Cl- induces endothelium-dependent mesenteric arteriolar vasorelaxation through the NKCC1/TRPV4/NCX axis

Aims: Although absorbed NaCl increases intestinal blood flow to facilitate absorption and transportation, it is unclear if it can directly mediate mesenteric arterial relaxation. We aimed to investigate and test our hypothesis that Cl- induces mesenteric arterial vasorelaxation via endothelium-dependent hyperpolarization (EDH). Main methods: We used wire myograph to study NaCl-induced vasorelaxation of mesenteric arteries isolated from mice. Cl-, Ca2+ and K+ imaging was performed in human vascular endothelial cells pre-treated with pharmacological agents. Key findings: The Cl- concentration-dependently induced vasorelaxation of mesenteric arteries likely through EDH. The Cl--induced vasorelaxation was attenuated in TRPV4 KO mice and inhibited by selective blockers of Na+-K+-2Cl(-) cotransporter 1 (NKCC1) (bumetanide, 10 mu M), transient receptor potential vanilloid 4 (TRPV4) (RN-1734, 40 mu M), and small conductance Ca2+-activated K+ channels (SKCa) (apamin, 3 mu M)/ intermediate conductance Ca2+-activated K+ channels (IKCa) (TRAM-34, 10 mu M) and myoendothelial gap junction (18a-glycyrrhetinic acid, 10 mu M), but enhanced by a selective activator of IKCa/SKCa (SKA-31, 0.3 mu M). Cl- decreased intracellular K+ concentrations in endothelial cells, which was reversed by apamin (200 nM) plus TRAM-34 (500 nM). Extracellular Cl- raised intracellular Cl- concentrations in endothelial cells, which was attenuated by bumetanide (10 mu M). Finally, Cl- induced a transient Ca2+ signaling via TRPV4 in endothelial cells, which became sustained when the Ca2+ exit mode of Na+-Ca2+ exchanger (NCX) was blocked. Significance: Cl- induces a pure EDH-mediated vasorelaxation of mesenteric arteries through activation of endothelial NKCC1/TRPV4/NCX axis. We have provided a novel insight into the role of Cl--induced vasorelaxation via EDH mechanism.