The effects of ruthenium red on the inhibitory actions of noladin ether, THC & HU210 on sensory neurotransmission in the rat isolated mesenteric arterial bed M. Duncan, D.A. Kendall & V. Ralevic. School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH.	Print Abstract Search PubMed for: Duncan M Kendall DA Ralevic V

In the rat mesenteric arterial bed calcitonin gene-related peptide (CGRP) is released upon activation of sensory nerves producing vasodilatation (Kawasaki et al., 1988). We have previously reported that the endocannabinoid noladin ether, ⁹-tetrahydrocannabinol (THC) and HU210 (Ralevic & Kendall, 2001; Duncan et al, 2002a,b) inhibit sensory neurotransmission via a non-CB₁/CB₂ mechanism in this preparation. Zygmunt et al(2002) recently reported that THC released CGRP from sensory nerves in rat mesenteric arterial segments and this release was sensitive to the vanilloid receptor channel inhibitor ruthenium red but was independent of TRPV1. In this study we investigated whether ruthenium red could reverse the inhibitory actions of these cannabinoid receptor agonists on sensory neurotransmission in the rat isolated mesenteric arterial bed.

Male Wistar rats (250-300g) were killed by exposure to CO₂ and decapitation. Mesenteric beds were isolated and perfused with oxygenated Krebs' solution containing guanethidine (5µM) to block sympathetic neurotransmission (Ralevic & Kendall, 2001). After 30 min equilibration, preparations were preconstricted with methoxamine (10-100µM) and three consecutive frequency-response curves to electrical field stimulation (EFS, 1-12Hz, 60V, 0.1ms, 30s) (EFS control, EFSI and EFSII) were constructed in each preparation. The agonist or vehicle (0.01% ethanol) was added after EFS control, 15 min before EFSI. Ruthenium red (1µM) was added at the start of the equilibration period. Data are expressed as mean±s.e.m. and analysed by ANOVA with Tukey's post-hoc test or by Student's unpaired t test.

EFS produced frequency-dependent relaxation (1-12Hz) of the rat mesenteric bed. In the presence of 1µM noladin ether, the sensory neurogenic relaxation response, at a submaximal frequency of 8Hz was reduced from 57.33±6.83%, EFS control, to 23.3±3.78%, EFSII (n=4, P<0.05). In the presence of 1µM ruthenium red, the response at 8Hz was reduced from 46.82±6.44%, EFS control, to 17.96±2.47%, EFSII (n=6, P<0.01). THC (1µM) inhibited the response at 8Hz from 53.08±4.98%, EFS control, to 11.14±1.58%, EFSII (n=4, P<0.001); ruthenium red attenuated this effect (8Hz EFS control 54.51±5.78% to EFSII 25.26±4.86%, n=12, P<0.01). HU210 (1mM) also inhibited the response at 8Hz; it was reduced from 57.97±5.37%, EFS control, to 18.37±4.08%, EFSII (n=7, P<0.001). In the presence of ruthenium red, the inhibitory effect of HU210 at 8Hz was abolished; 49.15±6.96%, EFS control, to 40.63±3.65%, EFSII (n=4, P>0.05).

These data show that the inhibitory actions of noladin ether and THC on sensory neurotransmission are largely resistant to ruthenium red. The inhibition of sensory neurotransmission by HU210, however, is ruthenium red sensitive. The site of action for noladin ether and THC is different from that of HU210 and does not appear to require the presence of functional vanilloid receptors.

We are grateful to Servier for financial support.

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