Abstract 12915: The Contribution of Transient Receptor Potential Vanilloid 4 (TRPV4) to Endothelium-Dependent Dilation is Enhanced by Shear Stress

Abstract

Shear stress on the endothelial surface of blood vessels influences vasodilator mechanisms. We tested the hypothesis that shear enhances the contribution of TRPV4 ion channels to vasodilation and investigated whether there was a functional interaction with the muscarinic receptor 3 (MR3). In HEK293 cells we established that the MR3 signals to and opens TRPV4. Using pressure myography, dilator responses to the TRPV4 agonist GSK1016790A and to the MR3 agonist acetylcholine (ACh) were examined in rat cremaster arterioles which had either experienced no shear stress or within 15 min of exposure to high shear stress (200 μl/min flow, 6 min, n=8-12). In control vessels GSK1016709A caused vasodilation (pEC 50 7.85 ± 0.15 M, R max 109 ± 6 %) which was attenuated by the TRPV4 antagonist GSK2193874 (100 nM) (pEC 50 6.26 ± 0.14 M, p<0.05, R max 84 ± 5 %) and abolished by endothelium denudation. When responses were assessed in shear treated arterioles there was an increase in sensitivity to GSK1016790A (pEC 50 8.39 ± 0.11, p<0.05, R max 107 ± 3 %). Vasodilation to ACh (pEC 50 7.23 ± 0.06 M, R max 99 ± 1 %) was not affected by exposure to shear forces (pEC 50 6.94 ± 0.08 M, R max 98 ± 1 %). Dilator responses to ACh were not affected by the TRPV4 antagonist in control arterioles (pEC 50 7.24 ± 0.07 M, R max 102 ± 2 %) but, in shear treated vessels, the ACh response was attenuated by GSK2193874 (pEC 50 6.26 ± 0.12 M, p<0.05, R max 95 ± 3 %) indicating that shear forces stimulate a functional interaction between TRPV4 and MR3. TRPV4 antibody immunofluorescence measured in the arterioles indicated localisation of ion channels to the endothelium, but the induction of shear stress on the endothelium had no effect on the localisation of TRPV4 within endothelial cells of the intact arterioles. Expression of TRPV4 channels at the cell surface was not affected by shear forces as immunofluorescence of an antibody for the extracellular TRPV4 domain was similar in both groups of arterioles. Shear stress increases the sensitivity of endothelial TRPV4 ion channels to a selective agonist and reveals their contribution to muscarinic receptor mediated endothelium-dependent vasodilation. Investigating dilator responses in isolated arterioles without exposure to shear forces may under estimate the contribution of TRPV4.