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Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells

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Abstract

Transient receptor potential vanilloid (TRPV) channels respond to polymodal stresses to induce pain, inflammation and tissue fibrosis. In this study, we probed for their functional expression in human conjunctival epithelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome experience the same type of symptomology induced by TRPV channel activation in other ocular tissues. TRPV gene and protein expression were determined by RT-PCR and immunohistochemistry in HCjE cells and human conjunctivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca2+ transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 localization was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5–20 μM) -induced Ca2+ transients were blocked by capsazepine (CPZ) (10 μM), the TRPV4 activator 4α-PDD (10 μM) -induced Ca2+ increases were reduced by ruthenium-red (RuR) (20 μM). Different heating (<40°C or >43°C) led to Ca2+ increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca2+ transients and nonselective cation channel currents. In conclusion, conjunctiva express TRPV1, TRPV2, and TRPV4 channels which may provide novel drug targets for dry eye therapeutics. Their usage may have fewer side effects than those currently encountered with less selective drugs.

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Acknowledgments

The human, spontaneously immortalized epithelial cell line from normal human conjunctiva (HCjE; IOBA-NHC) was kindly provided by Yolanda Diebold, University Institute of Applied Ophthalmobiology [IOBA], University of Valladolid, Valladolid, Spain). The SV40-transformed human corneal epithelial (HCE) cells were kindly provided by Kaoru Araki-Sasaki (Tane Memorial Eye Hospital, Osaka, Japan). The authors thank Norbert Kociok for helpful discussions as well as Gabriele Fels for technical assistance. This study was supported by Charité research founds and in part by DFG Pl 150/14-1 and PA 738/9-2 as well as by Pharm-Allergan GmbH (Ettlingen, Germany). The planar patch-clamp setup was supported in part by Berliner Sonnenfeld-Stiftung. Peter Reinach was supported by EY04795 and Department of Defense (W81XWH-09-2-0162). Finally, we appreciate very much the technical assistance provided by fellow students Frauke Harlis, Lena Paschke, Arina Riabinska and Nefeli Slavi during their lab rotation studies.

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Authors and Affiliations

  1. Department of Ophthalmology, Campus Virchow-Clinic, Charité, Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany

    Stefan Mergler, Monika Sahlmüller & Uwe Pleyer

  2. Department of Anatomy II, University Erlangen-Nuremberg, Universitätsstraße 19, Erlangen, Germany

    Fabian Garreis & Friedrich Paulsen

  3. International Graduate Program Medical Neurosciences, Charité, Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany

    Ekaterini-Maria Lyras

  4. Department of Biological Sciences, State University of New York, College of Optometry, 33 West 42nd Street, New York, NY, 10036, USA

    Peter S. Reinach

  5. Graduate School of Systemic Neurosciences (Master’s Program), Grosshaderner Str. 2, 82152, Planegg-Martinsried, Munich, Germany

    Abhilash Dwarakanath

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  1. Stefan Mergler
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  2. Fabian Garreis
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  4. Ekaterini-Maria Lyras
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  6. Abhilash Dwarakanath
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  7. Friedrich Paulsen
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Correspondence to Stefan Mergler.

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F. Garreis and S. Mergler contributed equally to the work.

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Mergler, S., Garreis, F., Sahlmüller, M. et al. Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells. Histochem Cell Biol 137, 743–761 (2012). https://doi.org/10.1007/s00418-012-0924-5

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