ASIC1 contributes to endothelium-dependent, hyperpolarization-induced vasodilation, colocalizes with IK _Ca and SK _Ca channels, and localizes within myoendothelial junctions. (A) ACh (10⁻⁹–10⁻⁵ M) induced vasodilatory responses in mesenteric arteries, in the absence or presence of the NOS-specific inhibitor L-NNA (100 µM), the cyclooxygenase specific inhibitor indomethacin (Indo; 10 µM), and either PcTX1 (20 nM) or Tram-34 and Apamin (Tram/Ap; 1 µM and 100 nM, respectively). Data represent five animals (biological replicates)/group and were analyzed by two-way ANOVA. The two-way ANOVA revealed that there was a statistically significant interaction (P < 0.0001) between factors. Post hoc pairwise comparisons were analyzed between individual groups using Tukey’s multiple comparisons test. *P < 0.05 vehicle vs. L-NNA, Indo; #P < 0.05 L-NNA, Indo alone vs. PcTX1 or Tram/Ap. P values for data in A are shown in Table 2. (B and C) Immunofluorescence in mesenteric endothelial tubes showing ASIC1a (red) and (B) IK_Ca (green) or (C) SK_Ca (green) distribution. Nuclei are stained with Sytox (blue). (D) Representative images showing the Duolink proximity ligation interaction between ASIC1-IK_Ca, ASIC1-SK_Ca, or no primary antibodies as a negative control. (E) Autofluorescence (magenta) of the IEL and immunofluorescence of ASIC1 (green) in an intact, whole-mount mesenteric artery. Areas without magenta demonstrate fenestrations in IEL associated with myoendothelial junctions. Magnified images (a-f; yellow boxes) show the orthogonal view of the IEL layers (arrows), with the smooth muscle on the outside and endothelium between the IEL layers.