Involvement of non‐CB1 anandamide receptor and Slit‐Robo signaling in Anandamide‐mediated Cell migration and Angiogenesis

Abstract

Previous studies from our laboratory have shown that anandamide and its metabolically stable analog methanandamide activate endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) via the activation of Gi protein and phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We have further shown that methanandamide produced angiogenic responses and evoked cell migration in endothelial cells via increase in matrix metalloprotease2/9 (MMP-2 and MMP-9) activity. These responses were not mimicked by the cannabinoid receptor agonists WIN55212 or CP55940 and could not be blocked by CB1 receptor antagonist SR141716A suggesting the involvement of a non-CB1 “anandamide receptor”. In the present study we showed that knocking down CB1 receptor in EAhy926 cells or human umbilical vein endothelial cells (HUVEC) enhanced the MMP2 and MMP 9 activity as measured by zymography. Further, we found that preincubation of EAhy926 endothelial cells with anti-slit (a secreted protein known to function through the Roundabout (Robo) receptor and involved in endothelial and neuronal cell migration), or anti-Robo antibody inhibits methanandamide-induced cell migration and angiogenesis. This suggests that methanandamide-acting on a non-CB1 anandamide receptor activates the slit-Robo signaling pathway in the regulation of cell migration and angiogenesis in endothelial cells and CB1 receptor exerts a inhibitory tone in this pathway. (This study was supported by AHA 0060377Z to SM; NIDA U24 DA 12385)