title: Requirement of translocated lysosomal V1 H(+)-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells.
abstract: Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H(+)-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase-mediated production of O(2)(-.) in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H(+)-ATPase apparently attenuated FasL-caused pH decrease. V1 H(+)-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H(+)-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H(+)-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.forename: Min
authors.initials: M
authors.lastname: Xia
authors.forename: Xiao-Xue
authors.initials: XX
authors.lastname: Li
authors.forename: Wei-Qing
authors.initials: WQ
authors.lastname: Han
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Boini
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Joseph K
authors.initials: JK
authors.lastname: Ritter
authors.forename: Pin-Lan
authors.initials: PL
authors.lastname: Li
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D060833:Cellular Microenvironment / Q000502:physiology*; D002518:Ceramides / Q000096:biosynthesis; D003331:Coronary Vessels / Q000166:cytology*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D053222:Fas Ligand Protein / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002518:Ceramides; D053222:Fas Ligand Protein; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D019255:NADPH Oxidases; D013108:Sphingomyelin Phosphodiesterase; D025262:Vacuolar Proton-Translocating ATPases; D010100:Oxygen