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Characterization of VIP- and PACAP-Receptors in a Trigeminovascular Model – Relevance for Migraine

Michael Baun


Neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide-38 (PACAP-38) have previously both been assessed for migraine-inducing properties in a human provocation model. Though eliciting comparable vasodilating properties, only PACAP-38 reliably triggered migraine attacks. VIP has a high affinity to VPAC1 and VPAC2 receptors. PACAP-38 has a similar high affinity to VPAC1 and VPAC2, but furthermore to a dedicated PACAP-receptor, PAC1. PACAP exists naturally in two isoforms, the most abundant PACAP-38 and a C-truncated 27-amino acid form, PACAP-27. The two forms have similar affinities to the three known receptors. The high similarity between VIP and PACAP-38 combined with their different migraine-inducing potential gives a rare opportunity to isolate which property causes one to induce migraine and the other not. This can lead to new insights in migraine pathophysiology. 

At the present, there are different hypothesis for neuropeptide-induced migraine. This thesis is divided in three projects all focusing on the VIP/PACAP peptides but addressing different anatomical compartments and mechanisms of action. Through this approach, it was attempted to elucidate differences in action that might shed light on the basis of PACAP-induced migraine. 

Paper I focuses on the vascular compartment, through in vitro studies of vasodilating properties of VIP, PACAP-27 and PACAP-38, blockade with selective receptor antagonists and expression studies. The tissues used in this paper were migraine-relevant intracranial arteries from the rat: Basilar artery (BA), middle cerebral artery (MCA) and middle meningeal artery (MMA). The functional assays wire myography and perfusion myography were combined with in situ hybridization for expression studies. Neither VIP nor PACAP induced relaxation of the MMA in the wire myograph. Pure PAC1-agonist maxadilan induced no relaxation in any of the tested vessels. Luminal administration of VIP in the perfusion myograph did not cause relaxation, excluding an endothelial mechanism of action. Potency of the pure agonists was approximately equal, with VIP being slightly more potent in the BA. Experiments with the VPAC1 antagonist PG 97-269 and the VPAC2 antagonist PG 99-265 indicated that relaxation induced by VIP and PACAP was mediated mainly by the VPAC1 receptor and to a lesser degree by the VPAC2 receptor. In situ hybridization demonstrated the presence of mRNA transcripts of all three receptors in the smooth muscle cells of the vessels. The conclusions are that PACAP is not a stronger vasodilator than VIP in tested intracranial arteries of the rat, and that VPAC1 is the dominating receptor in mediating vasodilatation in intracranial vessels induced by VIP and PACAP. Thus, the headache-inducing properties of PACAP compared to VIP cannot be explained through a purely vascular hypothesis. 

Paper II examines the effect of neuropeptides on mast cells of the rat. Mast cells are widely present in the perivascular areas of the dura mater and release inflammatory mediators on activation. Extracted peritoneal mast cells of the rat and intact dura mater of the rat in hemisected skull preparation were used as models. We investigated the degranulating effects of naturally occurring peptides VIP, PACAP-27 and PACAP-38 as well as N-truncated fragments of PACAP-38. In attempt to determine which intracellular pathway facilitated degranulation, transduction mechanism inhibitors for adenylyl cyclase (AC) and phospholipase C (PLC) were co-administered with the agonists. It was found that PACAP-38 and N-truncated fragments up to and including PACAP(16- 38) induce a much stronger degranulatory response on peritoneal mast cells than PACAP-27 and VIP. The EC50 for “strong degranulators” (PACAP-38, PACAP(6-38) and PACAP(16-38) was on average approximately 30 times lower than for the “weak degranulators” (VIP, PACAP-27 and PACAP(28-38)). The degranulation was significantly inhibited by co-treatment with PLC inhibitor, but was not affected by AC inhibition. In the intact dura mater, the same order of potency for the natural peptides, PACAP38>PACAP-27=VIP, was observed. In conclusion, the difference in headache-inducing potential between PACAP-38 and VIP may be explained by mast cell activation by PACAP-38. 

Paper III investigates some of the putative mechanisms for peptide-induced migraine induction in the migraine-related tissues trigeminal ganglion (TG), trigeminal nucleus caudalis (TNC) and dura mater of the rat. In these tissues, we performed assays for nitric oxide (NO) induction and calcitonin gene related peptide (CGRP) release against VIP and PACAP-38. Expression of relevant receptors was explored by qPCR. For nitric oxide synthases, CGRP and PACAP, co-localization studies were done by immunostaining. For PACAP-38 and not the other peptides investigated, a concentrationdependent release of CGRP was observed in the TNC. mRNA for the PAC1 receptor was expressed in all tissues, with TNC>TG>dura mater. mRNA for VPAC2 was most expressed in the TG. No significant effect on nitric oxide synthase activity was observed. In conclusion, the most profound differences observed in the employed models were the effects of the peptides on mast cells. This suggests that degranulation of mast cells may be among the causative factors for PACAP-induced migraine. Studying these mechanisms in depth may eventually yield novel treatment options for migraine.