title: Role of acid sphingomyelinase in the regulation of mast cell function.
abstract: BACKGROUND
  Degranulation of mast cells is stimulated by store-operated Ca(2+) -entry (SOCE). In other cell types, Ca(2+) -entry is modified by ceramide. Exogenously added ceramide has been shown to trigger mast cell apoptosis. Effects of endogenously produced ceramide in mast cells remained, however, elusive. Ceramide may be produced from sphingomyelin by acid sphingomyelinase (Asm).
  
  
  OBJECTIVE
  This study explored the impact of Asm on mast cell functions.
  
  
  METHODS
  Mast cells were isolated from bone marrow (BMMCs) or peritoneal lavage of gene-targeted mice lacking Asm (asm(-/-)) and their wild-type littermates (asm(+/+)). BMMC maturation and apoptosis-associated annexin V binding were determined by flow cytometry. Asm activity was assessed enzymatically, cytosolic Ca(2+) activity ([Ca(2+)]i) utilizing Fura-2 fluorescence, current across the cell membrane by whole-cell patch clamp, degranulation from hexosaminidase-release and migration utilizing a transwell chamber. In vivo anaphylaxis was derived from decrease in body temperature.
  
  
  RESULTS
  Peritoneal mast cell number, BMMC phenotype, spontaneous BMMC apoptosis as well as BMMC CD117, CD34 and FcεRI expression were similar in both genotypes. In asm(+/+) BMMCs, stimulation with antigen resulted in a fast ~2.5-fold increase in Asm activity. Release of Ca(2+) from internal stores and hence several Ca(2+) -dependent functions were strongly impaired in asm(-/-) BMMCs. Thus, antigen-induced increase in [Ca(2+)]i in IgE-sensitized cells, antigen- but not ionomycin-induced currents through Ca(2+) -activated K(+) -channels (KCa 3.1), IgE/antigen-triggered β-hexosaminidase release, and antigen-induced migration were all lower in asm(-/-) BMMCs than in asm(+/+) BMMCs. Pharmacological inhibition of Asm by amitriptyline (500 nm, 3 h) in asm(+/+) BMMCs similarly decreased antigen-induced increase in [Ca(2+)]i , KCa 3.1 currents, ß-hexosaminidase release and migration. The decrease in body temperature upon the induction of systemic anaphylaxis was significantly less pronounced in asm(-/-) mice than in asm(+/+) mice, an observation pointing to in vivo significance of Asm.
  
  
  CONCLUSIONS
  Asm is a novel, powerful regulator of mast cell function and thus a potential target in the treatment of allergic reactions.
authors.affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: E
authors.initials: E
authors.lastname: Schmid
authors.forename: M K
authors.initials: MK
authors.lastname: Nurbaeva
authors.forename: K
authors.initials: K
authors.lastname: Szteyn
authors.forename: C
authors.initials: C
authors.lastname: Leibrock
authors.forename: J
authors.initials: J
authors.lastname: Yan
authors.forename: M
authors.initials: M
authors.lastname: Schaller
authors.forename: E
authors.initials: E
authors.lastname: Gulbins
authors.forename: E
authors.initials: E
authors.lastname: Shumilina
authors.forename: F
authors.initials: F
authors.lastname: Lang
mesh_terms: D000707:Anaphylaxis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000941:Antigens / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation / Q000187:drug effects; D016130:Immunophenotyping; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism; D008407:Mast Cells / Q000276:immunology* / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D018345:Mice, Knockout; D010641:Phenotype; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000941:Antigens; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496925:Kcnn4 protein, mouse; D013108:Sphingomyelin Phosphodiesterase; D002118:Calcium
keywords: Ca2+ channels; K+ channels; KCa3.1; acid sphingomyelinase; allergy; anaphylaxis; degranulation