Corneal mechanotransduction drives TRPV4-dependent release of the transmitter ATP

Abstract

Purpose : Corneal epithelium - the outermost corneal layer that protects the cornea from environmental stressors – can nonetheless be injured by various mechanical and chemical stimuli. This may diminish its function and lead to discomfort, pain and disease. It is not known how mechanical impact drives corneal physiology, hyperalgesia/nociception and dysfunction. We thus localized mechano-transducing cation permeable ion channels in corneal epithelial cells, characterized their functional properties and their role in release of nociceptive transmitters.

Methods : Corneas from C57BL/6, TRPV4-/- and TRPV4^eGFP mice were enzymatically detached from the stroma and used for in situ experiments. In vitro, epithelial cells were dissociated, plated onto silicon membranes and cultured for 4-7 days. Cells were stimulated with cyclic biaxial strain in the presence/absence of mechanosensitive ion channel inhibitors. Immunolabeling and qRT-PCR were performed to determine the changes in gene and protein expression, and localization. Whole cell recording and optical imaging assessed the properties of mechanosensitive currents and their role in Ca²⁺ homeostasis.

Results : Mechanical strain induced time-dependent changes in transcript levels of putative mechanosensitive channels from TRP and Piezo families. Both endothelial and epithelial cells strongly expressed TRPV4 channel. Accordingly, the selective agonist GSK101 increased [Ca²⁺]_i in corneal epithelial cells, whereas pressure and swelling -induced calcium signals were averted by the selective TRPV4 inhibitor, HC06. TRPV4-induced [Ca²⁺]_i increases were suppressed by the pannexin-1 blocker probenecid and purinergic P2 receptor blocker suramin. The epithelial cells responded to TRPV4 activation with massive release of ATP, that was sensitive to probenecid. Interestingly, swelling but not stretch-evoked Ca²⁺ responses were antagonized by TRPV4 ablation or pharmacological block.

Conclusions : Our data suggest that corneal mechanotransduction involves activation of multiple force-activated channels. A principal mechanosensor is TRPV4, which is activated by osmotic stress, contributes to [Ca²⁺]_i homeostasis as calcium-dependent release of ATP that requires purinergic auto-feedback and hemichannel (pannexin) activation. Overall, this study identifies TRPV4 as a potential mechanosensor and regulator of stimulus-dependent hyperalgesia that could contribute to corneal inflammation and pain.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.