New Insights Into Electrophysiology and Functional Transient Receptor Potential (Trp) Channel Expression in the Corneal Endothelium

Abstract

Purpose: : The corneal endothelium plays a crucial role in the physiology and tranparency of the human cornea. During life, the number of endothelial cells decreases. Moreover, endothelial cell loss is accelerated after various impairments such as surgical trauma (e.g. cataract extraction) and following corneal transplantation. Importantly, this cell loss is associated with apoptosis and changed ion channel activity. So far, little is known about the electrophysiology and ion channel expression (in particular TRP channels) in corneal endothelial cells. Here, we identified TRPV1 and TRPV4 expression and characterized its functional activity in human corneal endothelial cells (HCEC) for the first time.

Methods: : Ca²⁺ transients were measured by single cell fluorescence imaging. The whole-cell patch-clamp technique was used to monitor selective agonist-mediated stimulation of non-selective cation channel activity. Putative TRPV1 channels were activated with the selective agonists capsaicin (CAP) (0.5-20 µM). The phorbol ester 4α-PDD (5 µM) was used to selectively activate TRPV4.

Results: : CAP clearly induced a calcium increase, which was affected by the selective TRPV1 channel blocker capsazepine (CPZ). The fura-2 fluorescence ratio (f340/f380) was 1,1992 ± 0,0004 in the resting state (base line) (n = 6). After application of 20 µM CAP, it increased to 1.2579 ± 0.0159 (n = 6; p < 0.01). This increase was completely abolished by 10 µM CPZ or by a Ca²⁺-free bath substitution. On the other hand, 5 µM 4α-PDD also induced a calcium increase (1.24423 ± 0.012160; n = 5; p < 0.01), which was significantly reduced by 10 µM ruthenium red (RuR) (1.2094 ± 0.0040; n = 4; p < 0.05) or by a Ca²⁺-free bath substitution. In addition, 2 µM 4α-PDD-induced a transient inward and outward rectifying current-voltage relationship.

Conclusions: : There is functional expression of TRPV1 and TRPV4 thermo-sensitive channels in human corneal endothelial cells. This realization may have direct clinical implications. For example, in an eye bank setting, rinse solutions and incubator temperature ranges can be optimized for appropriate modulation of TRPV1 and TRPV4 activity. These protocol changes may help to better retain viability of stored human corneas prior to transplantation.

Keywords: cornea: endothelium • ion channels • calcium