Diadenosine tetraphosphate induces tight junction disassembly thus increasing corneal epithelial permeability



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Loma Lozano, Patricia and Guzmán Aránguez, Ana Isabel and Pérez de Lara, María Jesús and Pintor, Jesús (2015) Diadenosine tetraphosphate induces tight junction disassembly thus increasing corneal epithelial permeability. British Journal of Pharmacology, 172 (4). pp. 1045-1058. ISSN 0007-1188

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Official URL: http://dx.doi.org/10.1111/bph.12972


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.

Item Type:Article
Additional Information:

Received 26 May 2014 / Revised 26 September 2014 / Accepted 29 September 2014
5-MCA-NAT, 5-methoxycarbonylamino-N-acetyltryptamine; Ap4A, P1, P4-Di (adenosine-5′) tetraphosphate; HCLE, human corneal epithelial cells; IOP, intraocular pressure; NGS, normal goat serum; PPADS, pyridoxal phosphate-6-azo (benzene-2′,4′-disulfonic acid); RB2, reactive blue 2; TEER, transepithelial electrical resistance; TJ, tight junctions; U0126, 1,4- diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene ethanolate; ZO-1, zonula occludens-1

Uncontrolled Keywords:Adenine nucleotides ; Tight junctions (Cell biology) ; Epithelial cells ; Permeability (Biology) ; Drug delivery systems ; In vitro studies ; Cornea
Subjects:Medical sciences > Medicine > Ophtalmology
Medical sciences > Optics > Eyes anatomy
Medical sciences > Pharmacy > Pharmaceutical chemistry
ID Code:40884
Deposited On:20 Jan 2017 12:21
Last Modified:23 Jan 2017 08:46

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