Mechanisms of GPR55-mediated relaxation in mouse mesenteric arteries

C Sudlow, E McNaughton, V Ho. St George's University of London, London, UK

It has been suggested that the orphan G-protein-coupled receptor GPR55 is a novel vascular target for the endogenous cannabinoid, N-arachidonoyl ethanolamine (AEA). AEA is an agonist for cannabinoids receptors, but it is also known to mediate vasorelaxation that is independent of both cannabinoid CB1 and CB2 receptors (1). We have previously shown that relaxation response to AEA in mouse mesenteric arteries is reduced by the putative GPR55 antagonist O-1918 or in arteries from GPR55 KO mice, suggesting a role for GPR55 in the vascular action of AEA (2). In this study, we further investigated the GPR55-mediated relaxation in mouse mesenteric arteries.

Male wild-type (C57BL/6J) and age-matched GPR55 KO mice (14-21 weeks) were killed by cervical dislocation and mesenteric arteries were isolated and mounted in a wire myograph for isometric tension recording. Unless otherwise stated, vessels were contracted by 3µM methoxamine and 600nM U46619 before cumulative additions of AEA (0.1-30µM). Rmax represents % relaxation of precontracted tone attained at the maximum concentration of AEA used (30µM). pEC50 values were determined from Rmax of individual log concentration-response curves. Data are shown as mean±sem (n=4-10) and analysed by two-way analysis of variance, followed by a Bonferroni post-hoc test.

Relaxation response to AEA was significantly reduced in GPR55 KO mice (WT: pEC50=6.0±0.2, Rmax=60±6%; KO: pEC50=5.4±0.2, Rmax=40±7%; P<0.01) but was independent of the endothelium (WT; +endothelium: Rmax=55±11%; -endothelium: Rmax=62±10%). The AEA relaxation was not affected by CB1 and CB2 antagonists (WT control: pEC50=6.1±0.3, Rmax=59±7%; +1µM JTE907 and 1µM AM281 for 30min: pEC50=6.1±0.4, Rmax=58±10%), an adenylyl cyclase inhibitor (+20µM SQ22356 for 1h: pEC50=6.1±0.3, Rmax=47±11%) or the Gi/o protein uncoupler pertussis toxin (500ng/ml for 1h: pEC50=6.2±0.3, Rmax=62±8%). In contrast, AEA relaxations were inhibited by precontraction with 60mM KCl (pEC50=4.7±0.1, Rmax=38±19%; P<0.01) or the presence of the large-conductance Ca2+-activated K+ channel (BKCa) inhibitor, iberiotoxin (50nM for 30min; pEC50=5.5±0.2, Rmax=55±12%; P<0.01). Inhibition of integrin receptor function by RGDS peptide (100µM for 30min) also significantly reduced relaxation to AEA (WT; control peptide: pEC50=5.5±0.1, Rmax=68±10%; +RGDS: pEC50=5.1±0.1, Rmax=58±6%; P<0.01). Iberiotoxin or RGDS peptide had no effect on AEA responses in GPR55 KO mice (data not shown). RGDS peptide also had no effect on relaxation induced by the BKCa activator, NS11021 in WT mice (data not shown).

In conclusion, our data suggest that AEA induces mesenteric relaxation via a GPR55-integrin-BKCa pathway. This relaxation pathway does not involve the endothelium, Gi/o proteins or adenylyl cyclase. Further investigations are needed to elucidate how GPR55 is linked to integrins and the integrin subtypes involved.

(1) Begg M et al (2005). Evidence for novel cannabinoid receptors. Pharmacol Ther 206: 33-145.

(2) McNaughton E and Ho WSV (2013). Proceedings of British Pharmacological Society at http://www.pa2online.org/abstracts/vol11issue3abst074p.pdf