title: Diadenosine tetraphosphate induces tight junction disassembly thus increasing corneal epithelial permeability.
abstract: OBJECTIVE
  Here, we have studied the effects of the dinucleotide P(1), P(4)-Di (adenosine-5') tetraphosphate (Ap4 A) on corneal barrier function conferred by the tight junction (TJ) proteins and its possible involvement in ocular drug delivery and therapeutic efficiency.
  
  
  METHODS
  Experiments in vitro were performed using human corneal epithelial cells (HCLEs) treated with Ap4 A (100 μM) for 5 min. Western blot analysis and transepithelial electrical resistance (TEER) were performed to study the TJ protein levels and barrier function respectively. Intracellular pathways involved were determined using an ERK inhibitor and P2Y(2) receptor siRNAs. In in vivo assays with New Zealand rabbits, TJ integrity was examined by zonula occludens-1 (ZO-1) staining. The hypotensive compound 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) was used to assess improved delivery, measuring its levels by HPLC and measuring intraocular pressure using 5-MCA-NAT, P2Y receptor antagonists and P2Y2 siRNAs.
  
  
  RESULTS
  Two hours after Ap4 A pretreatment, TJ protein levels in HCLE cells were reduced around 40% compared with control. TEER values were significantly reduced at 2 and 4 h (68 and 52% respectively). TJ reduction and ERK activation were blocked by the ERK inhibitor U012 and P2Y(2) siRNAs. In vivo, topical application of Ap4 A disrupted ZO-1 membrane distribution. 5-MCA-NAT levels in the aqueous humour were higher when Ap4 A was previously instilled and its hypotensive effect was also increased. This action was reversed by P2Y receptor antagonists and P2Y(2) siRNA.
  
  
  CONCLUSIONS
  Ap4 A increased corneal epithelial barrier permeability. Its application could improve ocular drug delivery and consequently therapeutic efficiency.
authors.affiliation: Department of Biochemistry and Molecular Biology IV, Faculty of Optics and Optometry, Universidad Complutense de Madrid, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Loma
authors.forename: A
authors.initials: A
authors.lastname: Guzman-Aranguez
authors.forename: M J
authors.initials: MJ
authors.lastname: Pérez de Lara
authors.forename: J
authors.initials: J
authors.lastname: Pintor
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D057167:Claudins / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000494:pharmacology*; D004847:Epithelial Cells; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D009570:Nitriles / Q000494:pharmacology; D062793:Occludin / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D011817:Rabbits; D058470:Receptors, Purinergic P2Y / Q000235:genetics; D019108:Tight Junctions / Q000187:drug effects*; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D002070:Butadienes; C473255:CLDN7 protein, human; D057167:Claudins; D015226:Dinucleoside Phosphates; D009570:Nitriles; C568985:OCLN protein, human; D062793:Occludin; D034741:RNA, Small Interfering; D058470:Receptors, Purinergic P2Y; C569042:TJP1 protein, human; C113580:U 0126; D062826:Zonula Occludens-1 Protein; C020733:diadenosine tetraphosphate; D048049:Extracellular Signal-Regulated MAP Kinases