June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells.
Author Affiliations & Notes
  • Ana Guzman-Aranguez
    Department of Biochemistry and Molecular Biology, Faculty of Optics and Optometry, Complutense University, Madrid, Madrid, Spain
  • Maria J Perez de Lara
    Department of Biochemistry and Molecular Biology, Faculty of Optics and Optometry, Complutense University, Madrid, Madrid, Spain
  • Jesus J Pintor
    Department of Biochemistry and Molecular Biology, Faculty of Optics and Optometry, Complutense University, Madrid, Madrid, Spain
  • Footnotes
    Commercial Relationships   Ana Guzman-Aranguez, None; Maria J Perez de Lara, None; Jesus Pintor, None
  • Footnotes
    Support   Spanish Ministry of Economy (SAF2013-44416-R, SAF2016-77084R) and the Institute Carlos III (RETICS RD12/0034/0003, RD16/0008/0017).
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1385. doi:
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      Ana Guzman-Aranguez, Maria J Perez de Lara, Jesus J Pintor; Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1385.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Tear hyperosmolarity is a key event in dry eye. In this study, we analyzed whether hyperosmolar challenge induces ATP release on the ocular surface. Moreover, as extracellular ATP can activate P2X7 receptor, the changes in P2X7 protein levels and its involvement in pathological process triggered by hypertonic treatment were also examined.

Methods : Human corneal and conjunctival epithelial cells were exposed to hyperosmotic challenge. The presence of ATP in the conditioned media of cells exposed to hypertonic treatment as well as in tears of dry eye patients was examined by high performance liquid chromatography analysis. Cell viability was evaluated by MTT assay and protein expression of vesicular nucleotide transporter (VNUT) and P2X7 receptor was examined by western blot. Potential involvement of P2X7 receptor in cell death detected after hyperosmolar exposure was analyzed using specific P2X7 agonists and antagonists.

Results : ATP concentration in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge was 6.82 and 5 times, respectively, higher than in control cells. Likewise, a 8.65-fold increase in ATP levels for dry eye patients with low tear secretion was detected as compared to control subjects. A significant reduction in cell viability was detected after hyperosmolar treatment, indicating that the rise in ATP release was mainly due to cell lysis/death. Additionally, VNUT was identified in both cell lines and cells exposed to hypertonic media showed a 3-fold increase in VNUT expression. P2X7 receptor truncated form together with the full-length form were identified in both cell lines and experiments using specific antagonist and agonist for P2X7 revealed that this receptor did not mediate cell death induced by hyperosmolar stress.

Conclusions : Hyperosmotic stress induces ATP release on the ocular surface. The presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior in human corneal and conjunctival epithelial cells.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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