title: Extracellular release of ATP mediated by cyclic mechanical stress leads to mobilization of AA in trabecular meshwork cells.
abstract: OBJECTIVE
  To investigate the mechanisms that mediate the release of ATP induced by cyclic mechanical stress (CMS) and the role of extracellular ATP in the mobilization of arachidonic acid (AA) and prostaglandin secretion.
  
  
  METHODS
  Porcine trabecular meshwork (pTM) cells were subjected to CMS. Extracellular ATP was detected with a luciferin-luciferase assay in the presence or absence of transport inhibitors and a lipid raft disrupter. ATP vesicles were visualized with quinacrine. The release of AA (AA 1-14C) was measured with and without ATP, ATP inhibitors, and phospholipase-A and -C inhibitors. Prostaglandin E2 (PGE2) and viability were measured with ELISA and a lactate dehydrogenase assay, respectively.
  
  
  RESULTS
  CMS induced ATP release that was inhibited by the vesicle inhibitors N-ethylmaleimide (NEM) and monensin. Lipid raft disruption significantly increased the extracellular ATP induced by CMS. CMS induced AA release (1-4-fold increase) and its metabolic product PGE2 (3.9-fold increase). The AA mobilization induced by CMS could be mimicked by the addition of extracellular ATP and was partially inhibited by a P2 antagonist, by an ATP inhibitor, and by inhibitors of phospholipase-A2 and -C. Addition of PGE2 (10 microM) to the media exerted cytoprotective effects against long-term CMS.
  
  
  CONCLUSIONS
  Extracellular release of ATP induced by CMS in TM cells is mediated by exocytosis of ATP-enriched vesicles into lipid rafts. The resulting activation of purinergic receptors leads to mobilization of AA from the plasma membrane. The subsequent release of PGE could exert protective effects by preventing TM cell loss that may result from chronic exposure to CMS.
authors.affiliation: Department of Ophthalmology, Duke University, Durham, North Carolina, USA.
authors.forename: Coralia
authors.initials: C
authors.lastname: Luna
authors.forename: Guorong
authors.initials: G
authors.lastname: Li
authors.forename: Jianming
authors.initials: J
authors.lastname: Qiu
authors.forename: Pratap
authors.initials: P
authors.lastname: Challa
authors.forename: David L
authors.initials: DL
authors.lastname: Epstein
authors.forename: Pedro
authors.initials: P
authors.lastname: Gonzalez
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D064801:Phospholipase A2 Inhibitors; D054467:Phospholipases A2 / Q000378:metabolism; D013314:Stress, Mechanical; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D064801:Phospholipase A2 Inhibitors; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; D008985:Monensin; D054467:Phospholipases A2; D010738:Type C Phospholipases; D015232:Dinoprostone; D005033:Ethylmaleimide