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Ana Guzman-Aranguez, Patricia Loma, Maria Jesus Perez de Lara, Jesus Pintor; Diadenosine tetraphosphate modifies corneal epithelial tight junction assembly and barrier permeability.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5510.
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The corneal epithelium is sealed with intercellular tight junctional complexes and it provides a defensive barrier preventing the entrance of potentially harmful substances but also limiting helpful ocular drug delivery. The purpose of this study was to determine the effects of the diadenosine tetraphosphate (Ap4A) on corneal barrier function provided by tight junction proteins.
Human corneal epithelial cells were treated with Ap4A (100 µM) for 5 minutes. After nucleotide removal, cells were incubated to different times and tight junction protein levels and function were evaluated by western blot and transepithelial electrical resistance (TEER) measurement, respectively. Intracellular pathways mediating Ap4A effect were determined using ERK inhibitors and P2Y2 receptor siRNAs. In in vivo assays with New Zealand rabbits, tight junction integrity after Ap4A application was examined by ZO-1 staining. The hypotensor compound 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) (10 µM) was topically applied to rabbit eyes alone or 2 hours after Ap4A pre-treatment. The presence of the hypotensor compound in the aqueous humour was assessed by high-performance liquid chromatography and its effect on intraocular pressure was measured using a Tonopen® tonometer.
Treatment with Ap4A induced a significant decrease in tight junction protein levels as compared to control cells. Concomitantly, TEER values were dramatically reduced at 2 and 4 hours (% reduction: 68 and 52%, respectively). The Ap4A-induced down regulation of tight junction protein levels was accompanied by phosphorylation of extracellular signal-regulated kinase and both of these effects were blocked by the ERK inhibitor U0126 and P2Y2 siRNAs, indicating that Ap4A reduces tight junction protein levels through P2Y2 stimulation and subsequent ERK activation. In in vivo assays topical application of Ap4A disrupts the membrane distribution of ZO-1 along cell-cell contact regions. The presence of 5-MCA-NAT in the aqueous humour was 2.75 higher when Ap4A was previously instilled and its hypotensive effect was also increased.
Ap4A increased corneal barrier permeability by modifying tight junction protein levels and function. Ap4A application could contribute to improving ocular drug delivery and consequently therapeutic efficiency.
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