TRP-Dependent Calcium Regulation in HCEC-12 Cells: Involvement of Ascorbic Acid and Cannabinoid Receptor Signaling

The human corneal endothelium (HCE) is critical for maintaining corneal transparency. Dysfunctions due to cell loss are linked to altered intracellular calcium ([Ca²⁺]_i) homeostasis. Transient receptor potential channels (TRPs) are key regulators of [Ca²⁺]_i, and both L-ascorbic acid (Asc) and cannabinoid receptor (CB) agonists have been implicated in modulating TRP activity. This study investigated the effects of 1 mM Asc and the CB agonist WIN 55,212-2 (WIN) (10 µM) on [Ca²⁺]_i regulation in human corneal endothelial cells (HCECs). HCEC-12 was used as the established HCE cell model. [Ca²⁺]_i dynamics were assessed by fura-2/AM fluorescence imaging, and membrane currents were analyzed using planar patch-clamp recordings. Adding 1 mM Asc increased [Ca²⁺]_i, which was partially suppressed by the TRPV1 blocker AMG-9810 (AMG) (20 µM) and the TRPV4 blocker GSK2193874 (GSK219) (10 µM). Furthermore, 1 mM Asc increased whole-cell currents. WIN also induced [Ca²⁺]_i transients that were partially attenuated by AMG, the TRPM8 blocker AMTB (20 µM), GSK219, and the CB1 inverse agonist AM251 (10 µM). In addition, combined treatment with Asc and WIN enhanced [Ca²⁺]_i elevations compared with either treatment alone. These findings provide the first evidence for a functional interaction between TRP channel activity and CB signaling in HCECs. The inhibitory effect of AM251 suggests a predominant contribution of CB1 receptors. Given the central role of Ca²⁺ homeostasis in corneal endothelial function and disease, these results may contribute to a better understanding of endothelial pathophysiology and support further investigation of TRPs and cannabinoid signaling as potential targets in corneal disorders.