Kenneth Beri Ploug

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 VPAC1, VPAC2 and PAC1 receptors. The objective of the present study was to investigate the in vivo effect of VIP, PACAP-27, PACAP-38, the selective VPAC1 agonist ([Lys15, Arg16, Leu27]-VIP(1–7)-GRF(8–27)) and a PAC1 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 VPAC1 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 VPAC1 antagonist PG97–269 on PACAP-38-induced dilation of MMA. The VPAC2 antagonist PG99–465 and PAC1 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 VPAC1 receptor seems to be predominant in mediating MMA dilation. A selective VPAC1 antagonist may be a candidate molecule in the treatment of migraine headache.

Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes

OBJECTIVE The ATP-dependent K+-channel (K(ATP)) is critical for glucose sensing and normal glucagon and insulin secretion from pancreatic endocrine alpha- and beta-cells. Gastrointestinal endocrine L- and K-cells are also glucose-sensing cells secreting glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP) respectively. The aims of this study were to 1) investigate the expression and co-localisation of the K(ATP) channel subunits, Kir6.2 and SUR1, in human L- and K-cells and 2) investigate if a common hyperactive variant of the Kir6.2 subunit, Glu23Lys, exerts a functional impact on glucose-sensing tissues in vivo that may affect the overall glycaemic control in children with new-onset type 1 diabetes. DESIGN AND METHODS Western blot and immunohistochemical analyses were performed for expression and co-localisation studies. Meal-stimulated C-peptide test was carried out in 257 children at 1, 6 and 12 months after diagnosis. Genotyping for the Glu23Lys variant was by PCR-restriction fragment length polymorphism. RESULTS Kir6.2 and SUR1 co-localise with GLP-1 in L-cells and with GIP in K-cells in human ileum tissue. Children with type 1 diabetes carrying the hyperactive Glu23Lys variant had higher HbA1C at diagnosis (coefficient = 0.61%, P = 0.02) and 1 month after initial insulin therapy (coefficient = 0.30%, P = 0.05), but later disappeared. However, when adjusting HbA1C for the given dose of exogenous insulin, the dose-adjusted HbA1C remained higher throughout the 12 month study period (coefficient = 0.42%, P = 0.03). CONCLUSIONS Kir6.2 and SUR1 co-localise in the gastrointestinal endocrine L- and K-cells. The hyperactive Glu23Lys variant of the K(ATP) channel subunit Kir6.2 may cause defective glucose sensing in several tissues and impaired glycaemic control in children with type 1 diabetes.

Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation.

Background and aim Infusion of glyceryltrinitrate (GTN), a nitric oxide (NO) donor, in awake, freely moving rats closely mimics a universally accepted human model of migraine and responds to sumatriptan treatment. Here we analyse the effect of nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP) systems on the GTN-induced neuronal activation in this model. Materials and methods The femoral vein was catheterised in rats and GTN was infused (4 µg/kg/min, for 20 minutes, intravenously). Immunohistochemistry was performed to analyse Fos, nNOS and CGRP and Western blot for measuring nNOS protein expression. The effect of olcegepant, L-nitro-arginine methyl ester (L-NAME) and neurokinin (NK)-1 receptor antagonist L-733060 were analysed on Fos activation. Results GTN-treated rats showed a significant increase of nNOS and CGRP in dura mater and CGRP in the trigeminal nucleus caudalis (TNC). Upregulation of Fos was observed in TNC four hours after the infusion. This activation was inhibited by pre-treatment with olcegepant. Pre-treatment with L-NAME and L-733060 also significantly inhibited GTN induced Fos expression. Conclusion The present study indicates that blockers of CGRP, NOS and NK-1 receptors all inhibit GTN induced Fos activation. These findings also predict that pre-treatment with olcegepant may be a better option than post-treatment to study its inhibitory effect in GTN migraine models.

A naturalistic glyceryl trinitrate infusion migraine model in the rat

Background: and aim Glyceryl trinitrate (GTN) infusion is a reliable method to provoke migraine-like headaches in humans. Previous studies have simulated this human model in anaesthetized or in awake rodents using GTN doses 10,000 times higher than used in humans. The relevance of such toxicological doses to migraine is not certain. Anaesthesia and low blood pressure caused by high GTN doses both can affect the expression of nociceptive marker c-fos. Therefore, our aim was to simulate the human GTN migraine model in awake rats using a clinically relevant dose. Methods: Awake rats were infused with GTN (4 µg/kg/min, for 20 min, i.v.), a dose just 8 times higher than in humans. mRNA and protein expression for c-fos were analysed in the trigeminal vascular system at various time points using RT-PCR and immunohistochemistry, respectively. Results: A significant upregulation of c-fos mRNA was observed in the trigeminal nucleus caudalis at 30 min and 2 h that was followed by an upregulation of Fos protein in the trigeminal nucleus caudalis at 2 h and 4 h after GTN infusion. Pre-treatment with sumatriptan attenuated the activation of Fos at 4 h, demonstrating the specificity of this model for migraine. Conclusion: We present a validated naturalistic rat model suitable for screening of acute anti-migraine drugs.

MRNA expression of 5-hydroxytryptamine 1B, 1D, and 1F receptors and their role in controlling the release of calcitonin gene-related peptide in the rat trigeminovascular system

Summary The 5‐hydroxytryptamine (5‐HT)1F receptor and 5‐HT1D receptor are important for calcitonin gene–related peptide (CGRP) release in peripheral terminals in dura mater and in central terminals in trigeminal nucleus caudalis. If CGRP release inhibition is the aim of migraine treatment, then combined agonism on the 5‐HT1D and 5‐HT1F receptors would be optimal and without vascular effects. Abstract Triptans, a family of 5‐hydroxytryptamine (5‐HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5‐HT1 receptor that mediate the antimigraine effect have still to be identified. This study investigated the mRNA expression of these receptors and the role of 5‐HT1 receptor subtypes in controlling the release of calcitonin gene–related peptide (CGRP) in rat dura mater, trigeminal ganglion (TG), and trigeminal nucleus caudalis (TNC). The mRNA for each receptor subtype was quantified by quantitative real‐time polymerase chain reaction. A high potassium concentration was used to release CGRP from dura mater, isolated TG, and TNC in vitro. The immunoreactive CGRP (iCGRP) release was measured by enzyme‐linked immunoassay. The mRNA transcripts of the 3 5‐HT1 receptor subtypes were detected in the trigeminovascular system. Sumatriptan inhibited iCGRP release by 31% in dura mater, 44% in TG, and 56% in TNC. This effect was reversed by a 5‐HT1B/1D antagonist (GR127395). The 5‐HT1F agonist (LY‐344864) was effective in the dura mater (26% iCGRP inhibition), and the 5‐HT1D agonist (PNU‐142633) had a significant effect in the TNC (48%), whereas the 5‐HT1B agonist (CP‐94253) was unable to reduce the iCGRP release in all tissues studied. We found that sumatriptan reduced the iCGRP release via activation of 5‐HT1D and 5‐HT1F receptor subtypes. The 5‐HT1F receptor agonist was effective only in peripheral terminals in dura mater, whereas the 5‐HT1D agonist had a preferential effect on central terminals in the TNC.

Presence and vascular pharmacology of KATP channel subtypes in rat central and peripheral tissues

K(ATP) channel openers are vasodilators and induce headache in normal subjects. We previously identified the Kir6.1/SUR2B K(ATP) channel subtype in major cerebral and dural arteries of rat, pig and man. We hypothesized that craniovascular Kir6.1/SUR2B K(ATP) channels mediate the headache-inducing effects of K(ATP) channel openers and that a Kir6.1/SUR2B specific blocker might be effective in the treatment of primary headaches such as migraine. Since K(ATP) channels are ubiquitous, we characterized the K(ATP) channel subtypes in major rat cranial and peripheral arteries and organs in order to understand the possible adverse effects of a Kir6.1/SUR2B blocker. We studied the mRNA expression of K(ATP) channel subunits in rat femoral, mesenteric, renal, coronary, basilar, middle cerebral and middle meningeal arteries and in tissue from rat heart, brain, liver, colon, lung, kidney and pancreas. We also studied the effects and potencies of a panel of synthetic K(ATP) channel openers and their potential inhibition by the Kir6.1 subunit-specific K(ATP) channel blocker PNU-37883A in segments of the arteries mounted in a wire myograph. Our studies suggest that Kir6.1/SUR2B forms the major functional K(ATP) channel complex in rat cranial and peripheral arteries. The mRNA transcripts of SUR1 and Kir6.2 subunits were predominantly found in brain, pancreas and heart, while SUR2A mRNA was merely detected within the heart. K(ATP) channel blockers highly specific for the SUR2B subunit may have no adverse CNS and cardiac effects and will not affect insulin release in the pancreas. However, a SUR2B blocker may not discriminate between cranial and peripheral arteries.

Functional and molecular characterization of prostaglandin E2 dilatory receptors in the rat craniovascular system in relevance to migraine

Introduction: Migraine pain is thought to involve an increase in trigeminal nerve terminal activity around large cerebral and meningeal arteries, leading to vasodilatation. Because prostaglandin E2 (PGE2) is elevated in cephalic venous blood during migraine attacks, and is also capable of inducing headache in healthy volunteers, we hypothesize that PGE2 dilatory receptors, EP2 and EP4, mediate the response. Materials and methods: By the use of specific agonists and antagonists, the dilatory effect of PGE2 was characterized in rat cranial arteries by use of in vivo and in vitro methods. Furthermore, EP2 and EP4 quantitative messenger RNA (mRNA) receptor expression was studied in the rat craniovascular system. Results: Our results suggest that EP4, and to a lesser degree EP2, receptors mediate the dilatory effect of PGE2 in the craniovascular system in rats. Thus, antagonism of these receptors might be of therapeutic relevance in migraine.

Role for voltage gated calcium channels in calcitonin gene-related peptide release in the rat trigeminovascular system

Clinical and genetic studies have suggested a role for voltage gated calcium channels (VGCCs) in the pathogenesis of migraine. Release of calcitonin gene-related peptide (CGRP) from trigeminal neurons has also been implicated in migraine. The VGCCs are located presynaptically on neurons and are involved in the release of these peptides to different stimuli. We have examined the presence and importance of VGCCs in controlling the CGRP release from rat dura mater, freshly isolated trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC). Each of the four VGCCs, P/Q-, N-, and L- and T-type are abundantly found in TG and TNC relative to the dura mater and each mediates a significant fraction of high potassium concentration induced CGRP release. In dura mater, blockade of P/Q-, N- and L-type VGCCs by ω-agatoxin TK, ω-conotoxin GVIA and nimodipine at 1 μM respectively, significantly decreased the potassium induced CGRP release. In the absence of calcium ions (Ca2+) and in the presence of a cocktail of blockers, the stimulated CGRP release from dura mater was reduced almost to the same level as basal CGRP release. In the TG ω-conotoxin GVIA inhibited the potassium induced CGRP release significantly. In the absence of Ca2+ and in the presence of a cocktail of blockers the stimulated CGRP release was significantly reduced. In the TNC only the cocktail of blockers and the absence of Ca2+ could reduce the potassium induced release significantly. These results suggest that depolarization by high potassium releases CGRP, and the release is regulated by Ca2+ ions and voltage-gated calcium channels.

KATP channel expression and pharmacological in vivo and in vitro studies of the KATP channel blocker PNU‐37883A in rat middle meningeal arteries

Background and purpose: Dilatation of cerebral and dural arteries causes a throbbing, migraine‐like pain, indicating that these structures are involved in migraine.

KATP 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.