Mechanical Stretch Activates TRPV4 and Hemichannel Responses in the Nonpigmented Ciliary Epithelium

Abstract

Previously, we reported a mechanosensitive ion channel, TRPV4, along with functional connexin hemichannels on the basolateral surface of the ocular nonpigmented ciliary epithelium (NPE). In the lens, TRPV4-mediated hemichannel opening is part of a feedback loop that senses and respond to swelling. The present study was undertaken to test the hypothesis that TRPV4 and hemichannels in the NPE respond to a mechanical stimulus. Porcine NPE cells were cultured on flexible membranes to study effects of cyclic stretch and ATP release was determined by a luciferase assay. The uptake of propidium iodide (PI) was measured as an indicator of hemichannel opening. NPE cells subjected to cyclic stretch for 1–10 min (10%, 0.5 Hz) displayed a significant increase in ATP release into the bathing medium. In studies where PI was added to the bathing medium, the same stretch stimulus increased cell PI uptake. The ATP release and PI uptake responses to stretch both were prevented by a TRPV4 antagonist, HC067047 (10 µM), and a connexin mimetic peptide, Gap 27 (200µm). In the absence of a stretch stimulus, qualitatively similar ATP release and PI uptake responses were observed in cells exposed to the TRPV4 agonist GSK1016790A (10 nM), and Gap 27 prevented the responses. Cells subjected to an osmotic swelling stimulus (hypoosmotic medium: 200 mOsm) also displayed a significant increase in ATP release and PI uptake and the responses were abolished by TRPV4 inhibition. The findings point to TRPV4-dependent connexin hemichannel opening in response to mechanical stimulus. The TRPV4-hemichannel mechanism may act as a mechanosensor that facilitates the release of ATP and possibly other autocrine or paracrine signaling molecules that influence fluid (aqueous humor) secretion by the NPE.