ORIGINAL PAPER Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells Stefan Mergler • Fabian Garreis • Monika Sahlmu ¨ ller • Ekaterini-Maria Lyras • Peter S. Reinach • Abhilash Dwarakanath • Friedrich Paulsen • Uwe Pleyer Accepted: 19 January 2012 / Published online: 12 February 2012 Ó Springer-Verlag 2012 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 epi- thelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome expe- rience 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 conjunc- tivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca 2? transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 locali- zation was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5–20 lM) -induced Ca 2? transients were blocked by capsazepine (CPZ) (10 lM), the TRPV4 activator 4a-PDD (10 lM) -induced Ca 2? increases were reduced by ruthenium-red (RuR) (20 lM). Different heating ( \ 40°C or [ 43°C) led to Ca 2? increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca 2? 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 encoun- tered with less selective drugs. Keywords Human conjunctiva Á TRPV channels Á Intracellular Ca 2? Á Dry eye Á Planar patch-clamp Introduction The conjunctiva is highly vascularized mucous membrane covering the sclera and lines the inside of the eyelids. It is made up of a stratified squamous epithelium with an underlying lamina propria. Depending on the conjunctival region, its epithelial layer thickness varies from 2 to 10 layers (Gipson 1983) and in humans comprises almost 85% of the exposed ocular surface area, which is 17-fold larger than that of the cornea. There are two types of epithelial cells in the conjunctiva, stratified squamous cells and goblet cells. The latter can occur as single cells or form intraepithelial mucous glands. The conjunctiva extends to the corneal limbus on the surface of the globe. Compared F. Garreis and S. Mergler contributed equally to the work. S. Mergler (&) Á M. Sahlmu ¨ller Á U. Pleyer Department of Ophthalmology, Campus Virchow-Clinic, Charite ´, Universita ¨tsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany e-mail: stefan.mergler@charite.de F. Garreis Á F. Paulsen Department of Anatomy II, University Erlangen-Nuremberg, Universita ¨tsstraße 19, Erlangen, Germany E.-M. Lyras International Graduate Program Medical Neurosciences, Charite ´, Universita ¨tsmedizin Berlin, Charite ´platz 1, 10117 Berlin, Germany P. S. Reinach Department of Biological Sciences, State University of New York, College of Optometry, 33 West 42nd Street, New York, NY 10036, USA A. Dwarakanath Graduate School of Systemic Neurosciences (Master’s Program), Grosshaderner Str. 2, 82152 Planegg-Martinsried, Munich, Germany 123 Histochem Cell Biol (2012) 137:743–761 DOI 10.1007/s00418-012-0924-5