RT Journal Article
T1 Diadenosine tetraphosphate induces tight junction disassembly thus increasing corneal epithelial permeability
A1 Loma Lozano, Patricia
A1 Guzmán Aránguez, Ana Isabel
A1 Pérez de Lara, María Jesús
A1 Pintor Just, Jesús Jerónimo
AB 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.
PB The British Pharmacological Society / Wiley-Blackwell
SN 0007-1188
YR 2015
FD 2015-02
LK https://hdl.handle.net/20.500.14352/23083
UL https://hdl.handle.net/20.500.14352/23083
LA eng
NO Loma Lozano, P., Guzmán Aránguez, A. I., Pérez de Lara, M. J., Pintor Just, J. J. «Diadenosine Tetraphosphate Induces Tight Junction Disassembly Thus Increasing Corneal Epithelial Permeability». British Journal of Pharmacology, vol. 172, n.o 4, febrero de 2015, pp. 1045-58. DOI.org (Crossref), https://doi.org/10.1111/bph.12972.
NO Abbreviations5-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
NO Ministerio de Ciencia, Innovación y Universidades (España)
NO Redes Temáticas de Investigación Cooperativa en Salud (España)
DS Docta Complutense
RD 8 abr 2025