Activation of TRPV4 channels reduces IOP and improves outflow facility by regulating eNOS dependent nitric oxide release from the trabecular meshwork

Purpose: Nitric oxide (NO) is known to reduce intraocular pressure (IOP) by relaxation of the trabecular meshwork (TM) and distal vessels of the conventional outflow pathway. However, the intrinsic mechanisms by which outflow pathway tissues regulate NO production is yet to be elucidated. In vascular endothelium, activation of mechanosensory transient receptor potential vanilloid 4 (TRPV4) channels results in endothelial nitic oxide synthase (eNOS) mediated NO release, which in turn promotes vasodilation. Here, we determined whether activation of TRPV4 regulates IOP and conventional outflow via NO release in the TM. Methods: In wildtype (WT) and glucocorticoid-induced ocular hypertensive (OHT) C57BL/6J mice, the effect of TRPV4 agonist GSK1016790A on IOP and outflow facility was determined using rebound tonometry and constant-flow infusion method respectively. Effect of TRPV4 agonist on eNOS activation and NO production was determined using Western blot and fluorometric DAF-FM assay in primary human TM cells and ex vivo cultured human TM donor tissues. We report for the first time a method for electrochemical measurement of NO in human anterior segment donor tissues using NO microsensors. Results: Topical administration of TRPV4 agonist GSK1016790A significantly reduced IOP (Pin WT and OHT mice compared to contralateral control eyes. In OHT mice, treatment with GSK1016790A resulted in increased outflow facility (P=0.02)compared to contralateral vehicle treated eyes. We further demonstrate that TRPV4 activation by GSK1016790A resulted in increased eNOS phosphorylation in GTM3 cells, primary human TM cells, and cultured human TM donor tissues. Activation of TRPV4 in primary TM cells and ex vivocultured human TM donor tissues resulted in increased DAF-FM fluorescence, which signifies increase in TRPV4-mediated NO production. Treatment of human anterior segments with TRPV4 agonist resulted in increased production of NO as detected electrochemically using NO microsensors. Nonselective inhibition of NOS by L-NAME abrogated the IOP lowering effect of TRPV4 agonist in mice and reduced TRPV4-mediated NO production in outflow pathway cells and donor tissues. Conclusion: TRPV4 activation improves IOP and outflow facility, perhaps by regulation of eNOS dependent NO release.