Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens

Abstract

1. This study was directed at exploring the structure-activity relationship for anandamide and certain of its analogues at the rat VR1 receptor in transfected cells and at investigating the relative extent to which anandamide interacts with CB(1) and vanilloid receptors in the mouse vas deferens. 2. pK(i) values for displacement of [(3)H]-resiniferatoxin from membranes of rVR1 transfected CHO cells were significantly less for anandamide (5.78) than for its structural analogues N-(4-hydroxyphenyl)-arachidonylamide (AM404; 6.18) and N-(3-methoxy-4-hydroxy)benzyl-arachidonylamide (arvanil; 6.77). 3. pEC(50) values for stimulating (45)Ca(2+) uptake into rVR1 transfected CHO cells were significantly less for anandamide (5.80) than for AM404 (6.32) or arvanil (9.29). Arvanil was also significantly more potent than capsaicin (pEC(50)=7.37), a compound with the same substituted benzyl polar head group as arvanil. 4. In the mouse vas deferens, resiniferatoxin was 218 times more potent than capsaicin as an inhibitor of electrically-evoked contractions. Both drugs were antagonized to a similar extent by capsazepine (pK(B)=6.93 and 7.18 respectively) but were not antagonized by SR141716A (1 microM). Anandamide was less susceptible than capsaicin to antagonism by capsazepine (pK(B)=6.02) and less susceptible to antagonism by SR141716A (pK(B)=8.66) than methanandamide (pK(B)=9.56). WIN55212 was antagonized by SR141716A (pK(B)=9.02) but not by capsazepine (10 microM). 5. In conclusion, anandamide and certain of its analogues have affinity and efficacy at the rat VR1 receptor. In the mouse vas deferens, which seems to express vanilloid and CB(1) receptors, both receptor types appear to contribute to anandamide-induced inhibition of evoked contractions.

Figure 1

Structures of anandamide (arachidonyl ethanolamide), methanandamide, AEA (22 : 4, n-6) (docosatetraenyl ethanolamide), AEA (18 : 2, n-6) (linoleyl ethanolamide), MAFP (methyl arachidonyl fluorophosphonate), AM404 (4-hydroxyphenyl arachidonyl ethanolamide), capsaicin (3-methoxy-4-hydroxy benzyl-8-methyl-6-nonenamide) and arvanil (3-methoxy-4-hydroxy benzyl arachidonyl amide).

Figure 2

Displacement of [³H]-RTX (100 pM) by (a) anandamide (AEA) in the presence and absence of PMSF (b) AM404 in the presence and absence of PMSF and (c) arvanil and capsaicin in the presence of PMSF (200 μM). Each symbol represents the mean percent displacement±the s.e.mean (n=4 – 6).

Figure 3

The stimulation of ⁴⁵Ca²⁺ uptake by (a) anandamide (AEA) in the presence and absence of PMSF (b) AM404, AEA (22 : 4) and AEA (18 : 2) in the presence of 100 μM PMSF and (c) arvanil and capsaicin in the presence of 100 μM PMSF. The per cent stimulation is calculated from an RTX maximum for each experiment. Each symbol represents the mean per cent stimulation±the s.e.mean (n=4 – 6).

Figure 4

Mean concentration-response curves for capsaicin constructed in the presence of capsazepine or DMSO. Each symbol represents the mean value±s.e.mean for inhibition of electrically-evoked contractions of the mouse isolated vas deferens expressed as a percentage of the amplitude of the twitch response measured immediately before the first addition of capsaicin to the organ bath (n=6). Capsazepine and DMSO (10 μl) were added 30 min before the first addition of capsaicin. To minimize the effects of desensitization, responses to no more than two concentrations of capsaicin were measured in any one tissue. When 10 μl DMSO was added instead of capsaicin to tissues pretreated with 10 μl DMSO, mean twitch amplitude was not significantly affected by the first addition of DMSO and decreased by 12.4±3.9% after the second DMSO addition (n=6). pEC₅₀ and E_max values of capsaicin were 9.61±0.36 and 66.0±3.2% respectively in the presence of DMSO and 7.64±0.78 and 57.6±7.6% respectively in the presence of capsazepine (GraphPad Prism).