TRPV channels mediate temperature-sensing in human corneal endothelial cells

Abstract

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1–3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1–3 functional activity was evident based on capsaicin-induced Ca 2+ transients and induction of these responses through rises in ambient temperature from 25 °C to over 40 °C. The currents underlying Ca 2+ transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10–20 μM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 °C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La 3+) (100 μM) and ruthenium-red (RuR) (10 μM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 μM) led to a reversible increase in intracellular Ca 2+ indicating putative TRPV1–3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions.