RESUMEN
The major endocannabinoids (ECs) arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) and related N-ethanolamines act as full and partial agonists at CB(1), CB(2), GPR55, PPAR and TRPV1 receptors to various degrees. These receptors are also expressed in immune cells like monocytes/macrophages where they regulate different cellular processes. In this study, potentially bioactive lipids in fetal bovine sera (FBS) were quantified by GC/MS. We found that several commercial FBS contain ECs and bioactive amounts of 2-AG (250-700 nM). We show that residual 2-AG from FBS can activate primary macrophages and increase migration and RANKL-stimulated osteoclastogenesis. Furthermore, 2-AG high-content sera specifically upregulated LPS-stimulated IL-6 expression in U937 cells. Polymyxin B beads may be used to selectively and efficiently remove 2-AG from sera, but not arachidonic acid and N-ethanolamines. In conclusion, 2-AG in cell culture media may significantly influence cellular experiments. CD14+ mononuclear cells which strongly express surface CB receptors may be particularly sensitive towards residual 2-AG from FBS. Therefore, the EC content in culture media should be controlled in biological experiments involving monocytes/macrophages.
Asunto(s)
Moduladores de Receptores de Cannabinoides/metabolismo , Moduladores de Receptores de Cannabinoides/farmacología , Endocannabinoides , Sangre Fetal/química , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Animales , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Bovinos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Cromatografía de Gases y Espectrometría de Masas , Glicéridos/metabolismo , Glicéridos/farmacología , Humanos , Interleucina-6/metabolismo , Lípidos/análisis , Lípidos/aislamiento & purificación , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Ligando RANK/farmacología , Receptores de Cannabinoides/metabolismo , Células U937RESUMEN
The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.