DHE Repression of ATP‐Mediated Sensitization of Trigeminal Ganglion Neurons

Objective.— To investigate the mechanism by which adenosine triphosphate (ATP) causes sensitization of trigeminal neurons and how dihydroergotamine (DHE) represses this modulatory effect. Background.— Dihydroergotamine is an effective treatment of migraine. The cellular mechanisms of action of DHE in treating migraine attacks remain unclear. Methods.— In this study, neonatal rat trigeminal ganglia cultures were used to investigate effects of ATP, alpha, beta‐methyl ATP (α,β‐meATP), and DHE on intracellular calcium levels and calcitonin gene‐related peptide (CGRP) secretion. Results.— Pretreatment with ATP or α,β‐meATP caused sensitization of neurons, via P2X ₃ receptors, such that a subthreshold amount of potassium chloride (KCl) significantly increased intracellular calcium levels and CGRP secretion. Pretreatment with DHE repressed increases in calcium and CGRP secretion in response to ATP‐KCl or α,β‐meATP‐KCl treatment. Importantly, these inhibitory effects of DHE were blocked with an α ₂ ‐adrenoceptor antagonist and unaffected by a 5HT _1B/D receptor antagonist. DHE also decreased neuronal membrane expression of the P2X ₃ receptor. Conclusions.— Our findings provide evidence for a novel mechanism of action for DHE that involves blocking ATP‐mediated sensitization of trigeminal neurons, repressing stimulated CGRP release, and decreasing P2X ₃ membrane expression via activation of α ₂ ‐adrenoceptors.     

Selective Inhibition of 5‐HT7 Receptor Reduces CGRP Release in an Experimental Model for Migraine

Objective.— To investigate the role of 5‐HT₇ receptors on the release of calcitonin gene‐related peptide (CGRP) in an animal model of migraine. Background.— Calcitonin gene‐related peptide has been identified as a key neuropeptide in the pathophysiology of migraine. It is elevated in the external jugular vein during migraine attacks in humans and after stimulation of the trigeminal ganglion in animal models of migraine. This can be treated with the 5‐HT_1B/1D receptor agonist sumatriptan concomitant with headache relief. Nevertheless, triptans, the most effective agents for the treatment of acute migraine attacks, are not effective in more than 1/3 of migraineurs and less than 50% of migraineurs achieve complete pain freedom. This indicates other serotonin receptors may be involved in the pathophysiology of migraine. Increasing evidence has shown that 5‐HT₇ receptors may be involved in migraine pathogenesis. However, direct evidence for the role of 5‐HT₇ receptors in migraine is still lacking. Methods.— Unilateral electrical stimulation of the trigeminal ganglion (TGES) was performed in anesthetized male Sprague‐Dawley rats. Animals were pretreated with sumatriptan (300 µg/kg, i.v.), selective 5‐HT₇ receptor antagonist SB269970 (5, 10 mg/kg, s.c.), potential 5‐HT₇ receptor agonist AS19 (5, 10 mg/kg, s.c.) or co‐administration of SB269970 and AS19 (10 mg/kg, s.c.). Serum CGRP concentrations in the ipsilateral jugular vein were determined before and at 2 and 5 minutes after the start of TGES. Results.— Our results showed that sumatriptan almost completely inhibited the release of CGRP evoked by TGES. Pre‐administration of SB269970 (5, 10 mg/kg) caused a significant decrease in serum CGRP concentrations at 2 and 5 minutes following the onset of TGES, with a less inhibitory effect compared with sumatriptan. AS19 had no significant effect on CGRP release, while the SB269970‐induced inhibitory effect was reversed by AS19. Conclusions.— Selective inhibition of 5‐HT₇ receptors partly reduced CGRP release evoked by TGES. These findings suggest that 5‐HT₇ receptors may play a role in the pathophysiology of migraine. (Headache 2010;50:579‐587)     

Role of K ATP channels in cephalic vasodilatation induced by calcitonin gene-related peptide, nitric oxide, and transcranial electrical stimulation in the rat

Objective.— The objective of this study was to explore the role of K_ATP channels in vasodilatation induced by calcitonin gene‐related peptide (CGRP), nitric oxide (NO), and transcranial electrical stimulation (TES) in intracranial arteries of rat. Background.— Dilatation of cerebral and dural arteries causes a throbbing, migraine‐like pain. Both CGRP and NO are potent vasodilators that can induce migraine. Their antagonists are effective in the treatment of migraine attacks. K_ATP channel openers cause headache in the majority of healthy subjects suggesting a role for K_ATP channels in migraine pathogenesis. We hypothesized that vasodilatation induced by CGRP and the NO donor glyceryltrinitrate (GTN) is mediated via K_ATP channels. Methods.— We examined the effects of the K_ATP channel inhibitor glibenclamide on dural and pial vasodilatation induced by CGRP, NO, and endogenously released CGRP by TES. A rat genuine closed cranial window model was used for in vivo studies and myograph baths for studying the effect in vitro. In the closed cranial window model the diameter of dural vessels was measured directly in anesthetized animals to investigate the vascular effects of infused CGRP, NO, and endogenous CGRP after electrical stimulation. Also diameter changes of pial arteries, mean arterial blood pressure and local cerebral blood flow by Laser Doppler flowmetry (LCBF_Flux) were measured. Results.— CGRP, NO, and TES caused dilatation of the 2 arteries in vivo and in vitro. In anesthetized rats glibenclamide significantly attenuated CGRP induced dural and TES induced dural/pial artery dilatation (P = .001; P = .001; P = .005), but had no effect on dural/pial vasodilatation induced by GTN. In vitro glibenclamide failed to significantly inhibit CGRP‐ and GTN‐induced vasodilatation. Conclusions.— These results show that a K_ATP channel blocker in vivo but not in vitro inhibits CGRP, but not GTN‐induced dilatation of dural and pial arteries, a mechanism thought to be important in migraine.     

Sex differences in behavior and expression of CGRP-related genes in a rodent model of chronic migraine

(Headache 2011;51:674‐692) Objective.— The objectives of this study were to develop a preclinical rodent model that produces migraine‐like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene‐related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. Background.— Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate‐buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin‐like receptor (CLR), receptor activity‐modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. Methods.— Male and female Sprague‐Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post‐application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real‐time polymerase chain reactions were used to measure the expression of amplified constructs using gene‐specific primers for CGRP, RAMP1, CLR, and RCP. Results.— Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP‐encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP‐encoding mRNAs. In the medulla, females had higher baseline levels of CGRP‐encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP‐encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP‐related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP‐related genes were upregulated by both IS and PBS applications. Conclusions.— This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine‐like behaviors occur after meningeal inflammation.     

Distribution of Artemin and GFRα3 Labeled Nerve Fibers in the Dura Mater of Rat

(Headache 2010;50:442‐450) Objective.— We examined the distribution of artemin and its receptor, glial cell line‐derived neurotrophic factor family receptor α3 (GFRα3), in the dura mater of rats. Background.— Artemin, a member of the glial cell line‐derived neurotrophic factor family, is a vasculature‐derived growth factor shown to regulate migration of sympathetic neuroblasts and targeting of sympathetic innervation. The artemin receptor, GFRα3, is present in both sympathetic efferents and a subpopulation of nociceptive afferents. Recent evidence has shown that artemin may contribute to inflammatory hyperalgesia. The extent to which artemin is present in the dural vasculature and its relationship to GFRα3 containing fibers have yet to be investigated. Methods.— We used retrograde labeling, double and triple labeling with immunohistochemistry on the dura mater and trigeminal ganglia of female Sprague‐Dawley rats. Results.— Artemin‐like immunoreactivity (‐LI) was detected in the smooth muscle of dural vasculature. GFRα3‐LI was present in nerve fibers that closely associated with tyrosine hydroxylase or calcitonin gene‐related peptide (CGRP). CGRP‐LI and transient receptor potential ion channel 1 (TRPV1)‐LI were present in all GFRα3‐positive dural afferents, which constituted 22% of the total population of dural afferents. Conclusions.— These anatomical results support the hypothesis that artemin contributes to dural afferent activity, and possibly migraine pain, through modulation of both primary afferent and sympathetic systems.     

Involvement of Pro‐Nociceptive 5‐HT2A Receptor in the Pathogenesis of Medication‐Overuse Headache

(Headache 2010;50:185‐197) Objectives.— To determine the involvement of 5‐HT_2A (5‐HT_2A) receptor in the process of trigeminal plasticity induced by chronic analgesic exposure and in the process of inflammatory‐induced thermal hyperalgesia. Background.— Derangement in 5‐HT_2A serotonin receptor has been reported to implicate in pathogenesis of medication‐overuse headache. No clear explanation concerning the precise roles of these receptors in the process. Methods.— Wistar rats were daily administered with paracetamol (200 mg/kg) for 30 days. On the next day, ketanserin, a 5‐HT_2A antagonist, or saline was given prior to cortical spreading depression (CSD) induction. Electrocorticogram, cortical blood flow, Fos and 5‐HT_2A‐immunoreactivity in cortex and trigeminal pathway were studied. In the other experiment, complete Freund's adjuvant was injected into the rat hind paw to induce tissue inflammation. Three days later, ketanserin was given and noxious heat was applied to both inflamed and noninflamed paws. The response between 2 sides was compared by measuring paw withdrawal latency. Results.— Chronic paracetamol exposure led to an increase in CSD frequency and CSD‐evoked Fos expression in cerebral cortex indicating the increase in neuronal excitability. Prolonged medication exposure also facilitated trigeminal nociception as evident by an increase in CSD‐evoked Fos expression in trigeminal nucleus caudalis. The expression of 5‐HT_2A receptor in cerebral cortex and trigeminal ganglia was enhanced by chronic paracetamol administration. Pretreatment with ketanserin significantly attenuated these effects. The second experiment showed that ketanserin was able to lengthen the paw withdrawal latency in the inflamed side but did not alter nociceptive response in the noninflamed side. Conclusion.— These findings suggest that up‐regulation of pro‐nociceptive 5‐HT_2A receptor is an important step in the process of cortical hyper‐excitation and nociceptive facilitation induced by chronic analgesic exposure.     

Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro

To cite this article: Banfi C, Brioschi M, Lento S, Pirillo A, Galli S, Cosentino S, Tremoli E, Mussoni L. Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro. J Thromb Haemost 2011; 9 : 1608–19. Summary. Background: Protease‐activated receptors (PARs) are G‐protein‐coupled receptors that function in hemostasis and thrombosis, as well as in the inflammatory and proliferative responses triggered by tissue injury. We have previously shown that PAR1 or PAR2 occupancy by specific PAR‐agonist peptides (PAR‐APs) induces tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs), where TF regulation by PAR1 (but not by PAR2) requires intact endothelial caveolin‐enriched membrane microdomains in which PAR1 and caveolin‐1 associate. Objectives: The aim of this study was to determine the effects of cholesterol‐lowering agents (statins) and cholesterol‐loading lipoprotein on PAR1‐AP‐mediated and PAR2‐AP‐mediated TF induction in HUVECs. Results: Statins completely prevented TF induction by PAR‐APs in an isoprenoid‐independent manner, induced the delocalization of PAR1 from caveolin‐enriched membrane microdomains without affecting PAR1 mRNA, and decreased PAR2 mRNA and protein levels. Statins also prevented PAR‐AP‐mediated extracellular signal‐related kinase 1/2 activation, which is crucial for TF induction. The redistribution of PAR1 is accompanied by the relocation of the membrane microdomain‐associated G‐protein α, caveolin‐1, and Src, which we previously showed to play a key role in signal transduction and TF induction. Conversely, cholesterol loading potently amplified PAR1‐AP‐induced TF, probably as a result of the increased abundance of PAR1 and the Src and G‐protein α signaling molecules in the caveolin‐1‐enriched fraction, without affecting PAR1 mRNA. Conclusions: As PARs have important functions in hemostasis, cancer, thrombosis, and inflammatory processes, our findings that statins prevent TF induction by PAR‐APs altering the membrane localization of PAR1 and the expression of PAR2 suggest that they may provide health benefits other than reducing atherosclerosis.     

Calcitonin gene-related peptide does not cause migraine attacks in patients with familial hemiplegic migraine

(Headache 2011;51:544‐553) Background.— Calcitonin gene‐related peptide (CGRP) is a key molecule in migraine pathogenesis. Intravenous CGRP triggers migraine‐like attacks in patients with migraine with aura and without aura. In contrast, patients with familial hemiplegic migraine (FHM) with known mutations did not report more migraine‐like attacks compared to controls. Whether CGRP triggers migraine‐like attacks in FHM patients without known mutations is unknown. Objective.— In the present study we therefore examined the migraine‐inducing effect of CGRP in FHM patients without known mutations and healthy controls. Methods and design.— Eleven patients suffering from FHM without known mutations and 11 controls received an intravenous infusion of 1.5 µg/minute CGRP over 20 minutes. The study design was a balanced and controlled provocation study. Headache and other migraine symptoms were scored for 1 hour and self‐recorded hourly thereafter until 13‐hour postinfusion. Results.— We found no difference in the incidence of migraine‐like attacks between the 2 groups, with 9% (1 of 11) of patients and 0% (0 of 10) of controls reporting migraine‐like headache (P = 1.00). CGRP infusion did not induce aura symptoms in any of the participants. There was no difference in the incidence of CGRP‐induced delayed headaches between the groups (P = .18). Conclusion.— In contrast to patients suffering from migraine with aura and without aura, CGRP infusion did not induce more migraine‐like attacks in FHM patients without known mutations compared to controls. It seems that the majority of FHM patients with and without known mutation display no sensitivity to CGRP signaling compared to common types of migraine.     

Modulation of meningeal nociceptors mechanosensitivity by peripheral proteinase-activated receptor-2: the role of mast cells

Inflammatory-related activation and sensitization of meningeal nociceptors is believed to play a key role in promoting the intracranial throbbing pain of migraine. We have shown recently that mast cell activation and various mast cell-derived inflammatory mediators can promote activation and sensitization of meningeal nociceptors. Mast cell tryptase has also been proposed to promote pain hypersensitivity by activating the proteinase-activated receptor 2 (PAR2) that is expressed on nociceptive neurons. In this study using in vivo single-unit recording in the trigeminal ganglion of anaesthetized rats, we found that local meningeal activation of PAR2 using the specific agonist SLIGRL-NH₂ promoted sensitization of the threshold response while provoking desensitization of the suprathreshold responses. SLIGRL-NH₂ also excited a subpopulation of meningeal nociceptors. Chronic mast cell depletion enhanced the sensitizing effects of PAR2 activation while curbing its desensitizing effects. Mast cell depletion did not change the PAR2-mediated excitatory effect. We propose that by enhancing the mechanical sensitivity of meningeal nociceptors local PAR2 activation could play a role in promoting the throbbing pain of migraine and that local mast cell degranulation may modulate such an effect.     

Insulin sensitizes neural and vascular TRPV1 receptors in the trigeminovascular system

BackgroundClinical observations suggest that hyperinsulinemia and insulin resistance can be associated with migraine headache. In the present study we examined the effect of insulin on transient receptor potential vanilloid 1 (TRPV1) receptor-dependent meningeal nociceptor functions in rats.MethodsThe effects of insulin on the TRPV1 receptor stimulation-induced release of calcitonin gene related peptide (CGRP) from trigeminal afferents and changes in meningeal blood flow were studied. Colocalization of the insulin receptor, the TRPV1 receptor and CGRP was also analyzed in trigeminal ganglion neurons.ResultsInsulin induced release of CGRP from meningeal afferents and consequent increases in dural blood flow through the activation of TRPV1 receptors of trigeminal afferents. Insulin sensitized both neural and vascular TRPV1 receptors making them more susceptible to the receptor agonist capsaicin. Immunohistochemistry revealed colocalization of the insulin receptor with the TRPV1 receptor and CGRP in a significant proportion of trigeminal ganglion neurons.ConclusionsInsulin may activate or sensitize meningeal nociceptors that may lead to enhanced headache susceptibility in persons with increased plasma insulin concentration.     

CGRP and NO in the Trigeminal System: Mechanisms and Role in Headache Generation

Calcitonin gene‐related peptide (CGRP) and metabolic products of nitric oxide (NO) are increased in jugular venous plasma during migraine attacks and other primary headaches. Patients suffering from primary headaches are particularly sensitive to CGRP and NO donors responding with delayed headaches to an infusion of either of these substances. Accordingly, both CGRP and NO are considered as key mediators in migraine, and clinical trials have shown that inhibitors of CGRP receptors and NO synthase are effective in treating migraine. There is an implicit understanding that CGRP and NO systems interact, and here, we review the body of preclinical work on these systems focusing on the trigeminovascular system in migraine. NO derives from various cell types via 3 isoforms of NO synthase, whereas CGRP is produced from a subset of trigeminal afferents. In rodents, NO donors cause activity alterations on different levels of the trigeminal system including enhancement of CGRP release, which in turn results in arterial vasodilatation and possibly mast cell degranulation in the meninges. The activity of spinal trigeminal neurons, which is a sensitive integrative measure for trigeminal activity, is partly under the control of CGRP and NO. Both mediators facilitate nociceptive transmission, possibly via presynaptic mechanisms. These functions are supported by immunolocalization of CGRP receptor components on 3 trigeminovascular levels: cranial dura mater, trigeminal ganglion, and spinal trigeminal nucleus. Current data support a relationship of CGRP and NO actions on all levels of the trigeminovascular system and emphasize central CGRP receptors as possible therapeutic targets.     

Kynurenate Derivative Attenuates the Nitroglycerin‐Induced CamKIIα and CGRP Expression Changes

(Headache 2010;50:834‐843) Objective.— To examine the efficacy of L‐kynurenine and a novel kynurenic acid derivative on the nitroglycerin‐induced calmodulin‐dependent protein kinase II alpha (CamKIIα) and calcitonin gene‐related peptide (CGRP) expression changes in the rat caudal trigeminal nucleus. Background.— Systemic administration of the nitric oxide donor nitroglycerin can trigger an attack in migraineurs. In the rat, nitroglycerin activates second‐order neurons in the caudal trigeminal nucleus, and increases expression of the CamKIIα and decreases that of the CGRP there. As glutamatergic mechanisms may be crucial in trigeminal pain processing, the aim of our study was to examine the effects of L‐kynurenine, a metabolic precursor of the N‐methyl D‐aspartate receptor antagonist kynurenic acid, on the nitroglycerin‐induced changes in CamKIIα and CGRP immunoreactivity. Methods.— One hour before the nitroglycerin (10 mg/kg bodyweight, s.c.) injection, the animals were pretreated with L‐kynurenine (300 mg/kg bodyweight, i.p.) or 2‐(2‐N,N‐dimethylaminoethylamine‐1‐carbonyl)‐1H‐quinolin‐4‐one hydrochloride (300 mg/kg bodyweight, i.p.), a novel kynurenic acid derivative. Four hours later, the rats were perfused transcardially and the cervical spinal cord segments were removed for immunohistochemistry. Results.— L‐kynurenine and 2‐(2‐N,N‐dimethylaminoethylamine‐1‐carbonyl)‐1H‐quinolin‐4‐one hydrochloride pretreatment attenuated the nitroglycerin‐induced changes in CamKIIα and CGRP immunoreactivity in the rat caudal trigeminal nucleus. Conclusions.— These findings suggest a mechanism by which the inhibition of the excitatory amino acid receptors by kynurenic acid and its derivatives can alter trigeminal nociception.     

The soluble form of urokinase receptor promotes angiogenesis through its Ser88‐Arg‐Ser‐Arg‐Tyr92 chemotactic sequence

Summary. Background: The urokinase plasminogen activator receptor (u‐PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u‐PA) on the endothelial cell surface, thus promoting angiogenesis in a protease‐dependent manner. The u‐PAR may exist in a glycophosphatidylinositol‐anchored and in a soluble form (soluble u‐PAR [Su‐PAR]), both including the chemotactic Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² internal sequence. Objective: To investigate whether Su‐PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² sequence. Methods and Results: In this study, the formation of vascular‐like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su‐PAR protein promotes the formation of cord‐like structures, and that this ability is retained by the isolated Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² chemotactic sequence, the maximal effect being reached at 10 nmol L^?1 SRSRY peptide (SRSRY). This effect is mediated by the α_vβ₃ vitronectin receptor, is independent of u‐PA proteolytic activity, and involves the internalization of the G‐protein‐coupled formyl‐peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su‐PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su‐PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2. Conclusions: Our results show a new protease‐independent activity of Su‐PAR that stimulates in vivo angiogenesis through its Ser⁸⁸‐Arg‐Ser‐Arg‐Tyr⁹² chemotactic sequence.     

Two Mechanisms Involved in Trigeminal CGRP Release: Implications for Migraine Treatment

Objective/Background.— The goal of this study was to better understand the cellular mechanisms involved in proton stimulation of calcitonin gene‐related peptide (CGRP) secretion from cultured trigeminal neurons by investigating the effects of 2 antimigraine therapies, onabotulinumtoxinA and rizatriptan. Stimulated CGRP release from peripheral and central terminating processes of trigeminal ganglia neurons is implicated in migraine pathology by promoting inflammation and nociception. Based on models of migraine pathology, several inflammatory molecules including protons are thought to facilitate sensitization and activation of trigeminal nociceptive neurons and stimulate CGRP secretion. Despite the reported efficacy of triptans and onabotulinumtoxinA to treat acute and chronic migraine, respectively, a substantial number of migraineurs do not get adequate relief with these therapies. A possible explanation is that triptans and onabotulinumtoxinA are not able to block proton‐mediated CGRP secretion. Methods.— CGRP secretion from cultured primary trigeminal ganglia neurons was quantitated by radioimmunoassay while intracellular calcium and sodium levels were measured in neurons via live cell imaging using Fura‐2 AM and SBFI AM, respectively. The expression of acid‐sensing ion channel 3 (ASIC3) was determined by immunocytochemistry and Western blot analysis. In addition, the involvement of ASICs in mediating proton stimulation of CGRP was investigated using the potent and selective ASIC3 inhibitor APETx2. Results.— While KCl caused a significant increase in CGRP secretion that was significantly repressed by treatment with ethylene glycol tetraacetic acid (EGTA), onabotulinumtoxinA, and rizatriptan, the stimulatory effect of protons (pH 5.5) was not suppressed by EGTA, onabotulinumtoxinA, or rizatriptan. In addition, while KCl caused a transient increase in intracellular calcium levels that was blocked by EGTA, no appreciable change in calcium levels was observed with proton treatment. However, protons did significantly increase the intracellular level of sodium ions. Under our culture conditions, ASIC3 was shown to be expressed in most trigeminal ganglion neurons. Importantly, proton stimulation of CGRP secretion was repressed by pretreatment with the ASIC3 inhibitor APETx2, but not the transient receptor potential vanilloid‐1 antagonist capsazepine. Conclusions.— Our findings provide evidence that proton regulated release of CGRP from trigeminal neurons utilizes a different mechanism than the calcium and synaptosomal‐associated protein 25‐dependent pathways that are inhibited by the antimigraine therapies, rizatriptan and onabotulinumtoxinA.     

Increase of capsaicin-induced trigeminal Fos-like immunoreactivity by 5-HT(7) receptors

Objective.— To explore whether pharmacological stimulation of the 5‐hydroxytryptamine₇ (5‐HT₇) receptor modulates Fos‐like immunoreactivity in the trigeminal nucleus caudalis of rats. Background.— The serotonin 5‐HT₇ receptor was proposed to be involved in migraine pathogenesis and evidence suggests it plays a role in peripheral nociception and hyperalgesia through an action on sensory afferent neurons. Methods.— The potential activating or sensitizing role of 5‐HT₇ receptors on trigeminal sensory neurons, as visualized by Fos‐like immunoreactivity in the superficial layers of the trigeminal nucleus caudalis in rats, was investigated using the 5‐HT₇ receptor agonist, LP‐211, in the absence and the presence of intracisternal capsaicin, respectively. The agonist effect was characterized with the 5‐HT₇ receptor antagonist, SB‐656104. Male Wistar rats received a subcutaneous injection of LP‐211, SB‐656104, and SB‐656104 + LP‐211. They were then anesthetized and prepared to receive an intracisternal injection of capsaicin or its vehicle. Animals were perfused and brains removed; sections of the brain stem from the area postrema to the CI level were obtained and processed for Fos immunohistochemistry. Results.— Capsaicin but not its vehicle induced Fos‐like immunoreactivity within laminae I and II of trigeminal nucleus caudalis. Pretreatment with LP‐211 had no effect on Fos‐like immunoreactivity but strongly increased the response produced by capsaicin; this effect was abolished by SB‐656104. Interestingly, capsaicin‐induced Fos‐like immunoreactivity was abolished by SB‐656104 pretreatment thus suggesting involvement of endogenous 5‐HT. Conclusions.— Data suggest that 5‐HT₇ receptors increase activation of meningeal trigeminovascular afferents and/or transmission of nociceptive information in the brain stem. This mechanism could be relevant in migraine and its prophylactic treatment.     

The PKB inhibitor Akti‐1/2 potentiates PAR‐1‐mediated platelet function independently of its ability to block PKB

Summary. Background: The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small‐molecule inhibitors and limiting genetic models. Recently, a selective, non‐ATP‐competitive PKB inhibitor, Akti‐1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. Objective and methods: To determine the effect of the PKB inhibitor Akti‐1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Results: Akti‐1/2 potently inhibited thrombin‐mediated PKB phosphorylation on Thr³⁰⁸ and Ser⁴⁷³ and phosphorylation of its downstream substrate GSK3β, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti‐1/2 strongly potentiated PAR‐1‐mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti‐1/2 was associated with increased [Ca⁺⁺]_i, PKC activation and degranulation and was ablated by agents that antagonized this pathway. Conclusions: The PKB inhibitor Akti‐1/2 increases PAR‐1‐mediated platelet responses in a PKB‐independent, Ca⁺⁺/PKC‐dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti‐1/2 and structurally related compounds.     

Differentiation of Nerve Fibers Storing CGRP and CGRP Receptors in the Peripheral Trigeminovascular System

Primary headaches such as migraine are postulated to involve the activation of sensory trigeminal pain neurons that innervate intracranial blood vessels and the dura mater. It is suggested that local activation of these sensory nerves may involve dural mast cells as one factor in local inflammation, causing sensitization of meningeal nociceptors. Immunofluorescence was used to study the detailed distribution of calcitonin gene–related peptide (CGRP) and its receptor components calcitonin receptor–like receptor (CLR) and receptor activity–modifying protein 1 (RAMP1) in whole-mount rat dura mater and in human dural vessels. The relative distributions of CGRP, CLR, and RAMP1 were evaluated with respect to each other and in relationship to mast cells, myelin, substance P, neuronal nitric oxide synthase, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide. CGRP expression was found in thin unmyelinated fibers, whereas CLR and RAMP1 were expressed in thicker myelinated fibers coexpressed with an A-fiber marker. CLR and RAMP1 immunoreactivity colocalized with mast cell tryptase in rodent; however, expression of both receptor components was not observed in human mast cells. Immunoreactive substance P fibers coexpressed CGRP, although neuronal nitric oxide synthase and vasoactive intestinal peptide expression was very limited, and these fibers were distinct from the CGRP-positive fibers. Few pituitary adenylate cyclase-activating polypeptide immunoreactive fibers occurred and some colocalized with CGRP.     

Involvement of AMPA Receptors in CSD‐Induced Impairment of LTP in the Hippocampus

Objective.— To investigate the alteration of hippocampal long‐term plasticity and basal synaptic transmission induced by repetitive cortical spreading depressions (CSDs). Background.— There is a relationship between migraine aura and amnesia attack. CSD, a state underlying migraine attacks, may be responsible for hippocampus‐related symptoms. However, the precise role of CSD on hippocampal activity has not been investigated. Methods.— Male Wistar rats were divided into CSD and control groups. Repetitive CSDs were induced in vivo by topical application of solid KCl. Forty‐five minutes later, the ipsilateral hippocampus was removed, and hippocampal slices were prepared for a series of electrophysiological studies. Results.— Repetitive CSDs led to a decrease in the magnitude of long‐term potentiation in the hippocampus. CSD also reduced hippocampal synaptic efficacy, as shown by a reduction in post‐synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor responses. In contrast, the post‐synaptic N‐methyl‐d ‐aspartate receptor responses remained unchanged. In addition, there were no changes in paired‐pulse profiles between the groups, indicating that CSD did not induce any presynaptic alterations. Conclusion.— These findings suggest that a reduction of post‐synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor responses is the mechanism responsible for impaired hippocampal long‐term potentiation induced by CSD.     

Calcitonin gene-related peptide and size of the atrial septal defect in new-onset migraine after transcatheter closure: results of a preliminary study

Background.— New‐onset migraine headache attacks (MHAs) can occur after atrial septal device implantation in patients without previous migraine. Plasma calcitonin gene‐related peptide (CGRP), which plays a crucial role in migraine pathophysiology, has shown to be released from specific cardiac tissues. Methods and Results.— We prospectively collected patients before and after closure and measured plasma CGRP levels using enzyme‐linked immunosorbent assay. Forty atrial septal defect (ASD) patients who had no migraine previously were enrolled. Four (23.5%) of the 17 consecutive patients whose CGRP levels were checked before ASD closure had new‐onset MHAs. The patients with MHAs had bigger ASD size (20 ± 0.9 vs 16 ± 1 mm, P = .009) and lower CGRP levels before closure (21.1 ± 3.9 vs 90.1 ± 27.1 pg/mL, P = .042) than those without. Among the 5 patients with blood samplings both during and between attacks, a paired comparison revealed a significantly increased level during attack (257.2 ± 45.5 vs 45.6 ± 25.5 pg/mL, P = .03). Conclusion.— Bigger ASD size and lower plasma CGRP levels before closure can be a potential predictor of new‐onset MHAs. Furthermore, a significant increase of CGRP levels during migraine attack implies that the occurrences of new‐onset MHAs after ASD closure correlate with the release of CGRP. This suggests CGRP sensitization from a lower baseline may be involved in the occurrence of new‐onset MHAs after ASD closure.