title: Lung endothelial Ca2+ and permeability response to platelet-activating factor is mediated by acid sphingomyelinase and transient receptor potential classical 6.
abstract: BACKGROUND
  Platelet-activating factor (PAF) increases lung vascular permeability within minutes by activation of acid sphingomyelinase (ASM) and a subsequent nitric oxide (NO)-inhibitable and Ca(2+)-dependent loss in barrier function.
  
  
  OBJECTIVE
  To elucidate the molecular mechanisms underlying this response.
  
  
  METHODS
  In isolated perfused rat and mouse lungs, endothelial Ca(2+) concentration ([Ca(2+)](i)) was quantified by real-time fluorescence imaging, and caveolae of endothelial cells were isolated and probed for Ca(2+) entry channels. Regulation of transient receptor potential classical (TRPC) 6-mediated currents in lung endothelial cells was assessed by patch clamp technique.
  
  
  RESULTS
  PAF increased lung weight gain and endothelial [Ca(2+)](i). This response was abrogated by inhibitors of ASM or in ASM-deficient mice, and replicated by lung perfusion with exogenous ASM or C2-ceramide. PAF increased the caveolar abundance of TRPC6 channels, which was similarly blocked by ASM inhibition. PAF-induced increases in lung endothelial [Ca(2+)](i), vascular filtration coefficient, and edema formation were attenuated by the TRPC inhibitor SKF96365 and in TRPC6-deficient mice, whereas direct activation of TRPC6 replicated the [Ca(2+)](i) and edema response to PAF. The exogenous NO donor PapaNONOate or the cyclic guanosine 3',5'-monophosphate analog 8Br-cGMP blocked the endothelial [Ca(2+)](i) and permeability response to PAF, in that they directly blocked TRPC6 channels without interfering with their PAF-induced recruitment to caveolae.
  
  
  CONCLUSIONS
  The present findings outline a new signaling cascade in the induction of PAF-induced lung edema, in that stimulation of ASM causes recruitment of TRPC6 channels to caveolae, thus allowing for Ca(2+) influx and subsequent increases in endothelial permeability that are amplified in the absence of endothelial NO synthesis.
authors.affiliation: Institute for Physiology, Charité - Universitätsmedizin Berlin, Germany.
authors.forename: Rudi
authors.initials: R
authors.lastname: Samapati
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.forename: Christof
authors.initials: C
authors.lastname: Stoerger
authors.forename: Christoph
authors.initials: C
authors.lastname: Arenz
authors.forename: Alexander
authors.initials: A
authors.lastname: Dietrich
authors.forename: Thomas
authors.initials: T
authors.lastname: Gudermann
authors.forename: Dieter
authors.initials: D
authors.lastname: Adam
authors.forename: Songwei
authors.initials: S
authors.lastname: Wu
authors.forename: Marc
authors.initials: M
authors.lastname: Freichel
authors.forename: Veit
authors.initials: V
authors.lastname: Flockerzi
authors.forename: Stefan
authors.initials: S
authors.lastname: Uhlig
authors.forename: Wolfgang M
authors.initials: WM
authors.lastname: Kuebler
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002199:Capillary Permeability; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism*; D066298:In Vitro Techniques; D008168:Lung / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism*; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000378:metabolism*
chemical_list: D010972:Platelet Activating Factor; D050052:TRPC Cation Channels; D009569:Nitric Oxide; D013108:Sphingomyelin Phosphodiesterase; D002118:Calcium