Is there an endogenous cannabinoid tone in the guinea-pig and rat isolated ileum myenteric plexus-longitudinal muscle?

Makwana, R., Molleman, A., & Parsons, M.E. School of Life Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire, AL10 9AB, U.K.

In the myenteric plexus-longitudinal muscle (MPLM) preparation of the ileum of the guinea-pig and rat, the cannabinoid CB1 receptor antagonist SR 141716 enhances electrical field stimulation (EFS) induced contractions, suggesting either an endocannabinoid tone or inverse agonism. In the present study we investigated the possible existence of a endocannabinoid tone by determining the effect of three fatty acid amide hydrolase (FAAH) inhibitors; AA-5HT (arachidonoylserotonin), PMSF (phenylmethylsulphonyl fluoride) and URB-597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester) and of VDM-11 ( N -(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide), an inhibitor of the putative anandamide uptake transporter, by themselves and on the potency of exogenously applied anandamide and WIN 55212-2 to inhibit EFS evoked contractions.

Strips of MPLM were dissected from the ileum of Dunkin-Hartley guinea-pigs (500-800 g) of either sex or male Wistar rats (400-550 g) for recording of isometric contractile responses to EFS. Guinea-pig and rat MPLM strips were stimulated with single pulses of supramaximal voltage, 0.5 ms duration at a frequency of 0.1 Hz and 0.05 Hz respectively. The EFS induced contractions of both tissues were abolished by treatment with either tetrodotoxin (10-6 M) or atropine (10-6 M). All drugs with the exception of tetrodotoxin and atropine were dissolved in 100 % ethanol. Changes in the amplitude of the EFS evoked contractions are expressed as mean ± s.e.m ratio of the amplitude of the contractile response after each addition of the drug to the amplitude immediately before the first addition of the drug.

Both anandamide (10-11 - 10-5 M) or WIN 55212-2 (10-11 - 10-6 M) produced a concentration dependent inhibition of the EFS evoked contractions with similar maxima and pIC50 of 6.85 ± 0.01 (n= 6) and 8.52 ± 0.01 (n=6) respectively on the guinea-pig and 7.64 ± 0.03 (n= 6) and 8.21 ± 0.03 (n=6) respectively on the rat. This inhibition was antagonised by a 20 min pre-incubation with SR 141716 (10-7 or 10-6 M) with pA2 values of 7.90 and 7.97 respectively on the guinea pig and 7.68 and 8.21 respectively on the rat tissues. By themselves cumulative additions of AA-5HT (up to 10-5 M), PMSF (up to 10–4 M) or URB-597 (up to 10-5 M) had no effect on the EFS evoked contractions alone in either species. However 15 min pre-treatment of the tissues with AA-5HT (10-6 M), PMSF (10-4 M) or URB-597 (3 x 10-8 M) produced significant (P < 0.05, one way-ANOVA) leftward parallel displacements in the concentration response curves of anandamide. The dose ratios caused by each FAAH inhibitor on the guinea-pig tissues were 3.24, 32.44 and 48.81 respectively while in the rat tissues these values were 2.04, 22.14 and 20.24 respectively. WIN 55212-2 induced inhibition was not altered by the three FAAH inhibitors in either species. VDM-11 (10-9 – 10-5 M) had no effect on its own nor did a 15 min pre-incubation with a concentration of 10-5 M produce a displacement of the anandamide dose response curve.

Under the EFS conditions employed, the lack of effect of the FAAH inhibitors in the absence of the exogenous agonists suggests that the MPLM does not release anandamide or substrates that are susceptible to hydrolysis by FAAH. The potentiation of the action of exogenously applied anandamide suggests that FAAH is present in the guinea-pig and rat MPLM. The lack of effects of VDM-11 may support the absence of an endocannabinoid tone or simply indicate the absence of the uptake transporter for anandamide in the MPLM. Our results support the notion that an endocannabinoid tone is absent in the guinea-pig and rat MPLM and therefore that the action of SR 141716 is due to inverse agonism.