Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries

Abstract

Background and purpose: 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.

Experimental approach: 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.

Key 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 and implications: 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.

Figure 1

RMA-SMCs express CLR, RAMP1 and ECE-1 isoforms. (A) Expression of immunoreactive α-smooth muscle actin in cultures from rat mesenteric resistance arteries, indicating cultured cells are SMCs. Scale bar, 100 μm. (B) RT-PCR amplification of mRNA for CLR (442 bp), ECE-1a (177 bp), ECE-1b (289 bp), ECE-1c (288 bp), ECE-1d (255 bp) and RAMP1 (277 bp) in RMA-SMCs. bp, base pairs. (C) Immunoreactive ECE-1 was detected in vesicles in α-smooth muscle actin-positive cells. ECE-1 co-localizes with a marker for early endosomes, the early endosome antigen-1 (EEA1) in RMA-SMCs. Scale bar, 10 μm; n = 3.

Figure 2

ECE-1 regulates resensitization of CGRP-induced cAMP generation in RMA-SMCs. (A) Concentration–response analysis of CGRP-induced cAMP generation in RMA-SMCs. CGRP induced a biphasic response indicating the presence of more than one receptor type in RMA-SMCs. n = 3 wells. (B) Western blot analysis of CGRP-induced phosphorylation of ERK in RMA-SMCs. CGRP induced a prompt increase in levels of phosphorylated ERK1 and ERK2 (pERK1/2) at 2 and 10 min. n = 3 (C) RMA-SMCs were incubated with vehicle (control) or SM-19712, stimulated with vehicle (control) or CGRP (10 nM, 10 min), washed, incubated in CGRP-free medium (30 min) and cAMP accumulation to a second challenge of CGRP (10 nM) recorded. SM-19712 reduced resensitization of CGRP-induced cAMP generation (vehicle, 70 ± 12% and SM-19712, 35 ± 10%). n = 4, *P < 0.05.

Figure 3

Rat mesenteric arteries express ECE-1 and the CGRP receptor components, CLR and RAMP1. (A) RT-PCR amplification of mRNA for CLR (442 bp), RAMP1 (277 bp), ECE-1a (177 bp), ECE-1b (289 bp), ECE-1c (288 bp), ECE-1d (255 bp) in rat mesenteric arteries. (B) Localization of nuclei (DAPI), ECE-1 and PECAM-1 in whole mounts of rat mesenteric artery. Immunoreactive ECE-1 was detected in the endothelial and SMC layers of mesenteric arteries. PECAM-1-immunoreactivity was only detected in the endothelial cell layer. Scale bar, 20 μm. (C) Immunoreactive CLR, RAMP1 and ECE-1 was detected in the smooth muscle layer of sections of mesenteric arteries. Nuclei are shown by DAPI staining. Scale bar, 50 μm; n = 3.

Figure 4

Effect of the ECE-1 inhibitor SM-19712 on CGRP-induced relaxation in rat mesenteric arteries. Arteries were incubated with vehicle (control), contracted with phenylephrine (0.3–10 μM), exposed to increasing concentrations of CGRP (1 pM–3 nM), washed, contracted with phenylephrine and exposed again to increasing concentrations of CGRP (1 pM–3 nM). Arteries were then incubated with SM-19712 (10 μM) and the concentration-response curves repeated. (A) Incubation of arteries with SM-19712 had no effect on initial responses to CGRP. (B, C) SM-19712 shifted the pEC₅₀ to a second challenge of CGRP at both 5 and 30 min after the initial challenge. n = 4 arteries for each group.