Novel role of ‘entourage’ monoacylglycerols in function of human cerebral microvascular endothelial cells

Abstract

2-arachidonoyl-glycerol (2-AG) is one of the monoacylglycerols which is a known endocannabinoid produced in various organs including brain and gut. It is recognized as a neuromodulatory, cytoprotective and vasodilatory agent. The 2-AG (isolated from brain) was shown to be accompanied by other monoacyglycerols, primarily the 2-acyl-glycerol esthers, 2-linoleoyl-glycerol (2-Lino-G) and 2-palmitoyl-glycerol (2-Palm-G). These two monoacyglycerols, which were referred to as 'entourage' substances, were not shown to be active by themselves. However, they were demonstrated to enhance the function of 2-AG. Lately, we reported that 2-AG functioned as an agonist for both of the cannabinoid receptors CB1 and CB2, as well as the vanilloid receptor, TRPV1. All of these receptors were shown to be constitutively expressed on human cerebromicrovascular endothelial cells (HBEC). Here we characterize the effects of all three monoacylglycerols on intracellular Ca2+-levels on HBEC using a fluorescent probe Fluo-3/AM. It was observed that treatment with 2-AG stimulated Ca2+-levels in HBEC. Interestingly, treatment with 2-Lino-G and 2-Palm-G also stimulated Ca2+-levels in HBEC. The effects of all three of these endogenous fatty acid glycerol esters were almost completely abolished in the presence of Ca2+-free medium containing EDTA. In addition, 2-Lino-G (100 M) and 2-Palm-G (50 M) augmented (344.5 13% and 263.1 42%, respectively) the level of Ca2+ induced by 2-AG (10 M). Although inhibition of 2-AG-induced effects with selective receptor antagonists for CB1 (SR141716A) and TRPV1 (capsazepine) was previously shown, treatment with both antagonists also inhibited Ca2+ influx induced by 2-Palm-G (46 10% and 37 2%, respectively) and 2-Lino-G (75 4% and 44 2%, respectively). Significant inhibition by SR141716A and capsazepine of augmented Ca2+-influx was also observed in HBEC exposed to combined treatments (2-AG with either of these two monoacylglycerols). Treatment with inhibitors of protein kinase C (PKC; bisindolylmaleimide) and protein kinase A (PKA; H-89 Dihydrohloride) also blocked Ca2+-influx induced by 2-Lino-G and 2-Palm-G. These results indicate that 2-Lino-G and 2-Palm-G not only function as active entourage agents, but are also capable of directly affecting Ca2+-influx and modulating 2-AG-induced effects. These findings also implicate both PKC and PKA in the signal transduction mechanism(s) associated with this phenomenon. In conclusion, these substances may actively participate in the endothelium-dependent regulation of microcirculation in the brain.