Oxytocin as a regulatory neuropeptide in the trigeminovascular system: Localization, expression and function of oxytocin and oxytocin receptors.

BACKGROUND: Recent clinical findings suggest that oxytocin could be a novel treatment for migraine. However, little is known about the role of this neuropeptide/hormone and its receptor in the trigeminovascular pathway. Here we determine expression, localization, and function of oxytocin and oxytocin receptors in rat trigeminal ganglia and targets of peripheral (dura mater and cranial arteries) and central (trigeminal nucleus caudalis) afferents. METHODS: The methods include immunohistochemistry, messenger RNA measurements, quantitative PCR, release of calcitonin gene-related peptide and myography of arterial segments. RESULTS: Oxytocin receptor mRNA was expressed in rat trigeminal ganglia and the receptor protein was localized in numerous small to medium-sized neurons and thick axons characteristic of A∂ sensory fibers. Double immunohistochemistry revealed only a small number of neurons expressing both oxytocin receptors and calcitonin gene-related peptide. In contrast, double immunostaining showed expression of the calcitonin gene-related peptide receptor component receptor activity-modifying protein 1 and oxytocin receptors in 23% of the small cells and in 47% of the medium-sized cells. Oxytocin immunofluorescence was observed only in trigeminal ganglia satellite glial cells. Oxytocin mRNA was below detection limit in the trigeminal ganglia. The trigeminal nucleus caudalis expressed mRNA for both oxytocin and its receptor. K+-evoked calcitonin gene-related peptide release from either isolated trigeminal ganglia or dura mater and it was not significantly affected by oxytocin (10 µM). Oxytocin directly constricted cranial arteries ex vivo (pEC50 ∼ 7); however, these effects were inhibited by the vasopressin V1A antagonist SR49059. CONCLUSION: Oxytocin receptors are extensively expressed throughout the rat trigeminovascular system and in particular in trigeminal ganglia A∂ neurons and fibers, but no functional oxytocin receptors were demonstrated in the dura and cranial arteries. Thus, circulating oxytocin may act on oxytocin receptors in the trigeminal ganglia to affect nociception transmission. These effects may help explain hormonal influences in migraine and offer a novel way for treatment.

Characterization of binding, functional activity, and contractile responses of the selective 5-HT1F receptor agonist lasmiditan.

BACKGROUND AND PURPOSE: Triptans are 5-HT1B/1D receptor agonists (that also display 5-HT1F receptor affinity) with antimigraine action, contraindicated in patients with coronary artery disease due to their vasoconstrictor properties. Conversely, lasmiditan was developed as an antimigraine 5-HT1F receptor agonist. To assess the selectivity and cardiovascular effects of lasmiditan, we investigated the binding, functional activity, and in vitro/in vivo vascular effects of lasmiditan and compared it to sumatriptan. EXPERIMENTAL APPROACH: Binding and second messenger activity assays of lasmiditan and other serotoninergic agonists were performed for human 5-HT1A , 5-HT1B , 5-HT1D , 5-ht1E , 5-HT1F , 5-HT2A , 5-HT2B , and 5-HT7 receptors, and the results were correlated with their potency to constrict isolated human coronary arteries (HCAs). Furthermore, concentration-response curves to lasmiditan and sumatriptan were performed in proximal and distal HCA, internal mammary, and middle meningeal arteries. Finally, anaesthetized female beagle dogs received i.v. infusions of lasmiditan or sumatriptan in escalating cumulative doses, and carotid and coronary artery diameters were measured. KEY RESULTS: Lasmiditan showed high selectivity for 5-HT1F receptors. Moreover, the functional potency of the analysed compounds to inhibit cAMP increase through 5-HT1B receptor activation positively correlated with their potency to contract HCA. In isolated human arteries, sumatriptan, but not lasmiditan, induced contractions. Likewise, in vivo, sumatriptan decreased coronary and carotid artery diameters at clinically relevant doses, while lasmiditan was devoid of vasoconstrictor activity at all doses tested. CONCLUSIONS AND IMPLICATIONS: Lasmiditan is a selective 5-HT1F receptor agonist devoid of vasoconstrictor activity. This may represent a cardiovascular safety advantage when compared to the triptans.

Decreased expression of circ_0020397 in intracranial aneurysms may be contributing to decreased vascular smooth muscle cell proliferation via increased expression of miR-138 and subsequent decreased KDR expression.

Dysfunction of vascular smooth muscle cells (VSMCs) mediates intracranial aneurysm (IA). KDR is reported to alleviate IA progression via promoting VSMC proliferation, while the upstream regulators are still unclear. Arterial wall tissues at the aneurysm site from 12 patients were obtained. The real-time PCR result indicated that circRNA_0020397 was down-regulated, but miR-138 was up-regulated in artery wall tissues and cells of IA. Overexpressed circRNA_0020397 promoted proliferation of human umbilical artery SMCs. MiR-138 negatively regulated KDR via binding with 3'UTR of KDR mRNA. The expression of circRNA_0020397 was negatively correlated with miR-138. In conclusion, our findings demonstrated that decreased expression of circRNA_0020397 in IA may contribute to the decreased VSMC proliferation via increasing miR-138 expression and subsequently decreasing KDR expression.

Exploration of purinergic receptors as potential anti-migraine targets using established pre-clinical migraine models.

BACKGROUND: The current understanding of mechanisms behind migraine pain has been greatly enhanced with the recent therapies targeting calcitonin gene-related peptide and its receptor. The clinical efficacy of calcitonin gene-related peptide-blocking drugs indicates that, at least in a considerable proportion of patients, calcitonin gene-related peptide is a key molecule in migraine pain. There are several receptors and molecular pathways that can affect the release of and response to calcitonin gene-related peptide. One of these could be purinergic receptors that are involved in nociception, but these are greatly understudied with respect to migraine. OBJECTIVE: We aimed to explore purinergic receptors as potential anti-migraine targets. METHODS: We used the human middle meningeal artery as a proxy for the trigeminal system to screen for possible anti-migraine candidates. The human findings were followed by intravital microscopy and calcitonin gene-related peptide release measurements in rodents. RESULTS: We show that the purinergic P2Y13 receptor fulfills all the features of a potential anti-migraine target. The P2Y13 receptor is expressed in both the human trigeminal ganglion and middle meningeal artery and activation of this receptor causes: a) middle meningeal artery contraction in vitro; b) reduced dural artery dilation following periarterial electrical stimulation in vivo and c) a reduction of CGRP release from both the dura and the trigeminal ganglion in situ. Furthermore, we show that P2X3 receptor activation of the trigeminal ganglion causes calcitonin gene-related peptide release and middle meningeal artery dilation. CONCLUSION: Both an agonist directed at the P2Y13 receptor and an antagonist of the P2X3 receptor seem to be viable potential anti-migraine therapies.

PACAP and its receptors in cranial arteries and mast cells.

BACKGROUND: In migraineurs pituitary adenylate cyclase activating peptide1-38 (PACAP1-38) is a potent migraine provoking substance and the accompanying long lasting flushing suggests degranulation of mast cells. Infusion of the closely related vasoactive intestinal peptide (VIP) either induces headache or flushing. This implicates the pituitary adenylate cyclase activating peptide type I receptor (PAC1) to be involved in the pathophysiology of PACAP1-38 provoked headaches. Here we review studies characterizing the effects of mainly PACAP but also of VIP on cerebral and meningeal arteries and mast cells. DISCUSSION: PACAP1-38, PACAP1-27 and VIP dilate cerebral and meningeal arteries from several species including man. In rat cerebral and meningeal arteries the dilation seems to be mediated preferably via vasoactive intestinal peptide receptor type 1 (VPAC1) receptors while, in human, middle meningeal artery dilation induced via vasoactive intestinal peptide receptor type 2 (VPAC2) receptors cannot be ruled out. PACAP1-38 is a strong degranulator of peritoneal and dural mast cells while PACAP1-27 and VIP only have weak effects. More detailed characterization studies suggest that mast cell degranulation is not mediated via the known receptors for PACAP1-38 but rather via a still unknown receptor coupled to phospholipase C. CONCLUSION: It is suggested that PACAP1-38 might induce migraine via degranulation of dural mast cells via a yet unknown receptor.

Effect of PGD2 on middle meningeal artery and mRNA expression profile of L-PGD2 synthase and DP receptors in trigeminovascular system and other pain processing structures in rat brain.

BACKGROUND: Prostaglandins (PGs), particularly prostaglandin D2 (PGD2), E2 (PGE2), and I2 (PGI2), are considered to play a role in migraine pain. In humans, infusion of PGD2 causes lesser headache as compared to infusion of PGE2 and PGI2. Follow-up studies in rats have shown that infusion of PGE2 and PGI2 dilate the middle meningeal artery (MMA), and mRNA for PGE2 and PGI2 receptors is present in rat trigeminovascular system (TVS) and in the brain structures associated with pain. In the present study, we have characterized the dilatory effect of PGD2 on rat MMA and studied the relative mRNA expression of PGD2 receptors and lipocalin-type of PGD2 synthase (L-PGDS). METHOD: Rat closed-cranial window (CCW) model was used to study the effect of the DP1 receptor antagonist, MK-0524, on PGD2-induced vasodilation of middle meningeal artery. The qPCR technique was used for mRNA expression analysis. RESULTS: PGD2 infusion evoked a dose-dependent dilation of the rat MMA. The calculated mean pED50 value was 5.23±0.10 and Emax was 103±18% (n=5). MK-0524 significantly (∼61%, p<0.05) blocked the PGD2-induced dilation of MMA. mRNA for the DP1, DP2 and L-PGDS were expressed differentially in all tested tissues. DP1 receptor mRNA was expressed maximally in trigeminal ganglion (TG) and in cervical dorsal root ganglion (DRG). CONCLUSIONS: High expression of DP1 mRNA in the TG and DRG suggest that PGD2 might play a role in migraine pathophysiology. Activation of the DP1 receptor in MMA was mainly responsible for vasodilation induced by PGD2 infusion.

Immunohistochemical localization of the calcitonin gene-related peptide binding site in the primate trigeminovascular system using functional antagonist antibodies.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a neuromodulator implicated in the pathophysiology of migraine. It binds to the extracellular domains of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 1 that together form the CGRP receptor. Antagonist antibodies against CGRP and its binding site at the receptor are clinically effective in preventing migraine attacks. The blood-brain barrier penetration of these antagonist antibodies is limited, suggesting that a potential peripheral site of action is sufficient to prevent migraine attacks. To further understand the sites of CGRP-mediated signaling in migraine, we used immunohistochemical staining with recently developed antagonist antibodies specifically recognizing a fusion protein of the extracellular domains of RAMP1 and CLR that comprise the CGRP binding pocket at the CGRP receptor in monkey and man. We confirmed binding of the antagonist antibodies to human vascular smooth muscle cells (VSMCs) of dural meningeal arteries and neurons in the trigeminal ganglion, both of which are likely sites of action for therapeutic antibodies in migraine patients. We further used one of these antibodies for detailed mapping on cynomolgus monkey tissue and found antagonist antibody binding sites at multiple levels in the trigeminovascular system: in the dura mater VSMCs, in neurons and satellite glial cells in the trigeminal ganglion, and in neurons in the spinal trigeminal nucleus caudalis. These data reinforce and clarify our understanding of CGRP receptor localization in a pattern consistent with a role for CGRP receptors in trigeminal sensitization and migraine pathology.

Permeability Imaging as a Biomarker of Leptomeningeal Collateral Flow in Patients with Intracranial Arterial Stenosis.

Different methods of angiography are of great clinical utility; however, it still remains unstandardized as which method would be suitable to determine cerebral collateral circulation. Here we compared digital subtraction angiography (DSA), computer tomography angiography (CTA) and dynamic contrast-enhanced T1-weighted imaging magnetic resonance imaging (MRI) findings in seven patients with severe intracranial arterial stenosis, and determine whether volume transfer constant (K(trans)) maps of permeability imaging could be used as the biomarkers of cerebral collateral circulation. We retrospectively reviewed seven adult patients with severe intracranial arterial stenosis or occlusion with a complete parenchymal and vascular imaging work-up. DSA, CTA source imaging (CTA-SI), arterial spin labeling (ASL), and K(trans) maps were used to assess their collateral flow. Cohen's Kappa coefficient was calculated to test the consistency of their collateral scores. A reasonable agreement was found between DSA and K(trans) maps (Kappa = 0.502, P < 0.001) when all 15 regional vascular sites were included, and a better agreement found after exclusion of perforating artery territories (N = 10 sites, Kappa = 0.766, P < 0.001). The agreement between CTA-SI and DSA was moderate on all 15 sites (Kappa = 0.413, P < 0.001) and 10 sites (Kappa = 0.329, P < 0.001). The agreement between ASL and DSA was least favorable, no matter for all 15 sites (Kappa = 0.270, P < 0.001) or 10 sites (Kappa = 0.205, P = 0.002). K(trans) maps are useful and promising for leptomeningeal collateral assessment, when compared to CTA-SI or ASL. Further studies are requited for verify its validity in a large registry of patients.

Expression and characterization of purinergic receptors in rat middle meningeal artery-potential role in migraine.

The dura mater and its vasculature have for decades been central in the hypothesis of migraine and headache pathophysiology. Although recent studies have questioned the role of the vasculature as the primary cause, dural vessel physiology is still relevant in understanding the complex pathophysiology of migraine. The aim of the present study was to isolate the middle meningeal artery (MMA) from rodents and characterize their purinergic receptors using a sensitive wire myograph method and RT-PCR. The data presented herein suggest that blood flow through the MMA is, at least in part, regulated by purinergic receptors. P2X1 and P2Y6 receptors are the strongest contractile receptors and, surprisingly, ADPßS caused contraction most likely via P2Y1 or P2Y13 receptors, which is not observed in other arteries. Adenosine addition, however, caused relaxation of the MMA. The adenosine relaxation could be inhibited by SCH58261 (A2A receptor antagonist) and caffeine (adenosine receptor antagonist). This gives one putative molecular mechanism for the effect of caffeine, often used as an adjuvant remedy of cranial pain. Semi-quantitative RT-PCR expression data for the receptors correlate well with the functional findings. Together these observations could be used as targets for future understanding of the in vivo role of purinergic receptors in the MMA.

5-HT7 receptors are involved in neurogenic dural vasodilatation in an experimental model of migraine.

Neurogenic dural vasodilation has been demonstrated to play an important role in migraine. 5-HT(7) receptors have been found on trigeminal nerve endings and middle meningeal arteries and demonstrated involved in the dilatation of meningeal arteries. The aim of the present study was to demonstrate whether 5-HT(7) receptors are involved in neurogenic dural vasodilation in migraine. The neurogenic dural vasodilation model of migraine was used in this study. Unilateral electrical stimulation of dura mater was performed in anesthetized male Sprague-Dawley rats. Animals were pretreated with selective 5-HT(7) receptor agonist AS19, 5-HT(7) receptor antagonist SB269970, 5-HT1B/1D receptor agonist sumatriptan, or vehicles. Blood flow of the middle meningeal artery (MMA) was measured by a laser Doppler flowmetry. AS19 significantly increased the basal and stimulated blood flows of the middle meningeal artery following electrical stimulation of dura mater, and its effect was dose dependent at the early stage. SB269970 and sumatriptan significantly reduced the basal and stimulated blood flows of middle meningeal artery. The present study demonstrates for the first time that 5-HT(7) receptors are involved in neurogenic dural vasodilation evoked by electrical stimulation of dura mater and maybe of relevance in the pathophysiology and treatment of migraine.

Pituitary adenylate cyclase-activating polypeptide (PACAP) potently dilates middle meningeal arteries: implications for migraine.

Migraine is a debilitating neurological disorder characterized by mild to severe headache that is often accompanied by aura and other neurological symptoms. Among proposed mechanisms, dilation of the dural vasculature especially the middle meningeal artery (MMA) has been implicated as one component underlying this disorder. Several regulatory peptides from trigeminal sensory and sphenopalatine postganglionic parasympathetic fibers innervating these vessels have been implicated in the process including pituitary adenylate cyclase-activating polypeptide (PACAP). Although PACAP has been well described as a potent dilator in many vascular beds, the effects of PACAP on the dural vasculature are unclear. In the current study, we examined the ability of PACAP to dilate MMAs that were isolated from rats and pressurized ex vivo. PACAP38 potently dilated pressurized MMAs with an EC(50) of 1 pM. The PAC1 receptor antagonist, PACAP(6-38), abolished MMA dilation caused by picomolar concentrations of PACAP. In contrast, cerebellar arteries isolated from the brain surface were ~1,000-fold less sensitive to PACAP than MMAs. Although cerebellar arteries expressed transcripts for all three PACAP receptor subtypes (PAC1, VPAC1, and VPAC2 receptors) by RT-PCR analyses, MMA demonstrated only PAC1 and VPAC2 receptor expression. Further, multiple variants of the PAC1 receptor were identified in the MMA. The expression of PAC1 receptors and the high potency of PACAP to induce MMA vasodilation are consistent with their potential roles in the etiology of migraine.

Pharmacological and expression profile of the prostaglandin I(2) receptor in the rat craniovascular system.

Activation of the trigeminal nerve terminals around cerebral and meningeal arteries is thought to be an important patho-mechanism in migraine. Vasodilatation of the cranial arteries may also play a role in increasing nociception. Prostaglandin I(2) (PGI(2)) is capable of inducing a headache in healthy volunteers, a response that is likely to be mediated by the prostaglandin I(2) receptor (IP). This study investigates the functional and molecular characteristics of the IP receptor in the rat craniovascular system. In the closed cranial window model, iloprost, an IP receptor agonist, dilated the rat middle meningeal artery (MMA) (E(max)=170%±16%; pED(50)=6.5±0.2) but not the rat cerebral artery (CA) in vivo. The specific antagonist of the IP receptor, CAY10441, significantly blocked the iloprost-induced response dose-dependently, with the highest dose attenuating iloprost (1µgkg(-1)) induced dilatations by 70% (p<0.05). CAY10441 did not have any effect on the prostaglandin E(2)-induced vasodilatory response, thus suggesting no interaction with EP(2) and EP(4) receptors. IP receptor mRNA transcripts and protein were present in meningeal as well as in cerebral rat vasculature, and localized the IP receptor protein to the smooth vasculature of the cranial arteries (MMA, MCA and basilar artery). Together, these results demonstrate that the IP receptor mediates the dilatory effect of PGI(2) in the cranial vasculature in rats. Antagonism of this receptor might be of therapeutic relevance in acute migraine treatment.

Characteristics of dyshoric capillary cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) affects brain parenchymal and leptomeningeal arteries and arterioles but sometimes involves capillaries (capCAA) with spread of the amyloid into the surrounding neuropil, that is, dyshoric changes. We determined the relationship between capCAA and larger vessel CAA, ß amyloid (Aß) plaques, neurofibrillary changes, inflammation, and apolipoprotein E (APOE) in 22 cases of dyshoric capCAA using immunohistochemistry. The dyshoric changes contained predominantly Aß1-40, whereas dense bulblike deposits adjacent to the capillary wall contained mostly Aß1-42. There was an inverse local correlation between Aß plaque load and capCAA severity (p = 0.01), suggesting that Aß transport between the neuropil and the circulation may be mechanistically involved. Deposits of hyperphosphorylated tau and ubiquitin and clusters of activated microglia, resembling the changes around Aß plaques, were found around capCAA but were absent around larger vessel CAA. In 14 cases for which APOE genotype was available, there was a high APOE-ε4 allele frequency (54%; 43% homozygous). The severity of CapCAA increased with the number of ε4-alleles; and APOE4 seemed to colocalize with capCAA by immunohistochemistry. These results suggest that capCAA is pathologically and possibly pathogenetically distinct from larger vessel CAA, and that it is associated with a high APOE-ε4 allele frequency.

Effects of ionotropic glutamate receptor antagonists on rat dural artery diameter in an intravital microscopy model.

BACKGROUND AND PURPOSE: During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by alpha-CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats were anaesthetized and the overlying bone was thinned to visualize the dural artery. Then, vasodilator responses to exogenous (i.v. alpha-CGRP) and endogenous (released by i.v. capsaicin and periarterial electrical stimulation) CGRP were elicited in the absence or presence of the above antagonists. KEY RESULTS: alpha-CGRP, capsaicin and periarterial electrical stimulation increased dural artery diameter. Ketamine and MK801 inhibited the vasodilator responses to capsaicin and electrical stimulation, while only ketamine attenuated those to alpha-CGRP. In contrast, GYKI52466 only attenuated the vasodilatation to exogenous alpha-CGRP, while LY466195 did not affect the vasodilator responses to endogenous or exogenous CGRP. CONCLUSIONS AND IMPLICATIONS: Although GYKI52466 has not been tested clinically, our data suggest that it would not inhibit migraine via vascular mechanisms. Similarly, the antimigraine efficacy of LY466195 seems unrelated to vascular CGRP-mediated pathways and/or receptors. In contrast, the cranial vascular effects of ketamine and MK801 may represent a therapeutic mechanism, although the same mechanism might contribute, peripherally, to cardiovascular side effects.

The in vivo effect of VIP, PACAP-38 and PACAP-27 and mRNA expression of their receptors in rat middle meningeal artery.

The parasympathetic nervous system is probably involved in migraine pathogenesis. Its activation releases a mixture of signalling molecules including vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), which subsequently stimulate VPAC(1), VPAC(2) and PAC(1) receptors. The objective of the present study was to investigate the in vivo effect of VIP, PACAP-27, PACAP-38, the selective VPAC(1) agonist ([Lys15, Arg16, Leu27]-VIP(1-7)-GRF(8-27)) and a PAC(1) agonist, maxadilan on rat middle meningeal artery (MMA) diameter using the closed cranial window model. Selective antagonists were used for further characterization of the responses. Reverse transcriptase-polymerase chain reaction experiments were also conducted to determine expression of mRNA of PACAP receptors in the MMA. The results showed that VIP, PACAP-38, PACAP-27 and the VPAC(1) specific agonist evoked significant dilations with the rank order of potency; VIP = PACAP-38 > PACAP-27 = [Lys15, Arg16, Leu27]-VIP(1-7)-GRF(8-27). Significant inhibition of dilation was only observed for the VPAC(1) antagonist PG97-269 on PACAP-38-induced dilation of MMA. The VPAC(2) antagonist PG99-465 and PAC(1) antagonist PACAP(6-38) did not significantly block VIP- or PACAP-induced dilation. Expression of mRNA of all three receptors was detected in the MMA. In conclusion, the VPAC(1) receptor seems to be predominant in mediating MMA dilation. A selective VPAC(1) antagonist may be a candidate molecule in the treatment of migraine headache.

Amyloid beta peptides in human plasma and tissues and their significance for Alzheimer's disease.

BACKGROUND: We evaluated the amounts of amyloid beta (Abeta)) peptides in the central nervous system (CNS) and in reservoirs outside the CNS and their potential impact on Abeta plasma levels and Alzheimer's disease (AD) pathology. METHODS: Amyloid beta levels were measured in (1) the plasma of AD and nondemented (ND) controls in a longitudinal study, (2) the plasma of a cohort of AD patients receiving a cholinesterase inhibitor, and (3) the skeletal muscle, liver, aorta, platelets, leptomeningeal arteries, and in gray and white matter of AD and ND control subjects. RESULTS: Plasma Abeta levels fluctuated over time and among individuals, suggesting continuous contributions from brain and peripheral tissues and associations with reactive circulating proteins. Arteries with atherosclerosis had larger amounts of Abeta40 than disease-free vessels. Inactivated platelets contained more Abeta peptides than activated ones. Substantially more Abeta was present in liver samples from ND patients. Overall, AD brain and skeletal muscle contained increased levels of Abeta. CONCLUSIONS: Efforts to use plasma levels of Abeta peptides as AD biomarkers or disease-staging scales have failed. Peripheral tissues might contribute to both the circulating amyloid pool and AD pathology within the brain and its vasculature. The wide spread of plasma Abeta values is also due in part to the ability of Abeta to bind to a variety of plasma and membrane proteins. Sources outside the CNS must be accounted for because pharmacologic interventions to reduce cerebral amyloid are assessed by monitoring Abeta plasma levels. Furthermore, the long-range impact of Abeta immunotherapy on peripheral Abeta sources should also be considered.

K ATP channels in pig and human intracranial arteries.

Clinical trials suggest that synthetic ATP-sensitive K(+) (K(ATP)) channel openers may cause headache and migraine by dilating cerebral and meningeal arteries. We studied the mRNA expression profile of K(ATP) channel subunits in the pig and human middle meningeal artery (MMA) and in the pig middle cerebral artery (MCA). We determined the order of potency of four K(ATP) channel openers when applied to isolated pig MMA and MCA, and we examined the potential inhibitory effects of the Kir6.1 subunit specific K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the isolated pig MMA and MCA. Using conventional RT-PCR, we detected the mRNA transcripts of the K(ATP) channel subunits Kir6.1 and SUR2B in all the examined pig and human intracranial arteries. Application of K(ATP) channel openers to isolated pig MMA and MCA in myographs caused a concentration-dependent vasodilatation with an order of potency that supports the presence of functional SUR2B K(ATP) channel subunits. 10(-7) M PNU-37883A significantly inhibited the in vitro dilatory responses of the potent K(ATP) channel opener P-1075 in both pig MMA and MCA. In conclusion, our combined mRNA expression and pharmacological studies indicate that Kir6.1/SUR2B is the major functional K(ATP) channel complex in the pig MMA and MCA, and mRNA expression studies suggest that the human MMA shares this K(ATP) channel subunit profile. Specific blocking of Kir6.1 or SUR2B K(ATP) channel subunits in large cerebral and meningeal arteries may be a future anti-migraine strategy.

CGRP function-blocking antibodies inhibit neurogenic vasodilatation without affecting heart rate or arterial blood pressure in the rat.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) receptor antagonists effectively abort migraine headache and inhibit neurogenic vasodilatation in humans as well as rat models. Monoclonal antibodies typically have long half-lives, and we investigated whether or not function-blocking CGRP antibodies would inhibit neurogenic vasodilatation with a long duration of action and therefore be a possible approach to preventive therapy of migraine. During chronic treatment with anti-CGRP antibodies, we measured cardiovascular function, which might be a safety concern of CGRP inhibition. EXPERIMENTAL APPROACH: We used two rat blood flow models that measure electrically stimulated vasodilatation in the skin or in the middle meningeal artery (MMA). These vasomotor responses are largely dependent on the neurogenic release of CGRP from sensory afferents. To assess cardiovascular function during chronic systemic anti-CGRP antibody treatment, we measured heart rate and blood pressure in conscious rats. KEY RESULTS: Treatment with anti-CGRP antibodies inhibited skin vasodilatation or the increase in MMA diameter to a similar magnitude as treatment with CGRP receptor antagonists. Although CGRP antibody treatment had a slower onset of action than the CGRP receptor antagonists, the inhibition was still evident 1 week after dosing. Chronic treatment with anti-CGRP antibodies had no detectable effects on heart rate or blood pressure. CONCLUSIONS AND IMPLICATIONS: We showed for the first time that anti-CGRP antibodies exert a long lasting inhibition of neurogenic vasodilatation in two different rat models of arterial blood flow. We have provided strong preclinical evidence that anti-CGRP antibody may be a suitable drug candidate for the preventive treatment of migraine.

Role of BK(Ca) channels in cephalic vasodilation induced by CGRP, NO and transcranial electrical stimulation in the rat.

Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators that have been shown to induce headache in migraine patients. Their antagonists are effective in the treatment of migraine attacks. In the present study, we hypothesize that vasodilation induced by the NO donor glyceryltrinitrate (GTN) or by CGRP is partially mediated via large conductance calcium-activated potassium (BK(Ca)) channels. The effects of the BK(Ca) channel selective inhibitor iberiotoxin on dural and pial vasodilation induced by CGRP, GTN and endogenously released CGRP by transcranial electrical stimulation (TES) were examined. Iberiotoxin significantly attenuated GTN-induced dural and pial artery dilation in vivo and in vitro, but had no effect on vasodilation induced by CGRP and TES. Our results show that GTN- but not CGRP-induced dural and pial vasodilation involves opening of BK(Ca) channels in rat.