CB₁ cannabinoid receptor (CB₁R) belongfs to the G-protein-coupled receptor (GPCR) superfamily and couples to G_i/o heterotrimeric proteins. We have shown previously that CB₁R can be transactivated by stimulation of the calcium-mobilizing GPCR, AT₁ angiotensin receptor (AT₁R) coexpressed in the same cells. Since these two receptors can be expressed in adjacent cells, we asked if paracrine transactivation of CB₁R is possible. In Chinese hamster ovary (CHO) cells CB₁R activity can be monitored by bioluminescence resonance energy transfer (BRET) between G protein subunits (a-subunit tagged with renilla luciferase and ß-subunit with yellow fluorescent protein, YFP). Activation of CB₁ receptors leads to decrease of BRET signal, whereas treatment with inverse agonist causes an elevation. AT1R stimulation with angiotensin II (Ang II) caused CB₁R activation when the two receptors were expressed in separate set of CHO cells and mixed prior to measurement, indicating endocannabinoid release. Paracrine transactivation also occurred when M₁, M₃, M₅ muscarinic-, B₂ bradykinin-, a₁-adrenergic- or V₁ vasopressin receptors were expressed and stimulated. As activated GPCRs bind ß-arrestins, their activity can be also detected by translocation of ß-arrestins to the membrane detected by confocal microscopy. ß-arrestin2-GFP translocated to cell membrane after stimulation of CB₁Rs with agonist. ß-arrestin binding was increased, when DRY motif in second intracellular loop was mutated to AAY in the receptor. Cells transfected with CB₁R(DRY/AAY)-RFP and ß-arrestin-GFP were mixed with cells expressing AT₁R-YFP. When cells were stimulated with Ang II, ß-arrestin-GFP translocated to the membrane in CB₁R(DRY/AAY)-RFP expressing cells.

In order to investigate the link between GPCR-induced effects and cannabinoid system also in the vasculature, rat skeletal muscle arterioles (SMA) were isolated, pressurized and subjected to microangiometry. In SMA calcium-mobilizing hormones Ang II and noradrenaline (NE) induced vasoconstriction by ca. 30 % and 40 % for 10^-8 M Ang II and 10^-6 M NE, respectively. CB₁ receptor inhibition with a neutral antagonist significantly augmented Ang II-induced vasoconstriction (by ~60 % at 10^-8 M) and noradrenaline (NE)-induced vasoconstriction as well (by ~80 % at 10^-6 M); and also completely inhibited the CB₁-agonist WIN55-induced vasodilation (from 15±2 %).

These results show a link between calcium-mobilizing GPCR-induced signaling and the cannabinoid system. We found, that stimulation of AT₁Rs or other calcium mobilizing receptors leads to endocannabinoid release and paracrine transactivation of CB₁Rs in CHO cells. In skeletal muscle arterioles CB₁Rs are involved in the adjustment of Ang II- and NE-induced vascular tone.