Characterisation of cannabinoid receptors and their ligands in isolated smooth muscle preparations
In recent years it has been shown conclusively that at least two cannabinoid receptors, termed CB1 and CB2, exist in mammalian tissues. Previous studies using the mouse isolated vas deferens have yielded results which suggest that this tissue contains cannabinoid CB1 receptors which, when activated, can mediate inhibition of electrically-evoked contractions. However, there is evidence which indicates that several of the cannabinoid receptor agonists investigated in this study may exert their effects via non-CB, or even non- cannabinoid mechanisms. In the present study, this evidence was further investigated using the cannabinoid-mediated inhibition of electrically-evoked contractions in the mouse isolated vas deferens as a model of study. The results obtained from studies using the cannabinoid receptor antagonists O-1184 and the CB1-selective SR141716A highlighted the existence of a level of agonist-dependent antagonism in mouse isolated vas deferens. This was indicated by discrepancies obtained in the pKB values of these antagonists against the compounds under investigation. In this series of investigations it was observed that the endogenous cannabinoid receptor agonist, anandamide and the capsaicin-anandamide hybrid compound, arvanil were less potently antagonised by the CB1selective antagonist/inverse agonist, SR141716A than the highly CB1-selective agonist methanandamide. Such discrepancies in pKB values indicate that anandamide and arvanil may be acting on a receptor type distinct from the cannabinoid CB1 receptor. Additionally this series of studies indicated that anandamide and WIN55212-2 were more potently antagonised when non-cumulative responses to these compounds were constructed, indicating the possibility of tolerance developing to these compounds during the construction of cumulative concentration response curves. Several, more recent studies have indicated that anandamide and its metabolically more stable analogue methanandamide may exert their actions in part through vanilloid VR1 receptors. Upon further investigation using the vanilloid VR1 receptor antagonist capsazepine in addition to SR141716A, it was observed that the effects of anandamide, methanandamide, and the capsaicin-anandamide hybrid arvanil could be attenuated by both antagonists. These results indicate that these three agonists can act through both receptor types to mediate their effects in the mouse isolated vas deferens. In this study the putative water-soluble cannabinoid receptor agonist, O-1057 was shown to inhibit the of electrically-evoked contractions in the mouse isolated vas deferens when only water was used as a vehicle. This effect was inhibited by the cannabinoid receptor antagonists O-1184 and SR141716A, providing evidence that this novel water-soluble compound was acting through the CB1 receptor. In a further study the ability of the endogenous compound palmitoylethanolamide and a range of cannabinoids which can act on the CB2 in addition to the CB1 receptor, to downregulate mast cell degranulation was investigated. It was observed that PEA, CP55940 and WIN55212-2 but not the highly CB2 receptor-selective L759656 could exert this effect. It was not possible to investigate the effects of the CB2 receptor antagonist/inverse agonist SR144528 at this time.