Fig. 5. PI3K is involved in anandamide/GPR55-evoked Ca²⁺ signaling. (A) The effect of 10 µM anandamide on cytosolic free-Ca²⁺ concentration was tested in the nominal Ca²⁺-free solution in the absence (n=33) or presence (n=38) of 0.1 µM wortmannin. (B) In the presence of extracellular Ca²⁺, intracellular Ca²⁺ signaling was initiated by 10 µM O1602 in the presence of 0.1 µM wortmannin followed by an additional stimulation with 10 µM O1602 after washout of the PI3K inhibitor as indicated (n=14). (C) The effect of the PLC inhibitor U73122 (2 µM, n=26; control, n=30) on anandamide (10 µM) -trigged cytosolic Ca²⁺ signaling was tested in Ca²⁺-free buffer. (D) In Ca²⁺-containing buffer, intracellular Ca²⁺ signaling was initiated by 10 µM O1602 in the presence of 1 µM U73122 followed by an additional stimulation with 10 µM O1602 after washout of the PLC inhibitor as indicated (n=14). (E) Concentration-response relationship of 2APB on cytosolic Ca²⁺ signaling induced by either 100 µM histamine (n=4) or 10 µM anandamide (n=6) in nominally Ca²⁺-free solution. Cytosolic free-Ca²⁺ concentrations were recorded in single cells using fura-2. ^*P<0.001 versus the absence of the inhibitor.