title: SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium.
abstract: The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91(phox), and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Wei-Qing
authors.initials: WQ
authors.lastname: Han
authors.forename: Min
authors.initials: M
authors.lastname: Xia
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.forename: Ning-Jun
authors.initials: NJ
authors.lastname: Li
authors.forename: Pin-Lan
authors.initials: PL
authors.lastname: Li
mesh_terms: D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D053222:Fas Ligand Protein / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008561:Membrane Fusion / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000378:metabolism; D034622:RNA Interference; D013481:Superoxides / Q000378:metabolism; D013744:Tetanus Toxin / Q000494:pharmacology; D013997:Time Factors; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology; D050988:Vesicle-Associated Membrane Protein 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D053222:Fas Ligand Protein; D052118:Lysosomal-Associated Membrane Protein 1; D050765:Qa-SNARE Proteins; D013744:Tetanus Toxin; D014665:Vasodilator Agents; D050988:Vesicle-Associated Membrane Protein 2; D013481:Superoxides; D019255:NADPH Oxidases; D001920:Bradykinin