Is the Cannabinoid CB1 Receptor a 2-Arachidonoylglycerol Receptor? Structural Requirements for Triggering a Ca2+ Transient in NG108-15 Cells

Abstract

The effects of delta9-tetrahydrocannabinol and 2-arachidonoylglycerol on the intracellular free Ca2+ concentration ([Ca2+]i) in NG108-15 cells were examined in detail. We found that delta9-tetrahydrocannabinol induces a rapid, modest increase in [Ca2+]i. The response was detectable with 3 nM delta9-tetrahydrocannabinol. We also found that very low concentrations of 2-arachidonoylglycerol elicit a rapid, more prominent increase in [Ca2+]i. Such a response was observed not only in NG108-15 cells but also in N18TG2 cells. The response induced by 2-arachidonoylglycerol in either NG108-15 cells or N18TG2 cells was abolished by pretreatment of the cells with a cannabinoid CB1 receptor specific antagonist, SR141716A, suggesting that 2-arachidonoylglycerol interacts with the CB1 receptor to induce the response. The results of an experiment involving a phospholipase C inhibitor suggested that phospholipase C is involved in the rapid increase in [Ca2+]i induced by 2-arachidonoylglycerol. We also found that 1(3)-arachidonoylglycerol exhibits similar activity to that of 2-arachidonoylglycerol, although its activity at low concentrations was somewhat weak compared with that of 2-arachidonoylglycerol. We further confirmed that several structural analogues of 2-arachidonoylglycerol were less active compared with 2-arachidonoylglycerol. These results suggest that the structure of 2-arachidonoylglycerol is strictly recognized by the CB1 receptor, which raises the possibility that the CB1 receptor is originally a 2-arachidonoylglycerol receptor.

Evidence That the Cannabinoid CB1 Receptor Is a 2-Arachidonoylglycerol Receptor

“…In preceding studies, we investigated the mechanism and physiological meaning of 2-arachidonoylglycerol-induced transient increases in [Ca 2ϩ ] i in NG108 -15 cells (22,23). We established that the response induced by adding 2-arachidonoylglycerol to the cells is mediated through the cannabinoid CB1 receptor and G i or G o (22,23), yet the detailed mechanism is not yet fully understood.…”

“…We established that the response induced by adding 2-arachidonoylglycerol to the cells is mediated through the cannabinoid CB1 receptor and G i or G o (22,23), yet the detailed mechanism is not yet fully understood. One important issue to be verified is whether or not 2-arachidonoylglycerol itself, but not its metabolites, is actually involved in this rapid cellular response.…”

“…Mechoulam et al (21) also isolated 2-arachidonoylglycerol from canine gut as another candidate for an endogenous cannabinoid receptor ligand; they demonstrated that 2-arachidonoylglycerol possesses binding activity toward cannabinoid receptors expressed on COS-7 cells transfected with cannabinoid receptor genes and induces the inhibition of adenylate cyclase in mouse spleen cells and twitch response in mouse vas deferens. Moreover, in recent studies, we found that 2-arachidonoylglycerol induces a rapid, transient elevation of intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) through a cannabinoid CB1 receptor-dependent mechanism and proposed that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor (22,23). Thus, the physiologi-cal significance of 2-arachidonoylglycerol came to receive increased attention (19 -33).…”

2-Arachidonoylglycerol Enhances the Phagocytosis of Opsonized Zymosan by HL-60 Cells Differentiated into Macrophage-Like Cells

“…7,8) Previously, we investigated in detail the structure-activity relationship of cannabinoid receptor ligands and found that 2-AG is the most efficacious agonist among the various structural analogs and homologs thus far examined. [9][10][11] Importantly, 2-AG acted as a full agonist toward these cannabinoid receptors in various assay systems, [9][10][11][12][13][14] whereas anandamide often acted as a partial agonist. Based on these experimental results, we proposed that 2-AG, rather than anandamide, is the true natural ligand for the cannabinoid receptors.…”

“…[9][10][11] 2-AG elicits a variety of biological responses in various nervous tissues and cells. 15) For example, 2-AG induced a Ca 2ϩ transient in NG108-15 cells, 9,10) the inhibition of longterm potentiation 16) and the inhibition of synaptic transmission 17) in rat hippocampal slices, and the inhibition of voltage-gated Ca 2ϩ channels and the activation of inwardly rectifying K ϩ channels in rat sympathetic neurons 18) via CB1 receptor-dependent mechanisms. On the other hand, not much information is available concerning the effects of 2-AG on inflammatory cells and immune-competent cells which express the CB2 receptor.…”