CB1/CB2R-independent cannabinoid signaling in endothelium

Cannabinoids and their synthetic analogs affect a broad range of physiological functions. Vascular effects of anandamide and its derivative inactive against CB1 and CB2 receptors, N- arachidonoyl glycine (NAGly), are likely to involve yet unidentified G protein-coupled receptor(s) (GPCR) located on endothelial cells (so called endothelial atypical CB receptor, eCBR), which is not yet identified and cloned. Indication for the presence of a novel eCBR comes primarily from the reported sensitivity of the vasorelaxant responses to pertussis toxin (PTX). However, the PTX-insensitive vasodilation to the CB has also been described, questioning the involvement of eCBR and/or specificity of eCBR ligands. Although anandamide and NAGly bind GPR18, it is still unclear whether the eCBR-attributable signaling requires GPCR. Collectively, the molecular identity of eCBR, signaling mechanisms and functional implications of eCBR stimulation remain obscure. The main goal of the proposal is to unveil the mechanisms of eCBR-attributable action of CB on endothelial cells and downstream signaling, and its role in vascular function. Because endothelial cells alter their properties and the expression profile under culture conditions, in order to explore the mechanisms of eCBR-attributed electrical signalling with preserved functional eCBR, the associated G-proteins and signal transduction pathways, we will investigate the effects of anandamide and NAGly on the membrane potential and transmembrane currents at whole- cell and single channel levels in in situ endothelial cells from isolated arteries. We aim to identify and characterize the GPCR-dependent and -independent targets for CB in vascular endothelium, the role of GPR18 in the electrical signaling, and, by using G-protein-specific antibodies against the subunits of Gi/Go, Gq/G11 and Gs proteins, we aim to identify the G- proteins involved in the responses and their coupling to specific ion transport mechanisms in endothelial signalling to the CB. We will address the role of smooth muscle cells and myo- endothelial gap junctions in the endothelial electrical responses to CBs, the impact of CBs on production of reactive oxygen species (ROS) and their role in eCBR-attributed endothelial electrical signaling. Having identified the mechanisms of endothelial CB1/CB2R-independent CB signaling, their functional implications will be addressed, including endothelial cell viability, migration and angiogenesis. Identification of currently elusive targets for CB in the endothelium and their functional role is vital for elaboration of new pharmacological strategies with improved efficacy against a wide range of diseases associated with deregulation of endocannabinoid system. The project can be continued in the future in various pathophysiological models.

Cannabis-derived compounds lacking psychoactive properties are emerging potential therapeutic option in a number of disorders including cardiovascular ones. For two decades, the vasoactive properties of cannabinoids have been largely attributed to their binding to a third type of cannabinoid receptor distinct from the classical cannabinoid receptors types 1 and type 2. The third type of cannabinoid receptor was thought to be located on the inner surface of the vascular wall comprising very thin and flat cells called endothelium. To understand the molecular events responsible for sensing of cannabinoids by vascular wall and we examined and characterized the electrical signalling generated by opening of single ion channels located within the endothelium following its interaction with cannabinoid molecules. We also assessed the requirement of specific receptor for these events. In vascular endothelial cells, we have identified several molecular targets for cannabinoids that directly and bidirectionally influence endothelial cell signalling. and determined functional consequences of their activation. We found that interaction with vascular endothelial cells with the endocannabinoid anandamide, its derivative N-arachydonoyl glycerol and a synthetic analogue of phytocannabinoid cannabidiol abnormal cannabidiol stimulate the large conductance calcium-dependent potassium channels. The effect appeared to occur independently of the presence of specific receptor and essentially depends on the cholesterol level in cell membrane. Interaction of cannabinoid molecules with endothelium results in activation of Na+- and Ca2+ -permeable channels coupled to stimulation of Ca2+- dependent potassium channels. As a result, the endothelium hyperpolarizes and triggers vasodilation. Furthermore, we found that cannabinoinds capable to inhibit endothelial cell hyperpolarization and endothelium-dependent vascular relaxation to acetylcholine and histamine via inhibition of Na+-Ca2+ exchanger. We found that these effects do not require the presence of specific cannabinoid receptor. Our studies reveal novel mechanisms of vascular cannabinoid sensing that account for the regulation of vasoactivity. Selective targeting of the mechanisms involved may be therapeutically effective in mimicking the beneficial effect of CB1/CB2-inactive cannabinoids in cardiovascular abnormalities.