JCS020958 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Arachidonic acid fails to initiate Ca²⁺ signaling in endothelial cells used in this study. In the absence of extracellular Ca²⁺ endothelial cells were stimulated by 10 µM arachidonic acid and 10 µM anandamide as indicated (n=14).

• Supplemental Figure S2A-E
• Supplemental Figure S2F-H -

  Fig. S2. Pharmacological characterization of the receptors involved in anandamide-/O-1602-triggered Ca²⁺ signaling in endothelial cells. (A) In the nominal absence of extracellular Ca²⁺ endothelial cells were stimulated with 10 µM anandamide in the absence (n=20) or presence of the CB₂R antagonist SR144528 (1 µM) (n=25). (B) The TRPV1 inhibitor SB366791 (10 µM) had no effect on anandamide-triggered Ca²⁺ signaling in Ca²⁺ free solution (control, n=23; SB366791, n=30). (C) Endothelial cells were stimulated with 10 µM HU210 in the presence and absence of extracellular Ca²⁺. To approve their viability, the effect of 100 µM histamine in Ca²⁺ containing solution was measured in the same cells (n=8). (D) The TRPV1 inhibitor SB366791 (10 µM) had no effect on O-1602-triggered Ca²⁺ signaling in the presence of extracellular Ca²⁺ (control, n=32; SB366791, n=20). (E) Real-time PCR for quantification of knock down efficiencies of siRNA against GPR55 (n=6). Data are expressed as percentage of GAPDH-based normalized expression in respect to mRNA content (non-functional siRNA). Experiments were performed in single cells that were co-transfected with the respective siRNA and a marker construct (e.g. mtDsRed) in order to assure effective transfection with siRNA. Thus, protein knock down in single cells used for functional studies most likely exceed that revealed in real-time PCR. *P<0.0001 vs anandamide. (F) Cellular localization of GPR55 in endothelial cells. EA.hy926 cells were transiently transfected with 3xHA-tagged GPR55 cDNA (I) and mtDsRed (II) and an antibody feeding experiment was conducted as described in Methods. GPR55 (I, III) is expressed both on the cell surface (arrows) and intracellulary. I, GPR55 (green), II, mtDsRed (red), III, overlay. (G) Effect of pretreatment with siRNA against GPR55 (n=22; control, n=24) on (10 µM) LPI-induced cytosolic Ca²⁺ signaling in Ca²⁺-containig solution. (H) Consequences of GPR55 overexpression on (10 µM) LPI-induced Ca²⁺ signaling in the nominal absence of extracellular Ca²⁺ (n=22; control, n=21; P>0.005).

• Supplemental Figure S3A-C
• Supplemental Figure S3DE -

  Fig. S3. Anandamide does not form a stable complex with Ca²⁺. (A) Chemical structure and numbering of anandamide. (B) Possible anadamide-Ca²⁺ complex that was tested using NMR. No changes in the chemical shifts of anandamide by Ca²⁺ was reported (TMS as internal standard; data not shown). (C) Binding and uptake of anandamide (AEA) by human endothelial cells. Experiments were performed by incubating 1.5 x 10⁶ cells for 10 min with 2 x 10⁶ dpm ³H-anandamide (NEN) at 37°C (uptake) or 2°C (binding) for in buffer containing either 1 mM EGTA (EGTA) or 2 mM Ca²⁺ (Ca²⁺) (n=6 for each point). Fibronectin and the integrin-binding peptide RGD prevented anandamide-induced Ca²⁺ signaling in endothelial cells. In the presence of either 20 µg/ml fibronectin (D) or 20 µg/ml RGD peptide (E) the effect of 10 µM anandamide on cytosolic free Ca²⁺ was tested in nominally Ca²⁺-free solution (left graphs) or in the presence of 2 mM extracellular Ca²⁺ (right graphs).

• Supplemental Figure S4 -

  Fig. S4. (A) Time course of anandamide (10 µM) triggered tyrosine phosphorylation in the presence (upper blot) and absence (lower blot) of extracellular Ca²⁺. (B) The tyrosine kinase inhibitor PP1 efficiently prevents protein phosphorylation. (A) Representative western blots on (10 µM) anandamide-induced tyrosine phosphorylation in endothelial cells in the presence (upper blot) and absence (lower blot) of extracellular Ca²⁺. (A) Representative western blots on the inhibitory effect of 10 µM PP1 on (10 µM) anandamide-induced tyrosine phosophorylation in endothelial cells in the presence (upper blot) and absence (lower blot) of extracellular Ca²⁺. Blots show representative blots of three independent experiments.

• Supplemental Figure S5 -

  Fig. S5. The PI3K inhibitor wortmannin (0.1 µM) efficiently prevents protein/tyrosine phosphorylation upon 10 µM anandamide in the presence (upper blot) and absence of extracellular Ca²⁺ (lower blot). Representative western blot on the effect of 0.1 µM wortmannin on tyrosine phosphorylation in endothelial cells in the presence (upper blot) and absence of extracellular Ca²⁺ (lower blot). Blots show representative blots of three independent experiments.

• Supplemental Figure S6 -

  Fig. S6. (A,B) Expression of Bmx/Etk and Syk is reduced by the respective siRNA constructs (A); in endothelial cells, the long form of SYK is expressed (B). (A) Real-time PCR for quantification of knock down efficiencies of siRNAs against Bmx/Etk (n=6) and Syk (n=6) used in this study. Data are expressed as percentage of the GAPDH-based normalized expression in respect to mRNA content in the respective controls (non-functional siRNA).(B) Using RT-PCR, the expression of the long but not short variant of Syk could be found in human endothelial cells. (C) Representative images out of 6 independent experiments of the cellular CB₁R distribution in human endothelial cells in Ca²⁺-free solution prior and after stimulation with anandamide. Endothelial cells were transiently transfected with GFP-tagged CB₁R and cellular distribution was verified prior and 3 min after addition of 10 µM anandamide.