The suitability of the electrically stimulated guinea-pig MP-LM preparation, mouse isolated vas deferens and urinary bladder for the study of cannabinoid receptor ligands was investigated. Cannabinoid receptor agonists produced concentration-related inhibition of the contractile responses in all three tissue preparations, demonstrating high potency, chemical- and stereo-selectivity. The rank order of the inhibitory potency of the cannabinoid agonists in all three tissue preparations correlated with their binding affinity for specific cannabinoid CB1 binding sites in rat brain tissue. These results suggested a receptor-mediated mechanism of action for cannabinoid receptor agonists via prejunctional functional cannabinoid CB1 receptors in these three models, in the absence of an antagonist. The endogenous cannabinoid receptor ligand anandamide, also produced concentration-related inhibitory effects in all three tissue preparations. However, anandamide was found to be metabolically less stable in the guinea-pig MP-LM preparation. SR141716A, a potent, CB1 selective cannabinoid receptor antagonist was found to attenuate the inhibitory effects of cannabinoid receptor agonists investigated in all three tissue preparations. This provided further evidence for a receptor-mediated mechanism of action for cannabinoid receptor ligands in these three tissue preparations. However, further studies with SR141716A suggests that, it may be acting as an inverse agonist rather than a pure antagonist in these three preparations. Finally, this study was further extended to characterise some novel cannabinoid receptor ligands in the guinea-pig ML-LM preparation and mouse isolated vas deferens.

There is significant sequence homology and anatomical co-distribution between cannabinoid (CB1) and sphingosine-1-phosphate (S1P) receptors in the CNS, but potential functional relationships between these lysolipid receptors have not been examined. Therefore, to investigate possible relationships between these two systems at the level of G-protein activation, agonist-stimulated [35S]GTPγS binding and autoradiography were conducted. Autoradiographic studies were first performed to localize receptor-mediated G-protein activation in mouse brain. Coronal brain slices were processed for stimulation of [35S]GTPγS binding using the synthetic cannabinoid agonist WIN 55,212-2 (WIN) or SIP. High levels of WIN- and S1P-stimulated [35S]GTPγS binding were observed in the caudate putamen, hippocampus, substantia nigra, and cerebellum. To further characterize the relationship between S1P-and CB1-mediated G-protein activation, spinal cords from adult male CB1 receptor knockout mice, CNS-deleted S1Pl receptor knockout mice and wild type C57 mice were collected, and assessed using agonist-stimulated [35S]GTPγS binding. Results from this experiment revealed that the S1Pl receptor is predominant in mouse spinal cord. To further investigate potential CBl and SIP receptor interactions spinal cords were collected from adult male ICR mice. Additivity studies were preformed using agonist-stimulated [35S]GTPγs binding. Results showed significantly less than additive stimulation when spinal cord tissue was treated with both WIN and SIP. These results suggest an interaction between the CB1 and S1P receptors in the mouse spinal cord. The effect of cannabinoid antagonists, SR141716A (CB1) and SR144528 (CB2) on S1P-and WIN-stimulated [35S]GTPγS binding were also examined in mouse spinal cord homogenates. These results showed that there was no significant difference between S1P-stimulated [35S]GTPγS binding in the presence of SR141716A or SR144528 compared to vehicle control. This shows that S1P produced stimulation independent of the CBl or CB2receptor. In addition WIN-stimulated [35S]GTPγS binding was not affected by SR144528, but was inhibited by SR141716A, confirming that this action is due to the CB1 receptor. The combined results of this project demonstrate an interaction between CB1 and S1P receptors in certain CNS regions where they are co-distributed, such as the caudate putamen, hippocampus, substantia nigra, cerebellum and spinal cord. These results may be due to convergence on a common pool of G-proteins via dimerization or co-localization in lipid rafts, or a possible direct ligand-receptor interaction.