Fig. 4. Anandamide initiates tyrosine phosphorylation. (A,B) The effect of anandamide (10 µM) -induced activation of tyrosine kinases was visualized in single endothelial cells in the presence (A) and absence (B) of extracellular Ca²⁺. As indicated, 10 µM PP1 was added to the media (n=4 under each condition). Tyrosine kinase activity was monitored by measuring the excitation ratio of F₅₃₅/F₄₈₀ nm (FRET) at 440 nm excitation. (C) The effect of 10 µM anandamide on cytosolic free-Ca²⁺ concentration was tested in nominally Ca²⁺-free solution in the absence (n=24) or presence of 10 µM PP1 (n=25). (D) In Ca²⁺-containing solution, Ca²⁺ intracellular Ca²⁺ signaling was initiated by 10 µM O1602 in the presence of 10 µM PP2, followed by an additional stimulation with 10 µM O1602 after washout of PP2 as indicated (n=14). For visualization of tyrosine kinase activity in single endothelial cells, cells were transiently transfected with Picchu 936X. Cytosolic free-Ca²⁺ concentration was recorded using fura-2. ^*P<0.001 versus the absence of PP1.