the dura, expressed in plasma extravasation and vasodilatation, putatively contributes to different types of headache. A novel in vitro preparation of the fluid-filled skull cavities was developed to measure mediator release from dura mater encephali upon antidromic electrical stimulation of the trigeminal ganglion and after application of a mixture of inflammatory mediators (serotonin, histamine and bradykinin, 10(-5) M each, pH 6.1) to the arachnoid side of rat dura. The release of calcitonin gene-related peptide, substance P and prostaglandin E2 from dura mater was measured in 5-min samples of superfusates using enzyme immunoassays. Orthodromic chemical and antidromic electrical stimulation of dural afferents caused significant release of calcitonin gene-related peptide (2.8- and 4.5-fold of baseline). The neuropeptide was found to be increased during the 5-min stimulation period and returned to baseline (20.9 +/- 12 pg/ml) in the sampling period after stimulation. In contrast, release of substance P remained at baseline levels (19.3 +/- 11 pg/ml) throughout the experiment. Prostaglandin E2 release was elevated during chemical and significantly also after antidromic electrical stimulation (6- and 4.2-fold of baseline, which was 305 +/- 250 pg/ml). Prostaglandin E2 release outlasted the stimulation period for at least another 5 min. The data support the hypothesis of neurogenic inflammation being involved in headaches and provide new evidence for prostaglandin E2 possibly facilitating meningeal nociceptor excitation and, hence, pain." [Abstract]

Jenkins DW, Feniuk W, Humphrey PP.
Characterization of the prostanoid receptor types involved in mediating calcitonin gene-related peptide release from cultured rat trigeminal neurones.
Br J Pharmacol. 2001 Nov;134(6):1296-302.
"1. Prostaglandins and the vasodilator neuropeptide, calcitonin-gene related peptide (CGRP), have both been implicated in the pathogenesis of migraine headache. We have used primary cultures of adult rat trigeminal neurones to examine the effects of prostanoids on CGRP release in vitro. 2. CGRP release was stimulated by prostaglandin E2 (PGE2) and the IP receptor agonist, carbaprostacyclin (cPGI2). These responses were extracellular calcium-dependent, and the PGE2-induced CGRP release was unaltered by inhibition of nitric oxide synthase (NOS), ATP receptor blockade, or the addition of adenosine deaminase. 3. Increases in CGRP levels were also observed in response to prostaglandin D2 (PGD2), and the EP2 receptor selective agonist, butaprost. No increases in CGRP release were observed in response to prostaglandin F2alpha (PGF2alpha) or the TP receptor selective agonist, U46619, or the EP3 receptor selective agonist, GR63799X. 4. The selective DP receptor antagonist, BWA868C, antagonized the PGD2-, but not PGE2- or cPGI2-induced release. Furthermore, the EP1 selective antagonist, ZM325802, failed to antagonize the PGE2-induced CGRP release from these cells. 5. These data indicate that activation of DP, EP and IP receptors can each cause CGRP release from trigeminal neurones, and suggest that the predominant EP receptor subtype involved may be the EP2 receptor. Together with evidence that the cyclo-oxygenase inhibitor, aspirin, particularly when administered intravenously is effective in treating acute migraine, these findings further suggest a role for prostaglandins in migraine pathophysiology." [Abstract]

Zimmermann K, Reeh PW, Averbeck B.
S+ -flurbiprofen but not 5-HT1 agonists suppress basal and stimulated CGRP and PGE2 release from isolated rat dura mater.
Pain. 2003 Jun;103(3):313-20.
"Neurogenic inflammation of the meninges, expressed in plasma extravasation and vasodilatation, putatively contributes to certain types of headache. Both, non-steroidal antiinflammatory drugs (NSAIDs) and serotonin-1 (5-HT1) receptor agonists are similarly effective antimigraine drugs but their mechanism of action is unclear. The clinical observation that sumatriptan lowered plasma levels of calcitonin gene-related peptide (CGRP), found increased during migraine attacks, drew attention to a possible inhibition of pro-inflammatory neuropeptide release from trigeminal afferents. An isolated preparation of fluid-filled rat skull cavities was used to study effects of NSAIDs and 5-HT(1B/D) agonists on the dura stimulated by inflammatory mediators (bradykinin, histamine and serotonin, 10(-5)M each). The release of immunoreactive CGRP (iCGRP) and immunoreactive PGE(2) (iPGE(2)) was measured in 5-min samples of superfusates using enzyme immunoassays. S(+)-flurbiprofen (10(-6)M) strongly reduced the basal and stimulated iCGRP release and abolished iPGE(2) release; R(-)-flurbiprofen showed much less effect on iPGE(2) liberation and did not influence iCGRP release. The 5-HT(1B/D) agonists naratriptan and CP93,129 were ineffective on both iCGRP and iPGE(2) release. Inspite of its weak COX blocking effect, R(-)-flurbiprofen is reported to exert antinociceptive effects, although it has not been tested in migraine. Only the potent COX blocker S(+)-flurbiprofen also suppressed iCGRP release while the 5-HT(1B/D) agonists were ineffective. Thus, inhibition of meningeal neuropeptide secretion is not a common action principle of the drugs that could be essential for their antimigraine effects." [Abstract]

Limmroth V, Katsarava Z, Liedert B, Guehring H, Schmitz K, Diener HC, Michel MC.
An in vivo rat model to study calcitonin gene related peptide release following activation of the trigeminal vascular system.
Pain. 2001 May;92(1-2):101-6.
"Calcitonin gene related peptide (CGRP) released from the C-fibers projecting from the trigeminal ganglion to the meninges has been suggested to play a crucial role in the pathophysiology of headache, particularly migraine. In humans it has been shown that CGRP is released during migraine-attacks, and this is attenuated by the administration of typical anti-migraine drugs such as dihydroergotamine or sumatriptan. We describe a new rat model which allows the study of CGRP release from the meninges into venous blood following activation of the trigeminal vascular system. The effects of classical and new anti-migraine drugs such as acetylsalicylic acid (ASA), sumatriptan and the new high efficacy 5-HT1B/1D agonist donitriptan (4-[4-[2-(2-aminoethyl)-1H-indol-5-yloxyl]acetyl]piperazinyl-1-yl]benzonitrile) were evaluated in comparison with the established model of neurogenic inflammation in the meninges. Sumatriptan and donitriptan inhibited CGRP release as well as neurogenic inflammation. ASA, however, attenuated neurogenic inflammation, but not CGRP release, confirming the concept of prejunctional inhibition of CGRP release by 5-HT1B/1D receptors. This new model allows the further study of prejunctional pharmacology and mechanisms of neuropeptide release in the trigeminal vascular system, which might be crucial for the further development of potent, more effective anti-migraine drugs." [Abstract]

Durham PL, Dong PX, Belasco KT, Kasperski J, Gierasch WW, Edvinsson L, Heistad DD, Faraci FM, Russo AF.
Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons.
Brain Res. 2004 Jan 30;997(1):103-10.
"We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures. Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co-expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists. Promoter activity was decreased two- to threefold by treatment with five 5-HT(1B/D) agonists, including the triptan drugs sumatriptan, eletriptan, and rizatriptan that are used for migraine treatment. As controls, CMV promoter activity was not affected, and 5-HT(1B/D) receptor antagonists blocked the repression caused by sumatriptan and eletriptan. Thus, adenoviral gene transfer can be used in trigeminal ganglia neurons for studying the mechanisms of triptan drug action on CGRP synthesis." [Abstract]

Oliver KR, Wainwright A, Edvinsson L, Pickard JD, Hill RG.
Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature.
J Cereb Blood Flow Metab. 2002 May;22(5):620-9.
"Calcitonin gene-related peptide and adrenomedullin belong to a structurally related neuropeptide family and are potent vasodilators expressed in the trigeminovascular system. The molecular identity of receptors for these proteins has only recently been elucidated. Central to functional binding of these neuropeptides is the G-protein-coupled receptor, the calcitonin receptor-like receptor (CRLR), whose cell surface expression and pharmacology is determined by coexpression of a receptor activity-modifying protein (RAMP). CRLR combined with RAMP binds calcitonin gene-related peptide with high affinity, whereas CRLR coexpression with RAMP2 or -3 confers high-affinity binding of adrenomedullin. The authors investigated the expression of these receptor components in human cerebral vasculature to further characterize neuropeptide receptor content and the potential functions of these receptors. Localization has been carried out using specific antisera raised against immunogenic peptide sequences that were subsequently applied using modern immunohistochemical techniques and confocal microscopy. The results are the first to show the presence of these receptor component proteins in human middle meningeal, middle cerebral, pial, and superficial temporal vessels, and confirm that both calcitonin gene-related peptide and adrenomedullin receptors may arise from the coassembly of RAMPs with CRLR in these vessel types. These novel data advance the understanding of the molecular function of the trigeminovascular system, its potential role in vascular headache disorders such as migraine, and may lead to possible ways in which future synthetic ligands may be applied to manage these disorders." [Abstract]

Smith D, Hill RG, Edvinsson L, Longmore J.
An immunocytochemical investigation of human trigeminal nucleus caudalis: CGRP, substance P and 5-HT1D-receptor immunoreactivities are expressed by trigeminal sensory fibres.
Cephalalgia. 2002 Jul;22(6):424-31.
"5-HT1D (but not 5-HT1B)-receptor immunoreactivity (i.r.) can be detected on trigeminal fibres within the spinal trigeminal tract of the human brainstem. The present study used immunohistochemical and morphometric techniques to determine the proportions of trigeminal fibres expressing substance P, CGRP or 5-HT1D-receptor immunoreactivities. Co-localization studies between 5-HT1D-receptor and substance P- or CGRP-i.r. were also performed. Brainstem material was obtained with consent (four donors) and the total number of immunoreactive fibres within the trigeminal tract was estimated using random field sampling. A greater proportion of fibres (>1 microm diameter) expressed CGRP-i.r. (80 +/- 6%) compared with substance P-i.r. (46 +/- 7%) or 5-HT1D-receptor-i.r. (25 +/- 1%). 5-HT1D-receptor-i.r. was co-localized on some CGRP- or substance P-i.r. fibres. This suggests that 5-HT1D-receptors can regulate the release of CGRP and substance P and may be relevant to the clinical effectiveness of 5-HT1B/1D-receptor agonists in the treatment of migraine and other cranial pain syndromes." [Abstract]

Hou M, Kanje M, Longmore J, Tajti J, Uddman R, Edvinsson L.
5-HT(1B) and 5-HT(1D) receptors in the human trigeminal ganglion: co-localization with calcitonin gene-related peptide, substance P and nitric oxide synthase.
Brain Res. 2001 Aug 3;909(1-2):112-20.
"5-Hydroxytryptamine (5-HT) is implicated in migraine and agonist directed against 5-HT(1B) and 5-HT(1D) receptors are commonly used as effective therapies. The antimigraine mechanisms involve the inhibition of intracranial sensory neuropeptide release. In order to determine which 5-HT(1) receptor subtypes are involved we have by immunocytochemistry examined the distribution of 5-HT(1B) and 5-HT(1D) receptors in the human trigeminal ganglia, and addressed which of them colocalize with calcitonin gene-related peptide (CGRP), substance P (SP) or nitric oxide synthase (NOS). We detected that 5-HT(1D) receptor immunoreactivity (i.r.) was predominantly expressed in medium-sized cells (86% of positive cells, 30-60 microm). About 9% of the 5-HT(1D) receptor i.r. cells were large in size (> 60 microm) and 5% were small in size (< 30 microm). In a similar pattern, 5-HT(1B) receptor i.r. was mainly expressed in medium-sized cells (81% in 30-60 microm, 15% in > 60 microm and 4% in < 30 microm). Double immunostaining was used to determine whether the 5-HT(1B) or 5-HT(1D) receptor immunoreactive cells co-localized with either CGRP, SP or NOS. Thus, 89% of the CGRP i.r. cells expressed 5-HT(1D) receptor i.r. and 65% of the CGRP positive cells were 5-HT(1B) receptor positive. Most of the 5-HT(1D) (95%) and the 5-HT(1B) (94%) receptor i.r. cells showed SP immunostaining and 83% of 5-HT(1D) receptor and 86% of 5-HT(1B) receptor i.r. cells contained NOS. In conclusion, both 5-HT(1B) and 5-HT(1D) receptors are expressed in the human trigeminal ganglion and they are mainly localized in medium-sized cells and they seem to colocalize with CGRP, SP and NOS." [Abstract]

Ma QP, Hill R, Sirinathsinghji D.
Colocalization of CGRP with 5-HT1B/1D receptors and substance P in trigeminal ganglion neurons in rats.
Eur J Neurosci. 2001 Jun;13(11):2099-104.
"Vasodilatation in the dura mater has been implicated in migraine pathogenesis. Anti-migraine triptan drugs block vasodilatation by binding to 5-HT1B/1D receptors localized on the peripheral sensory terminals and dural blood vessel smooth muscles. Previous studies suggest that calcitonin gene-related peptide (CGRP) released from Adelta-fibres plays a more important role than substance P (SP) released from C-fibres in inducing dural vasodilatation and that one of the antimigraine mechanisms of triptan drugs is inhibiting CGRP release. In the present study, the relationship between CGRP and 5-HT1B/1D receptors, and between CGRP and SP in the trigeminal ganglion neurons in rats was examined by double immunohistochemical staining. CGRP, 5-HT1B, 5-HT1D and SP-positive trigeminal ganglion neurons were all predominantly small and medium-sized. In the trigeminal ganglia, approximately 50% of CGRP-positive neurons were 5-HT1B positive. Similarly, approximately 55% of CGRP-positive neurons were 5-HT1D immunoreactive. Approximately 50% of CGRP-positive neurons were SP-positive, while 93% of SP-positive neurons were CGRP-positive, suggesting that nearly all SP-positive neurons also contain CGRP. The fibre types of the 5-HT1B- and 5-HT1D-positive neurons were further investigated with an antibody against the A-fibre marker 200-kDa neurofilaments (NF200). Approximately 46% of the 5-HT1B-positive and 43% of the 5-HT1D-positive trigeminal ganglion neurons were also NF200 positive, indicating that many A-fibre trigeminal neurons express 5-HT1B or 5-HT1D receptors. These results support the hypothesis that one important action of antimigraine drugs is the inhibition of CGRP release and that Adelta-fibres may play an important role in migraine pathogenesis." [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]

Goadsby PJ, Hoskin KL, Storer RJ, Edvinsson L, Connor HE.
Adenosine A1 receptor agonists inhibit trigeminovascular nociceptive transmission.
Brain. 2002 Jun;125(Pt 6):1392-401.
"There is a considerable literature to suggest that adenosine A1 receptor agonists may have anti-nociceptive effects, and we sought to explore the role of adenosine A1 receptors in a model of trigeminovascular nociceptive transmission. Cats were anaesthetized (alpha-chloralose 60 mg/kg, intraperitoneally), and prepared for physiological monitoring. The superior sagittal sinus (SSS) was stimulated electrically, and linked units were recorded in the trigeminocervical complex. Post-stimulus histograms were constructed to analyse the responses and the effect of drug administration. Blood was sampled from the external jugular vein to determine levels of calcitonin gene-related peptide (CGRP) release before and after drug administration. Intravenous administration of the highly selective adenosine A1 receptor agonist, GR79236 (3-100 microg/kg) had a dose-dependent inhibitory effect on SSS-evoked trigeminal activity. The maximal effect (80 +/- 6% reduction in probability of firing) was seen at 100 microg/kg. The neuronal inhibitory effect of GR79236 could be inhibited by the selective adenosine A1 receptor antagonist DPCPX (300 microg/kg; P < 0.05). SSS stimulation increased cranial CGRP levels from 33 +/- 2 pmol/l (n = 6) to 64 +/- 3 pmol/l, an effect substantially reduced by pre-treatment with GR79236 (30 microg/kg; P < 0.01). The selective low efficacy adenosine A1 receptor agonist, GR190178 (30-1000 microg/kg i.v.), also inhibited SSS-evoked neuronal activity in a dose-dependent fashion. In this model of trigeminovascular nociception, adenosine A1 receptor activation leads to neuronal inhibition without concomitant vasoconstriction, suggesting a novel avenue for the treatment of migraine and cluster headache." [Abstract]

Carruthers, Alan M., Sellers, Lynda A., Jenkins, David W., Jarvie, Emma M., Feniuk, Wasyl, Humphrey, Patrick P. A.
Adenosine A1 Receptor-Mediated Inhibition of Protein Kinase A-Induced Calcitonin Gene-Related Peptide Release from Rat Trigeminal Neurons
Mol Pharmacol 2001 59: 1533-1541
"Calcitonin gene-related peptide (CGRP), a potent vasodilator, has been implicated in the pathogenesis of migraine. Its release from adult rat trigeminal neurons in culture was shown to be markedly increased by the activation of adenylate cyclase with forskolin. Modulation of this secretion was investigated by a number of agents with known inhibitory effects on cAMP generation mediated via receptor coupling to G(i/o) proteins. Significantly, forskolin-stimulated CGRP release could be closely correlated with the phosphorylation of the protein kinase A (PKA) substrate cyclic AMP response element-binding protein (CREB). Forskolin-stimulated CGRP release could be potently and effectively inhibited by the adenosine A(1) receptor-selective agonist GR79236X (pIC(50) = 7.7 +/- 0.1, maximal inhibition 65 +/- 2.5% at 300 nM), whereas the A(2A) (CGS21680) and the A(3) (2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide) receptor-selective agonists were without effect. GR79236X-mediated inhibition was abolished by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Immunocytochemical studies and Western analysis revealed the presence of adenosine A(1) receptors on trigeminal neurons. However, despite the additional detection of 5-hydroxytryptamine (5-HT)(1B) receptors on these cells, the clinically effective antimigraine 5-HT(1B/1D) agonist sumatriptan did not inhibit forskolin-stimulated CGRP release nor did it show any effect on the concomitant CREB phosphorylation. In contrast, the mu-opioid agonist fentanyl elicited a 74 +/- 4% reduction in CGRP levels. Forskolin-stimulated CGRP release and CREB phosphorylation could be mimicked by incubation of the cells with chlorophenylthio-cAMP and blocked by pretreatment with the PKA inhibitor myrPKI(14-22). Taken together, the present data confirm the PKA-dependence of forskolin-stimulated CGRP release and suggest that A(1) adenosine agonists may warrant further investigation in models of migraine and neurogenic inflammation." [Full Text]

Durham PL, Cady R, Cady R.
Regulation of calcitonin gene-related peptide secretion from trigeminal nerve cells by botulinum toxin type A: implications for migraine therapy.
Headache. 2004 Jan;44(1):35-42; discussion 42-3. [Abstract]

Akerman S, Williamson DJ, Goadsby PJ.
Voltage-dependent calcium channels are involved in neurogenic dural vasodilatation via a presynaptic transmitter release mechanism.
Br J Pharmacol. 2003 Oct;140(3):558-66. Epub 2003 Aug 26.
"A missense mutation of the CACNA1A gene that encodes the alpha1A subunit of the voltage-dependent P/Q-type calcium channel has been discovered in patients suffering from familial hemiplegic migraine. This suggested that calcium channelopathies may be involved in migraine more broadly, and established the importance of genetic mechanisms in migraine. Channelopathies share many clinical characteristics with migraine, and thus exploring calcium channel functions in the trigeminovascular system may give insights into migraine pathophysiology. It is also known that drugs blocking the P/Q- and N-type calcium channels have been successful in other animal models of trigeminovascular activation and head pain. In the present study, we used intravital microscopy to examine the effects of specific calcium channel blockers on neurogenic dural vasodilatation and calcitonin gene-related peptide (CGRP)-induced dilation. The L-type voltage-dependent calcium channel blocker calciseptine significantly attenuated (20 microg kg(-1), n=7) the dilation brought about by electrical stimulation, but did not effect CGRP-induced dural dilation. The P/Q-type voltage-dependent calcium channel blocker omega-agatoxin-IVA (20 microg kg-1, n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP-induced dural dilation. The N-type voltage-dependent calcium channel blocker omega-conotoxin-GVIA (20 microg kg(-1), n=8 and 40 microg kg(-1), n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP-induced dural dilation. It is thought that the P/Q-, N- and L-type calcium channels all exist presynaptically on trigeminovascular neurons, and blockade of these channels prevents CGRP release, and, therefore, dural blood vessel dilation. These data suggest that the P/Q-, N- and L-type calcium channels may be involved in trigeminovascular nociception."
[Abstract]