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Fig. 7. Syk is the mediator of CB₁R-originated repression of anandamide-induced Ca²⁺ signaling. (A,B) The inhibition of Syk by either its inhibitor piceatannol (5 µM, n=24) or siRNA against Syk (n=7; control, n=4) allowed strong cytosolic Ca²⁺ elevation in response to 10 µM anandamide in the presence of 2 mM extracellular Ca²⁺. (C) The effect of the G_i protein inhibitor pertussis toxin (400 ng/ml for 3 hours, n=8; control, n=8) on anandamide (10 µM) -induced Ca²⁺ signaling in the presence of 2 mM extracellular Ca²⁺ was verified in human endothelial cells. (D) Immunoprecipitation using anti-β1-integrin antibody and subsequent staining with either anti-β1-integrin (upper blot) or anti-CB₁R (lower blot). Endothelial extracts were harvested from cells under basal conditions (-) and after stimulation with 10 µM anandamide (+) in the presence of 2 mM extracellular Ca²⁺ (Ca²⁺) or 2 mM extracellular Ca²⁺ plus 70 µM Mn²⁺ (Mn²⁺). Right panels shows precipitation with beads that were not preloaded with anti-β1-integrin were used. (E) Immunoprecipitation using anti-β1-integrin antibody and subsequent staining with either anti-β1-integrin (upper blot), anti-V-integrin (middle blot) or anti-CB₁R (lower blot). Endothelial extracts were harvested from cells under basal conditions (-) and after stimulation with 10 µM anandamide (+) in the and absence (EGTA) of extracellular Ca²⁺. Right panels shows precipitation with beads that were not preloaded with anti-β1-integrin were used. Standard immunoprecipitation was applied. In experiments with siRNA, cells were transiently transfected with a vector encoding approved siRNA against Syk 48 hours prior to the experiments. Cytosolic free-Ca²⁺ concentrations were recorded using fura-2. ^*P<0.0001 versus the absence of the inhibitor.

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