Inger Jansen-Olesen

Calcitonin Gene-Related Peptide is Released from Capsaicin-Sensitive Nerve Fibres and Induces Vasodilatation of Human Cerebral Arteries Concomitant with Activation of Adenylyl Cyclase

The vasomotor effects of calcilonin gene-related peptide (CGRP) analogues have been studied in circular segments of fresh human cereoral arteries obtained at neurosurgical operations using a sensitive in vitro system. Human a-CGRP, human b-CGRP, rat a-CGRP and rat b-CGRP induced strong and potent relaxation of precontracted circular vessel segments. The Imax (maximum relaxant effect) to human calcitonin was low and the pD2 (concentration for half maximum effect) 7.7 was much lower than that of CGRP. The CGRP-1, antagonist human a-CGRP8-37 blocked the response to human a-CGRP but not to human b-CGRP, while the putative antagonist [Tyr]CGRP28-37 did not. Capsaicin (10-15 - 10-8 M) caused relaxation of the cerebral arteries by 22% of precontract on. Pre-treatment with 10-6 M human a-CGRP8-37 inhibited this relaxation. Human a-CGRP increased the cyclic AMP content of human cerebral arteries in a concentration-dependent manner. This increase in adenylyl cyclase activity was blocked by human a-CGRP8-37. The results suggest that CGRP-1 receptors coupled to adenylyl cyclase are present in human cerebral arteries.

Inhibitory effect of BIBN4096BS, CGRP(8-37), a CGRP antibody and an RNA-Spiegelmer on CGRP induced vasodilatation in the perfused and non-perfused rat middle cerebral artery.

A new concept for the inhibition of CGRP signalling has been developed by interaction with the CGRP molecule per se by using a CGRP antibody or a CGRP binding RNA‐Spiegelmer (NOX‐C89). We have compared these CGRP scavengers with two known receptor antagonists (CGRP8–37 and BIBN4096BS) on CGRP‐induced relaxations in the rat middle cerebral artery (MCA). Furthermore, the role of the endothelial barrier has been studied.

Characterisation of the effects of a non-peptide CGRP receptor antagonist in SK-N-MC cells and isolated human cerebral arteries.

The cerebral circulation is innervated by calcitonin gene-related peptide (CGRP) containing fibers originating in the trigeminal ganglion. During a migraine attack, there is a release of CGRP in conjunction with the head pain, and triptan administration abolishes both the CGRP release and the pain at the same time. In the search for a novel treatment of migraine, a non-peptide CGRP antagonist has long been sought. Here, we present data on a human cell line and human and guinea-pig isolated cranial arteries for such an antagonist, Compound 1 (4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide). On SK-N-MC cell membranes, radiolabelled CGRP binding was displaced by both CGRP-(8-37) and Compound 1, yielding pK(i) values of 8.9 and 7.8, respectively. Functional studies with SK-N-MC cells showed that CGRP-induced cAMP production was antagonised by both CGRP-(8-37) and Compound 1 with pA(2) values of 7.8 and 7.7, respectively. Isolated human and guinea pig cerebral arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation in human cerebral arteries which was antagonized by both CGRP-(8-37) and Compound 1 in a competitive manner. In guinea pig basilar arteries, CGRP-(8-37) antagonised the CGRP-induced relaxation while Compound 1 had a weak blocking effect. The clinical studies of non-peptide CGRP antagonists are awaited with great interest.

Innervation of the human middle meningeal artery: immunohistochemistry, ultrastructure, and role of endothelium for vasomotility

The majority of nerve fibers in the middle meningeal artery and branching arterioles are sympathetic, storing norepinephrine and neuropeptide Y (NPY). A sparse supply of fibers contain acetylcholinesterase activity and immunoreactivity toward vasoactive intestinal peptide (VIP), peptidine histidine methionine (PHM), and calcitonin gene-related peptide (CGRP). Only few substance P and neuropeptide K immunoreactive fibers are noted. Electronmicroscopy shows axons and terminals at the adventitial medial border of the human middle meningeal artery, with a fairly large distance to the smooth muscle cells (>500 nM). Several axon profiles contain vesicles of different types, including putative sensory profiles. The perivascularly stored signal substances, norepinephrine and NPY induced vasoconstrictor. Relaxations were induced by acetylcholine and substance P, and these were significantly reduced in arteries without endothelium, while the responses to norepinephrine, NPY, VIP, PHM, and CGRP were not changed by endothelium removal. Blockade experiments showed that the vasomotor responses to norepinephrine were blocked by prazosin, to NPY by BIBP 3226, acetylcholine by atropin, substance P by RP 67580, and the human alpha-CGRP response by human alpha-CGRP(8-37).

CGRP release and c‐fos expression within trigeminal nucleus caudalis of the rat following glyceryltrinitrate infusion

Neuropeptide release and the expression of c-fos like immunoreactivity (c-fos LI) within trigeminal nucleus caudalis neurons (TNC) are activation markers of the trigeminal nerve system. Glyceryltrinitrate (GTN) is believed to stimulate the trigeminal nerve system, thereby causing headache. We examined the effects of a 30 min NO-donor infusion on CGRP release in jugular vein blood and c-fos LI within TNC of the rat. GTN (2 and 50 μg/kg/min) or NONOate infusion (25 nmol/kg/min) did not cause any CGRP release during and shortly after infusion, whereas administration of capsaicin resulted in strongly increased CGRP levels. GTN infusion (2 μg/kg/min for 30 min) did not lead to enhanced c-fos LI after 2 h and 4 h, whereas capsaicin infusion caused a time- and dose-dependent expression of c-fos LI within laminae I and II of the TNC. Surprisingly, GTN attenuated capsaicin-induced c-fos expression by 64%. The nitric oxide synthase (NOS) inhibitor L-NAME (5 and 50 mg/kg) reduced capsaicin-induced c-fos LI dose dependently (reduction by 13% and 59%). We conclude that GTN may lead to headaches by mechanisms independent of CGRP release from trigeminal nerve fibres. GTN doses comparable to those used in humans did not activate or sensitize the trigeminal nerve system. Both GTN and L-NAME reduced capsaicin-induced c-fos LI. This is most likely due to a feedback inhibition of nitric oxide synthases, which indicates that the c-fos response to capsaicin within TNC is mediated by NO dependent mechanisms.

The effect of nitric oxide synthase inhibition on histamine induced headache and arterial dilatation in migraineurs

We have previously proposed that histamine causes migraine via increased NO production. To test this hypothesis, we here examined if the NOS inhibitor, L-NG methylarginine hydrochloride (L-NMMA:546C88), could block or attenuate histamine induced migraine attacks and responses of the middle cerebral, temporal and radial arteries. In a double blind crossover design 12 patients were randomized to receive pretreatment with L-NMMA (6 mg/kg) or placebo i.v. over 15 min followed on both study days by histamine (0.5 μg/kg/min) i.v. for 20 min. Headache scores, mean maximal blood velocity (Vmean) in the middle cerebral artery (MCA) (transcranial doppler) and diameters of temporal and radial arteries (high resolution ultrasound) were repeatedly measured. Pre-treatment with L-NMMA, had no effect on histamine induced headache or migraine, but also had no effect on the magnitude of histamine induced – decrease in MCA blood velocity, or dilatation of neither the temporal nor the radial artery. L-NMMA constricted the temporal artery by 8% before histamine infusion, whereas the radial artery was unaffected. The temporal artery dilated 4-5 times more than the radial artery during histamine infusion. In conclusion the use of a NOS inhibitor in the highest possible dose did not block the histamine-induced headache response or arterial dilatation. Either the concentration of L-NMMA reaching the smooth muscle cell was insufficient or, histamine dilates arteries and causes headache via NO independent mechanisms. Our results showed for the first time a craniospecificity for the vasodilating effect of histamine and for the arterial effects of NOS inhibition.

Expression of calcitonin gene-related peptide1 receptor mRNA in human trigeminal ganglia and cerebral arteries

Reverse transcriptase polymerase chain reaction (RT-PCR) using primers for the recently cloned human CGRP1 receptor detected mRNA expression of CGRP1 receptors in trigeminal ganglia and cerebral vessels, obtained at autopsy or during neurosurgical tumor resections. An RT-PCR product of the expected size (339 bp) was seen in cerebral arteries, both in the presence and in the absence of endothelium and in trigeminal ganglia. Sequence analysis of the RT-PCR product of the published sequence showed 100% homology with the human CGRP1 receptor. The presence of the CGRP1 receptor mRNA in human trigeminal ganglia and cerebral blood vessels, indicates the occurrence of both prejunctional (trigeminal) and postjunctional location (blood vessels) of the CGRP1 receptor.

Dural mast cell degranulation is a putative mechanism for headache induced by PACAP-38

Background: Pituitary adenylate cyclase activating peptide-38 (PACAP-38) has been shown to induce migraine in migraineurs, whereas the related peptide vasoactive intestinal peptide (VIP) does not. In the present study we examine the hypothesis that PACAP-38 and its truncated version PACAP-27 but not VIP cause degranulation of mast cells in peritoneum and in dura mater. Methods: The degranulatory effects of PACAP-38, PACAP-27 and VIP were investigated by measuring the amount of N-acetyl-β-hexosaminidase released from isolated peritoneal mast cells and from dura mater attached to the skull of the rat in vitro. In peritoneal mast cells N-truncated fragments of PACAP-38 (PACAP(6–38), PACAP(16–38) and PACAP(28–38)) were also studied. To investigate transduction pathways involved in mast cell degranulation induced by PACAP-38, PACAP-27 and VIP, the phospholipase C inhibitor U-73122 and the adenylate cyclase inhibitor SQ 22536 were used. Results: The peptides induced degranulation of isolated peritoneal mast cells of the rat with the following order of potency: PACAP-38 = PACAP(6–38) = PACAP(16–38) » PACAP-27 = VIP = PACAP(28–38). In the dura mater we found that 10−5 M PACAP-38 was significantly more potent in inducing mast cell degranulation than the same concentration of PACAP-27 or VIP. Inhibition of intracellular mechanisms demonstrated that PACAP-38-induced degranulation is mediated by the phospholipase C pathway. Selective blockade of the PAC1 receptor did not attenuate degranulation. Conclusion: These findings correlate with clinical studies and support the hypothesis that mast cell degranulation is involved in PACAP-induced migraine. PACAP-38 has a much stronger degranulatory effect on rat peritoneal and dural mast cells than VIP and PACAP-27. The difference in potency between PACAP-38- and PACAP-27/VIP-induced peritoneal mast cell degranulation is probably not related to the PAC1 receptor but is caused by a difference in efficacy on phospholipase C.

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.