Abstract 632: Flow-induced TRPV4 Activation Mediates Calcium Influx In The Thick Ascending Limb.

Abstract

Background: The thick ascending limb is critical in the regulation of sodium balance and therefore blood pressure. We previously showed that nitric oxide (NO), which inhibits sodium chloride transport and therefore blood pressure is regulated by luminal flow in this segment. Transient receptor potential vanilloid 4 (TRPV4), a member of the TRPV family of cation channels, is expressed in the thick ascending limb. This calcium permeable channel can be activated by luminal flow in other cell types. Therefore, we hypothesized that in the thick ascending limb luminal flow induces TRPV4 activation thereby increasing calcium influx and NO production. METHODS: We used the TRPV4 antagonists Ruthenium red and RN 1734 and the TRPV4 agonist GSK1016790A and measured intracellular calcium and NO production in the absence and presence of luminal flow in isolated and perfused thick ascending limbs from Sprague Dawley rats. NO production was measured using the NO-sensitive dye DAF FM da. Intracellular calcium was measured using the ratiometric calcium sensitive dye Fura-2 AM. Results: Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min ( p <0.05, n =5). Increasing luminal flow in the presence of the TRPV4 antagonist Ruthenium red (15 μM) eliminated NO production (from 18 ± 5 to 16 ± 9 AU/min; n = 4). Flow-induced NO production was also prevented by the selective TRPV4 antagonist RN 1734 (10 μM) (from 11 ± 7 to 9 ± 2 AU/min; n = 4). Increasing luminal flow increased Fura 2 ratio units by 113 ± 20 %. However, in the presence of the TRPV4 antagonist Ruthenium red (15 μM) Fura 2 ratio units increased only by 55 ± 12 % in response to luminal flow ( p <0.04, n =6). In the absence of luminal flow, activating TRPV4 using the TRPV4 selective agonist GSK1016790A (1 ηM) increased Fura 2 ratio units by 204 ± 43 %. Conclusion: From these data we conclude that flow-induced TRPV4 activation mediates calcium influx and NO production in the thick ascending limb.