Cytochalasin e

As previously described, hippocampal cultures were treated with either Li₂CO₃ (hereinafter lithium, 1–50 mM_, (#255823, Sigma-Aldrich, St. Louis, MO, USA), cytochalasin B (Cyt B, 20 μM, #14930-96-2, Sigma-Aldrich), 2-deoxy-D-glucose (2-DG, 7 mM, #154-17-6, Sigma-Aldrich) or cytochalasin E (Cyt E, 20 μM, #C2149, Sigma-Aldrich) for 15 min and washed with incubation buffer (15 mM HEPES, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, and 0.8 mM MgCl₂) [92 (link)]. Cultures were then incubated for 0–180 s with 1–1.2 μCi ¹⁴C-glucose (D-[1-¹⁴C] glucose, #NEC043X, Perkin-Elmer, Waltham, MA, USA) at a final specific activity of 1–3 disintegrations/min/pmol (~1 mCi/mmol). Glucose uptake was then arrested with detention buffer (add 0.2 mM HgCl₂ to incubation buffer (pH 7.4)) and cultures were lysed in 1 mL of lysis buffer (10 mM Tris-HCL and 0.2% SDS, pH 8.0). In total, 3 mL of scintillating solution (#LS-270, National Diagnostics, Atlanta, GA, USA) were added to the cell lysates and radioactivity was measured using a liquid scintillation counter (TriCarb 2900TR analyzer).

HUVECs were incubated with 1 μmol L⁻¹ cytochalasin E (CCE) and 25 μmol L⁻¹ blebbistatin (Sigma‐Aldrich, St Louis, MO, USA) for 5–15 min before determining VWF or pro‐peptide release in the presence or absence of 100 ng mL⁻¹ phorbol 12‐myristate 13‐acetate (PMA) (Sigma‐Aldrich), 100 μmol L⁻¹ histamine or 100 μmol L⁻¹ histamine/10 μmol L⁻¹ adrenalin/100 μmol L⁻¹ 3‐isobutyl‐1‐methyl xanthine (IBMX) and/or the relevant drug for 30 min. VWF secretion assay and ELISAs have been described previously 30, 31. For VWF pro‐peptide secretion an ELISA kit (Mast Group Ltd, Bootle, Merseyside, UK) was used according to the manufacturer's instructions.

d‐glucose, sodium bisulfite, sodium phosphate dibasic anhydrous, sodium chloride, potassium chloride, magnesium chloride, potassium phosphate monobasic, sodium dodecyl sulfate (SDS), HEPES, dimethylsulfoxide, dithiothreitiol, and TRIS‐base were obtained from JT Baker. Cytochalasin B, cytochalasin E, d‐sorbitol, 2‐deoxy‐d‐glucose (2DG), 3‐O‐methyl‐d‐glucose (OMG), and nordihydroguaiaretic acid (NDGA) were obtained from Sigma Chemical Co, St. Louis, MO, USA. All radioisotopes (2‐[1,2‐³H(N)]‐deoxy‐d‐glucose, 36.2 Ci mmol⁻¹; 3‐O‐[methyl‐³H]‐methyl‐d‐glucose, 86.7 Ci mmol⁻¹; [4‐³H(N)]‐Cytochalasin B, 20 Ci mmol⁻¹) were from American Radiolabeled Chemicals, St. Louis, MO, USA. Trypan Blue, HyClone RPMI 1640 cell culture media and FBS were from Thermo Fisher Inc., Waltham, MA, USA.