Diadenosine tetraphosphate induces tight junction disassembly thus increasing corneal epithelial permeability
Background and Purpose Here, we have studied the effects of the dinucleotide P1, P4‐Di (adenosine‐5′) tetraphosphate (Ap4A) on corneal barrier function conferred by the tight junction (TJ) proteins and its possible involvement in ocular drug delivery and therapeutic efficiency. Experimental Approach...
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Published in | British journal of pharmacology Vol. 172; no. 4; pp. 1045 - 1058 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
England
Blackwell Publishing Ltd
01.02.2015
BlackWell Publishing Ltd |
Subjects | |
Online Access | Get full text |
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Summary: | Background and Purpose
Here, we have studied the effects of the dinucleotide P1, P4‐Di (adenosine‐5′) tetraphosphate (Ap4A) on corneal barrier function conferred by the tight junction (TJ) proteins and its possible involvement in ocular drug delivery and therapeutic efficiency.
Experimental Approach
Experiments in vitro were performed using human corneal epithelial cells (HCLEs) treated with Ap4A (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 P2Y2 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.
Key Results
Two hours after Ap4A 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 P2Y2 siRNAs. In vivo, topical application of Ap4A disrupted ZO‐1 membrane distribution. 5‐MCA‐NAT levels in the aqueous humour were higher when Ap4A was previously instilled and its hypotensive effect was also increased. This action was reversed by P2Y receptor antagonists and P2Y2 siRNA.
Conclusions and Implications
Ap4A increased corneal epithelial barrier permeability. Its application could improve ocular drug delivery and consequently therapeutic efficiency. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0007-1188 1476-5381 |
DOI: | 10.1111/bph.12972 |