Evidence for CGRP re-uptake in rat dura mater encephali

Abstract

Background and purpose: Calcitonin gene-related peptide (CGRP) is widely distributed in the trigeminovascular system and released from sensory fibres of the cranial dura mater upon noxious stimulation. Such release may be a mechanism underlying migraine headache. Based on data from guinea pig basilar artery preparations, we have here studied CGRP release and uptake in an organ preparation of the hemisected rat skull.

Experimental approach: CGRP release from the cranial dura was quantified by a commercial enzyme-linked immunoassay. CGRP was depleted using repetitive challenges of capsaicin. After incubating the tissue with CGRP for 20 min and extensive washing, another capsaicin challenge was performed. Immunohistochemistry was used to visualize CGRP immunofluorescence in dural nerve fibres.

Key results: Capsaicin-induced CGRP release was attenuated by the transient receptor potential vanilloid receptor type I antagonist capsazepine or by Ca(2+)-free solutions. After the CGRP-depleted preparation had been exposed to exogenous CGRP, capsaicin-induced CGRP release was increased compared to the challenge just prior to incubation. CGRP uptake was not influenced by Ca(2+)-free solutions. Olcegepant and CGRP(8-37) (CGRP receptor antagonists) did not affect uptake of CGRP. However, a monoclonal CGRP-binding antibody decreased CGRP uptake significantly. Release of CGRP after incubation was attenuated by Ca(2+)-free solutions and by capsazepine. Immunohistochemical assays indicated a weak trend towards CGRP uptake in rat dura mater.

Conclusion and implications: We have presented evidence for CGRP uptake in nerves and its re-release in rat dura mater. This may have implications for the pathophysiology and treatment of migraine.

Figure 8

Fluorescence images of calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers in the rat dura mater. Compared to an untreated control dura (A, panel a) the nerve fibers show a clear ‘string of pearls’ appearance after depletion of CGRP with capsaicin (B, panel b), which may indicate local clustering of CGRP within the axon. After incubation with CGRP (C, panel c) immunopositive varicosities seem to be more prominent as compared to depleted nerve fibers incubated with vehicle (B, panel b). Mast cells cross-reacting with the antibody as seen in the control (lower right in A) are no longer visible after capsaicin treatment. Magnification is the same in all images, size bar 100 µm. On the right panel, three equally magnified (3×) sections (a–c) of the corresponding left panels (A–C) (n = 4).

Figure 1

Calcitonin gene-related peptide (CGRP) release in response to capsaicin stimulation (10 min) at different concentrations in rat hemisected skull (A) and effect of capsazepine incubated in the skull for 10 min before capsaicin-induced CGRP release (B). **P < 0.01 and ***P < 0.0001, significantly different from basal value; ##P < 0.01, significantly different from capsaicin without capsazepine. One-way anova followed by Dunnett's multiple comparison test (n = 5−11) (A) and the non-parametric paired Wilcoxon signed-rank test was used (n = 4−7) (B).

Figure 2

Effect of Ca²⁺-free synthetic interstitial fluid (SIF) on basal calcitonin gene-related peptide (CGRP) levels (A) and on capsaicin-induced CGRP release (B). For statistical analysis the non-parametric paired Wilcoxon signed-rank test was used. **P < 0.01 and ***P < 0.0001, significantly different from normal SIF group (n = 10−12).

Figure 3

Calcitonin gene-related peptide (CGRP) release in response to four successive challenges of capsaicin (100 nM). This was followed by incubation with exogenous CGRP 100 nM (A) or synthetic interstitial fluid (SIF) (B). After extensive washing, a fifth capsaicin challenge was used to test if CGRP was taken up. For statistical analysis the non-parametric paired Wilcoxon signed-rank test was used. **P < 0.01 and ***P < 0.0001, significantly different from basal CGRP levels; $P < 0.05 and $$P < 0.01, significantly different from first challenge; #P < 0.05, significantly different from fourth challenge (n = 6).

Figure 4

Calcitonin gene-related peptide (CGRP) release in response to four successive capsaicin (100 nM) challenges to deplete CGRP followed by one KCl (60 mM) challenge (A), or eight successive KCl (60 mM) challenges to deplete CGRP followed by one capsaicin (100 nM) challenge (B). For statistical analysis the non-parametric paired Wilcoxon signed-rank test was used. *P < 0.05, significantly different from basal CGRP levels; $P < 0.05, significantly different from first capsaicin or first KCl challenge; #P < 0.05, significantly different from fourth capsaicin or eighth KCl challenge (n = 6). Contr., control; ns, not significant.

Figure 5

Effect of simultaneous incubation of the calcitonin gene-related peptide (CGRP) antibody (Ab) 12 µg·mL⁻¹ (A) and 48 µg·mL⁻¹ (B) with CGRP (100 nM) on capsaicin-induced CGRP release subsequent to uptake. For statistical analysis the non-parametric paired Wilcoxon signed-rank test was used. #P < 0.05, significantly different from fourth challenge and *P < 0.05, significantly different from fifth capsaicin challenge in absence of antibody incubation (n = 6−8). ns, not significant.

Figure 6

Effect of calcitonin gene-related peptide (CGRP) incubation in Ca²⁺-free synthetic interstitial fluid (SIF) on capsaicin-induced CGRP release in normal SIF (A) and capsaicin challenge in Ca²⁺-free SIF, subsequent to CGRP incubation in normal SIF (B). For statistical analysis the non-parametric paired Wilcoxon signed-rank test was used. ##P < 0.01 and ###P < 0.0001, significantly different from fourth challenge (n = 6−8).

Figure 7

Effect of capsazepine (CPZ) on capsaicin (Caps)-induced calcitonin gene-related peptide (CGRP) release (eighth challenge) subsequent to depletion by four capsaicin challenges (1–4) and three KCl challenges (fifth to seventh) followed by 20 min incubation with CGRP and washing. For statistical analysis the non-parametric paired Wilcoxon signed-rank test was used. #P < 0.05, significantly different from the seventh challenge; *P < 0.05, significantly different from capsaicin challenge in absence of capsazepine (n = 6). ns, not significant.

Figure 9

Quantitative data on calcitonin gene-related peptide (CGRP) immunofluorescence in dura mater of control preparations and preparations treated with capsaicin or capsaicin and CGRP. Each mean (±SEM) is from measurements of 48 images in four dura halves. Average size of fluorescent particles (A) and intensity of fluorescence are shown as inverse grey density (B). *P < 0.05, significant difference between control and capsaicin-treated but not capsaicin-/CGRP-treated preparations (Mann–Whitney U-test).

Figure 10

Schematic presentation of putative calcitonin gene-related peptide (CGRP) reuptake into vesicles in dural sensory nerves through unknown transporter. This uptake was not mediated by pre-synaptic CGRP receptors because CGRP receptor antagonists were not able to block the uptake of exogenous CGRP. TRPV1, transient receptor potential vanilloid receptor type I.