Temperature‐ and osmosensing by the human corneal endothelium through activation of TRPV channels

Abstract

Abstract Purpose The human corneal endothelium is essential for the physiology and transparency of the cornea. This is sustained by a number of different regulatory mechanisms and response to various stimuli. Exposure to hypotonic challenge and temperature changes may have major impact. This study was undertaken to identify such possible stimuli pathways in human corneal endothelial cells (HCEC). Methods The functional expression of putative temperature‐ and osmosensing ion channels was investigated by measurements of intracellular free Ca2+ ([Ca2+]i) with fura‐2 and automated patch‐clamping (microchip technology). Highly selective agents were used to identify TRPV channel subtypes in an immortalized HCEC population (HCEC‐SV40) and two subcloned cell lines (H9C1, B4G12). Results The TRPV1 selective agonist capsaicin (20 µM) increased [Ca2+]i and non‐selective cation channel outward currents. Cells pre‐treated with the antagonist capsazepine (10 µM) did not show any Ca2+ responses. Similar results were obtained with the TRPV4 selective agonist 4α‐PDD (5 µM) and the TRP channel blocker ruthenium red (10 µM). In addition, exposure to hypotonic challenge (150 mOsm) led to an increase in [Ca2+]i whereas the isotonic baseline (control) was stable. Furthermore, temperature rises from room temperature to 40‐43 °C led to an increase in [Ca2+]i in HCEC‐SV40 and H9C1. Conclusion There is functional expression of TRPV channels in HCEC. Therefore, these cells are able to react to temperature rises by activating of TRPV channels. In particular, TRPV1 and TRPV4 may be functionally expressed which are known as heat receptor and osmosensor respectively. These findings may have direct clinical implication (eye banking procedures, keratoplasty).