Oxysterol-induced macrophage apoptosis may have a role in atherosclerosis. Macrophages lacking the type 2 cannabinoid receptor (CB2) are partially resistant to apoptosis induced by 7-ketocholesterol (7KC). AM-251 and SR144528 are selective antagonists of CB1 and CB2 receptors, respectively. We observed that both compounds reduce 7KC-induced apoptosis in Raw 264.7 macrophages. As oxysterol-induced macrophage apoptosis requires acyl-coenzymeA:cholesterol acyltransferase (ACAT) activity, we tested their affects on ACAT activity. AM-251 and SR144528 both reduced cholesteryl ester synthesis in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages, CB2+/+ and CB2-/- peritoneal macrophages, as well as in vitro, in mouse liver microsomes. Consistent with inhibition of ACAT, the development of foam cell characteristics in macrophages by treatment with acetylated LDL was reduced by both compounds. This work is the first evidence that AM-251 and SR144528 are inhibitors of ACAT and as a result, might have anti-atherosclerotic activities independent of their affect on cannabinoid signaling.

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.