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Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells

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Abstract

Tear hyperosmolarity is a key event in dry eye. In this work, 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. High-performance liquid chromatography analysis revealed that ATP levels significantly increased in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge as well as in dry eye patients 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, vesicular nucleotide transporter was identified in both cell lines and their protein expression was upregulated in hypertonic media. P2X7 receptor truncated form together with the full-length form was identified in both cell lines, and experiments using specific antagonist and agonist for P2X7 indicated that this receptor did not mediate cell death induced by hyperosmolar stress. In conclusion, hyperosmotic stress induces ATP release. Extracellular ATP can activate P2X7 receptor leading to cytotoxicity in many cells/tissues; however, this does not occur in human corneal and conjunctival epithelial cells. In these cells, the presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior on the ocular surface.

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Acknowledgments

We thank Dr. Ilene Gipson, Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA, USA, for providing the HCLE cells. We are grateful to Leyre Nieto Roldan for her technical assistance. This work was supported by the Spanish Ministry of Economy (SAF2013-44416-R, SAF2016-77084R) and the Institute Carlos III (RETICS RD12/0034/0003, RD16/0008/0017).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology IV, Faculty of Optics and Optometry, Universidad Complutense de Madrid, C/Arcos de Jalón 118, 28037, Madrid, Spain

    Ana Guzman-Aranguez, María J. Pérez de Lara & Jesús Pintor

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  1. Ana Guzman-Aranguez
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  2. María J. Pérez de Lara
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  3. Jesús Pintor
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Correspondence to Ana Guzman-Aranguez.

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Ana Guzman-Aranguez declares that she has no conflict of interest.

María J. Pérez de Lara declares that she has no conflict of interest.

Jesús Pintor declares that he has no conflict of interest.

Ethical approval

This study was performed according to the Declaration of Helsinki. An informed consent was obtained from all participants and they were free to give up the session at any time.

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Guzman-Aranguez, A., Pérez de Lara, M.J. & Pintor, J. Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells. Purinergic Signalling 13, 249–258 (2017). https://doi.org/10.1007/s11302-017-9556-5

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