TRPV4 and mechanosensitive regulation of hemichannel function in ocular nonpigmented ciliary epithelium

Purpose: Previously we reported a mechanosensitive ion channel, TRPV4, along with functional connexin hemichannels on the NPE basolateral surface. In the lens, we have evidence of TRPV4-mediated hemichannel opening as part of a feedback mechanism that enables the lens to sense and respond to swelling. The present study was undertaken to test the hypothesis that TRPV4 and hemichannels might function as a mechanosensor in ocular nonpigmented ciliary epithelium (NPE). Methods: Porcine NPE cells were cultured on either plastic culture plates to study effects of osmotic swelling stretch, or on flexible membranes (Flexcell) to study effects of axial cyclic stretch. ATP release and the uptake of the large molecule propidium iodide (PI) from the bathing medium were taken as indicators of hemichannel opening and determined by luminescence and fluorescence. Results: NPE cells subjected to cyclic stretch for 1-10 min (10%, 0.5 Hz) significantly increased ATP release (CTRL 0.3±0.002 vs Stretch 0.7±0.004, nmoles/mg protein, n=6, p<.0001) into the bathing medium. The same stretch stimulus also increased uptake of large molecule propidium iodide (PI) from the bathing medium (CTRL 1.5±0.09 vs stretch 2.49±0.09, Fl unit/mg protein, n=6, p<0.001). The ATP release and PI uptake responses both were prevented by a TRPV4 antagonist, HC067047 (10 μM) and a connexin blocking peptide, GAP27 (200μm). Cells subjected to a hypoosmotic stimulus (200 mOsm) also displayed a significant increase in PI uptake (CTRL 4.65±0.24 vs Hypo 8.26±0.43 nmoles/mg protein, n=5, p<0.001) and the response was abolished by HC067047. The TRPV4 agonist GSK1016790A (10 nM) markedly increased ATP release (CTRL 0.56±0.03 vs GSK 1.24±0.06 nmoles/mg protein, n=12, p<0.001) and PI uptake (CTRL 1.83±0.05 vs GSK 3.69±0.09 Fl unit/mg protein) in the absence of any swelling or stretch stimulus and both responses were abolished by GAP27. Conclusion: The findings are consistent with TRPV4-dependent connexin hemichannel opening in response to mechanical stretch. The TRPV4-hemichannel mechanism may act as mechanosensor that facilitates release of ATP and other autocrine or paracrine signaling molecules that influence fluid (aqueous humor) secretion by the NPE. This research was funded by an NIH (NEI) grant, R01EY029171. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.