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Fig. 2. `Atypical' endothelial anandamide receptors are identified as GPR55 that mediates anandamide-initiated Ca²⁺ signaling, while activation of CB₁ receptors prevent anandamide-induced Ca²⁺ signaling. (A) Representative RT-PCR on CB₁R in a human endothelial cell line (lane 2) in comparison with human brain as positive control (lane 3). (B) In Ca²⁺-free solution, the effect of 10 µM anandamide on cytosolic free-Ca²⁺ concentration was analyzed in the absence (n=19) or presence of the CB₁R antagonist rimonabant (1 µM) (n=28). (C) In the presence of the CB₁R antagonist AM251 (10 µM), 10 µM anandamide triggered Ca²⁺ signaling even in the presence of extracellular Ca²⁺ (n=59). (D) In the presence of 2 mM extracellular Ca²⁺, the effect of the e-aR agonist O1602 (10 µM) on cytosolic free-Ca²⁺ concentration was tested (n=17). (E) In the absence of extracellular Ca²⁺, the effect of O1918 (10 µM), an inhibitor of the e-aR, on anandamide (10 µM, AEA) -induced Ca²⁺ signaling was assessed (n=8). (F) The effect of the CB₁R agonist HU-210 (10 µM) on O1602 (e-aR agonist, 10 µM) -induced Ca²⁺ signaling was tested in the presence of 2 mM extracellular Ca²⁺ (n=10). (G) Representative RT-PCR on GPR55 in the human endothelial cell line using various annealing temperatures. (H) Pretreatment with siRNA against GPR55 (n=49; control, n=48) on anandamide (10 µM) -induced cytosolic Ca²⁺ signaling was monitored in nominally Ca²⁺-free solution. (I) Pretreatment with siRNA against GPR55 (n=62; control, n=18) on O1602 (10 µM) -induced cytosolic Ca²⁺ signaling in Ca²⁺-containing solution. (J) Consequences of GPR55 overexpression on anandamide (AEA, 10 µM) -induced Ca²⁺ signaling in the nominal absence of extracellular Ca²⁺ (n=43; control, n=42). (K) Consequences of GPR55 overexpression on O1602 (10 µM) -induced Ca²⁺ signaling in the nominal absence of extracellular Ca²⁺ (n=21; control, n=25). Cytosolic free-Ca²⁺ concentrations were recorded using fura-2. ^*P<0.005 versus the absence of the respective receptor blocker/activator.

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