Flow‐Induced NO Production is Dependent on TRPV4 Activation and ATP Release in Thick Ascending limbs

We showed that mechanical stimulation by luminal flow enhances NO production in thick ascending limbs (TALs) by inducing ATP release. The Transient Receptor Potential Vanilloid 4 (TRPV4) channel mediates NO production induced by mechanical stimulation in endothelial cells. We hypothesized that TRPV4 activation enhances ATP release and stimulates NO production in TALs. We measured NO production in isolated rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nL/min stimulated the rate of NO production from 8 ± 3 to 45 ± 12 AU/min (p < 0.02). Flow did not affect NO levels in the presence of the selective TRPV4 antagonist RN 1734 (10 uM) (from 11 ± 7 to 9 ± 2 AU/min). In the absence of luminal flow and therefore mechanical stimulation, the TRPV4 activator 4αPDD (1 uM) stimulated NO production from 3 ± 1 to 9 ± 1 AU/min (p < 0.003) and this was prevented by pre‐treating TALs with the NO synthase inhibitor L‐NAME. Next, we measured luminal ATP release in isolated TALs using a bioluminescence assay. Increasing luminal flow from 0 to 20 nL/min stimulated ATP release from 9 ± 2 to 48 ± 11 arbitrary units AU/min (p< 0.03). When luminal flow was increased in the presence of the TRPV4 blocker RN1734 (10 uM), ATP release did not increase (from 11 ± 3 to 14 ± 3 AU/min). These data indicate that flow‐induced TRPV4 activation enhances ATP release which in turn stimulates NO production.