Elsevier

Neuroscience

Volume 169, Issue 2, 25 August 2010, Pages 683-696
Neuroscience

Cognitive, Behavioral, and Systems Neuroscience
Research Paper
Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion

https://doi.org/10.1016/j.neuroscience.2010.05.016Get rights and content

Abstract

Calcitonin gene related peptide (CGRP) has a key role in migraine and recently CGRP receptor antagonists have demonstrated clinical efficacy in the treatment of migraine. However, it remains unclear where the CGRP receptors are located within the CGRP signaling pathway in the human trigeminal system and hence the potential antagonist sites of action remain unknown. Therefore we designed a study to evaluate the localization of CGRP and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP) 1 in the human trigeminal ganglion using immunohistochemistry and compare with that of rat. Antibodies against purified CLR and RAMP1 proteins were produced and characterized for this study. Trigeminal ganglia were obtained at autopsy from adult subjects and sections from rat trigeminal ganglia were used to compare the immunostaining pattern. The number of cells expressing CGRP, CLR and RAMP1, respectively, were counted. In addition, the glial cells of trigeminal ganglion, particularly the satellite glial cell, were studied to understand a possible relation. We observed immunoreactivity for CGRP, CLR and RAMP1, in the human trigeminal ganglion: 49% of the neurons expressed CGRP, 37% CLR and 36% RAMP1. Co-localization of CGRP and the receptor components was rarely found. There were no CGRP immunoreactions in the glial cells; however some of the glial cells displayed CLR and RAMP1 immunoreactivity. Similar results were observed in rat trigeminal ganglia. We report that human and rat trigeminal neurons store CGRP, CLR and RAMP1; however, CGRP and CLR/RAMP1 do not co-localize regularly but are found in separate neurons. Glial cells also contain the CGRP receptor components but not CGRP. Our results indicate, for the first time, the possibility of CGRP signaling in the human trigeminal ganglion involving both neurons and satellite glial cells. This suggests a possible site of action for the novel CGRP receptor antagonists in migraine therapy.

Section snippets

Generation of antibodies

Antibodies were generated by Cambridge Research Biochemicals (Billingham, Cleveland, UK). Antigenic sequences were selected for human and rat CLR and RAMP1, purified, and coupled to keyhole limpet haemocyanin (KLH). Human CLR (acetyl-GYSHDCPSEHLNGK) was coupled to KLH using gluteraldehyde. Rat CLR (C-SIQDIENVALKPEKMYDLV), human RAMP1 (C-Ahx-QSKRTEGIV) and rat RAMP1 (C-Ahx-RSKRTEGIV) were coupled using m-Maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) to KLH via an N-terminal cysteine.

Specificity of CLR and RAMP1 antibodies

The specificity of the CLR and RAMP1 antibodies were evaluated by Western blotting and immunofluorescence using stably and transiently transfected cells. Antibody 3152 (human CLR) recognized a single protein of approximately 54 kDa in HEK293 cell membranes stably expressing the human CGRP receptor consistent with the predicted mass of the protein (Fig. 1A). Antibodies 3155 and 132 (rat CLR) recognized two proteins with molecular masses of approximately 49 and 60 kDa in HEK293 cells transiently

Discussion

The aim of the present study was to identify CGRP and its receptor components within the human trigeminal ganglion compared with the rat and to evaluate morphologically their relationship using immunohistochemistry. For this purpose, we developed new antibodies against human and rat CLR and RAMP1 and characterized these in detail. The study has, for the first time, demonstrated the detailed distribution and quantification of CGRP receptor components in the human trigeminal ganglion. The CGRP

Conclusion

The neuropeptide CGRP is implicated in the underlying pathology of migraine and the CGRP receptor has long been regarded as a useful target for the development of novel antimigraine therapies. Using immunohistochemistry and new antibodies specifically recognizing CLR and RAMP1, we studied in detail CGRP and its receptor components in the human trigeminal ganglion and compared with that of rat. Our study contributes to a better understanding of CGRP signaling in the trigeminal ganglion. In

Acknowledgments

Thanks to Professor Karin Warfvinge and Warfvinge Science Support for assistance and valuable comments on the manuscript.

References (52)

  • M. Hou et al.

    5-HT(1B) and 5-HT(1D) receptors in the human trigeminal ganglion: co-localization with calcitonin gene-related peptide, substance P and nitric oxide synthase

    Brain Res

    (2001)
  • S. Just et al.

    The role of CGRP and nicotinic receptors in centrally evoked facial blood flow changes

    Neurosci Lett

    (2005)
  • J. Li et al.

    Calcitonin gene-related peptide stimulation of nitric oxide synthesis and release from trigeminal ganglion glial cells

    Brain Res

    (2008)
  • V. Limmroth et al.

    An in vivo rat model to study calcitonin gene related peptide release following activation of the trigeminal vascular system

    Pain

    (2001)
  • Y. Liu et al.

    Central projections of sensory innervation of the rat superior sagittal sinus

    Neuroscience

    (2004)
  • Y. Liu et al.

    Central projections of the sensory innervation of the rat middle meningeal artery

    Brain Res

    (2008)
  • J.J. Mallee et al.

    Receptor activity-modifying protein 1 determines the species selectivity of non-peptide CGRP receptor antagonists

    J Biol Chem

    (2002)
  • M. Quartu et al.

    Calcitonin gene-related peptide in the human trigeminal sensory system at developmental and adult life stages: immunohistochemistry, neuronal morphometry and coexistence with substance P

    J Chem Neuroanat

    (1992)
  • J. Tajti et al.

    Messenger molecules and receptor mRNA in the human trigeminal ganglion

    J Auton Nerv Syst

    (1999)
  • R. Uddman et al.

    Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: trigeminal origin and co-existence with substance P

    Neurosci Lett

    (1985)
  • J.L. Bellamy et al.

    Salivary levels of CGRP and VIP in rhinosinusitis and migraine patients

    Headache

    (2006)
  • A. Capuano et al.

    Proinflammatory-activated trigeminal satellite cells promote neuronal sensitization: relevance for migraine pathology

    Mol Pain

    (2009)
  • G.S. Cottrell et al.

    Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat

    J Comp Neurol

    (2005)
  • P.L. Durham

    Calcitonin gene-related peptide (CGRP) and migraine

    Headache

    (2006)
  • P.L. Durham

    Inhibition of calcitonin gene-related peptide function: a promising strategy for treating migraine

    Headache

    (2008)
  • L. Edvinsson

    Blockade of CGRP receptors in the intracranial vasculature: a new target in the treatment of headache

    Cephalalgia

    (2004)

    • Cited by (0)

    View full text
    Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.