title: Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis.
abstract: Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.
authors.affiliation: MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.
authors.forename: Emma A
authors.initials: EA
authors.lastname: Cookson
authors.forename: Ianina L
authors.initials: IL
authors.lastname: Conte
authors.forename: John
authors.initials: J
authors.lastname: Dempster
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Hannah
authors.forename: Tom
authors.initials: T
authors.lastname: Carter
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology*; D055664:Electrochemical Techniques; D005071:Evoked Potentials / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects*; D015870:Gene Expression; D006632:Histamine / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism; D020302:Weibel-Palade Bodies / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: C493449:SLC18A1 protein, human; D050493:Vesicular Monoamine Transport Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D012701:Serotonin; D006632:Histamine; D002784:Cholesterol
keywords: Amperometry; Endothelial cell; Exocytosis; Serotonin; VMAT1; Weibel–Palade bodies