Enhanced vasorelaxant effects of Δ9-tetrahydrocannabinol (THC) in mesenteric arteries fromrats treated chronically with L-name

Saoirse E. O’Sullivan*, Michael D. Randall & Sheila Gardiner, School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH.

The aim of this study was to investigate whether vasorelaxation to Δ9-tetrahydrocannabinol (THC) is altered in mesenteric arteries isolated from rats treated chronically with the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME).

Male, Sprague-Dawley rats (200-250g) were assigned to either water (control) or L-NAME (0.1 mg ml-1 solution in water; ~7.5 mg kg-1 day-1) drinking groups. After 4 weeks, animals were killed by cervical dislocation and arteries were isolated and mounted on a Mulvany-Halpern Myograph. Briefly, 2 mm segments of third-order branches of the superior mesenteric artery (G3) were mounted on fine tungsten wires (40 µm diameter). Vessels were bathed in oxygenated Krebs’solution at 37oC. Tension was recorded on a MacLab 4e recording system ( ADInstruments, UK). All vessels were kept at 37°C in Krebs-Henseleit buffer and gassed with 5% CO2 in O2. Arteries were contracted with methoxamine and the thromboxane mimetic U46619 and once a stable contraction was achieved, the vasorelaxant effects of ligands were assessed as cumulative concentration-response curves. THC was dissolved in ethanol at 10 mM with further dilutions made in distilled water. The steady state response was taken at each concentration and expressed as the percentage relaxation of the pharmacologically-induced contraction .

Mesenteric resistance vessels (G3) from L-NAME-treated animals showed enhanced vasorelaxant responses to THC compared to controls (L-NAME-treated pEC50 = 6.13 ± 0.13, mean ± SEM, n =8 vs control pEC50 = 5.58 ± 0.12, n=8, P<0.01, ANOVA). Vasorelaxations to acetylcholine (L-NAME-treated pEC50 = 7.81 ± 0.13, n=5 vs control pEC50 = 7.64 ± 0.17, n=6) and verapamil (L-NAME-treated pEC50 = 7.72 ± 0.25, n=6 vs control pEC50 = 7.53 ± 0.19, n=7) were not different between the two groups. The response to THC in arteries obtained from either L-NAME-treated or control rats was not inhibited by the CB1 receptor antagonist, AM251 (1 μM). Pre-treatment with the TRPV1 receptor agonist capsaicin (10 μM, 1h), to deplete sensory nerves of neurotransmitters, caused an 8-fold reduction in the potency of THC in control rats (pEC50 = 5.58 ± 0.12, n=8 cf capsaicin pre-treatment pEC50 = 4.68 ± 0.30, n=6, P<0.05). However, in vessels from animals treated chronically with L-NAME, capsaicin pre-treatment caused a greater (38-fold) decrease in the potency of THC (pEC50 = 6.13 ± 0.13, n=8 vs capsaicin pre-treatment pEC 50 = 4.55 ± 0.25, n=6, P<0.01, ANOVA), and indomethacin (10 μM) inhibited the THC responses in arteries from L-NAME-treated rats (pEC50 = 6.13 ± 0.13, n=8 cf indomethacin pEC50 = 5.14 ± 0.20, n=6, P<0.001), but not controls.

In conclusion, we have shown that the vasorelaxant effect of THC is enhanced in isolated arteries obtained from rats treated chronically with L-NAME. The mechanisms underlying this effect appear not to be CB1-receptor mediated, but involve an increased sensory nerve component and prostanoid production stimulated by THC.

S.O’S is funded by an Early Career Fellowship from the Leverhulme Trust.