title: Endogenous production of extracellular adenosine by trabecular meshwork cells: potential role in outflow regulation.
abstract: OBJECTIVE
  To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility.
  
  
  METHODS
  Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes.
  
  
  RESULTS
  PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility.
  
  
  CONCLUSIONS
  These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma.
authors.affiliation: Department of Ophthalmology, Duke University, Durham, North Carolina, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Guorong
authors.initials: G
authors.lastname: Li
authors.forename: Coralia
authors.initials: C
authors.lastname: Luna
authors.forename: Ivan
authors.initials: I
authors.lastname: Spasojevic
authors.forename: David L
authors.initials: DL
authors.lastname: Epstein
authors.forename: Pedro
authors.initials: P
authors.lastname: Gonzalez
mesh_terms: D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000241:Adenosine / Q000096:biosynthesis* / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000493:pharmacokinetics; D000255:Adenosine Triphosphate / Q000493:pharmacokinetics; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001081:Apyrase / Q000235:genetics / Q000378:metabolism; D001082:Aqueous Humor / Q000502:physiology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000493:pharmacokinetics; D005110:Extracellular Space / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005901:Glaucoma / Q000378:metabolism* / Q000503:physiopathology; D007429:Intraocular Pressure / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D013314:Stress, Mechanical; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D015720:5'-Nucleotidase; D001081:Apyrase; C067869:CD39 antigen; D000241:Adenosine