Functional expression of thermo-sensitive transient receptor potential channels in cultivated human corneal endothelial cells (HCEC-12)

Abstract

Purpose: The human corneal endothelium (HCE) is indispensable for maintaining corneal transparency and clear vision. There is HCE cell loss during life and after corneal injury or diseases. A limited HCE cell density may cause opacity which can ultimately only be treated by keratoplasty. Even after penetrating keratoplasty, HCE cell loss of up to 50 % occurs. During graft storage, about 30 % of the few available corneal grafts have to be discarded due to HCE cell loss. In addition, the durability of corneal grafts depends on the storage temperature. The molecular mechanisms leading to HCE cell loss are still unknown. In this context, the functional expression of thermo-sensitive transient receptor potential channels (thermo-TRPs) in HCE cells (HCEC) was investigated.

Methods: Since access to corneal grafts is limited, mainly immortalized HCEC (HCEC-12) were used. Some primary cultivated HCEC were used additionally. The functional expression of thermo-TRPs was investigated using fluorescence calcium imaging (fura-2/AM) and planar patch-clamp recordings under various physical and pharmacological conditions.

Results: Cooling from room temperature (≈ 22 °C) to 14 - 18 °C caused an increase of intracellular calcium ([Ca²⁺]_i). In contrast, the cold receptor antagonist BCTC (10 µM) suppressed this cold-induced Ca²⁺ increase. The super-cooling agent icilin (50 µM) caused an increase of [Ca²⁺]_i and also slightly increased whole-cell outwardly rectifying currents (60 µM icilin). This icilin-induced Ca²⁺ increase could be suppressed by BCTC (10 µM). Primary cultivated HCEC responded with a similar icilin-induced increase of [Ca²⁺]_i, indicating that normal HCEC also express cold receptors. In addition, temperature rises over 43 °C as well as the application of capsaicin (20 µM) increased [Ca²⁺]_i by activating heat receptors such as the capsaicin receptor TRPV1.

Conclusions: These results demonstrate the functional expression of cold receptors (e.g. menthol receptor TRPM8) as well as heat receptors (e.g. TRPV1) in HCEC-12 cells. It is suggested that these thermo-TRPs are important for the function of HCE and that they might be linked to HCE cell loss. Consequently, thermo-TRPs might present targets for pharmacological modulation to prevent HCE cell loss, which is in the interest of eye banks and patients worldwide.

Keywords: 481 cornea: endothelium • 741 transplantation • 483 cornea: storage