title: Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries.
abstract: OBJECTIVE
  Calcitonin gene-related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR●RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin-converting enzyme-1 (ECE-1). However, it is not known if ECE-1 regulates the resensitization of CGRP-induced responses in functional arterial tissue.
  
  
  METHODS
  CLR, ECE-1a-d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA-SMCs) and mesenteric arteries was analysed by RT-PCR and by immunofluorescence and confocal microscopy. CGRP-induced signalling in cells was examined by measuring cAMP production and ERK activation. CGRP-induced relaxation of arteries was measured by isometric wire myography. ECE-1 was inhibited using the specific inhibitor, SM-19712.
  
  
  RESULTS
  RMA-SMCs and arteries contained mRNA for CLR, ECE-1a-d and RAMP1. ECE-1 was present in early endosomes of RMA-SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium-independent relaxation of arteries. ECE-1 inhibition had no effect on initial CGRP-induced responses but reduced cAMP generation in RMA-SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges.
  
  
  CONCLUSIONS
  ECE-1 regulated the resensitization of responses to CGRP in RMA-SMCs and mesenteric arteries. CGRP-induced relaxation did not involve endothelium-derived pathways. This is the first report of ECE-1 regulating CGRP responses in SMCs and arteries. ECE-1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine.
authors.affiliation: Reading School of Pharmacy, University of Reading, Whiteknights, Reading, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: McNeish
authors.forename: B T
authors.initials: BT
authors.lastname: Roux
authors.forename: S-B
authors.initials: SB
authors.lastname: Aylett
authors.forename: A M
authors.initials: AM
authors.lastname: Van Den Brink
authors.forename: G S
authors.initials: GS
authors.lastname: Cottrell
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000502:physiology*; D001794:Blood Pressure / Q000502:physiology; D015740:Calcitonin Gene-Related Peptide / Q000502:physiology*; D058286:Calcitonin Receptor-Like Protein / Q000502:physiology; D002478:Cells, Cultured; D011992:Endosomes / Q000502:physiology; D000072396:Endothelin-Converting Enzymes; D008297:Male; D008638:Mesenteric Arteries / Q000166:cytology / Q000502:physiology*; D008666:Metalloendopeptidases / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D059748:Proteolysis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D058262:Receptor Activity-Modifying Protein 1 / Q000502:physiology; D014664:Vasodilation / Q000502:physiology
chemical_list: D058286:Calcitonin Receptor-Like Protein; D012333:RNA, Messenger; C545508:Ramp1 protein, rat; D058262:Receptor Activity-Modifying Protein 1; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; D015740:Calcitonin Gene-Related Peptide