Freshly isolated third- or fourth-order mesenteric arteries (<180 μm) were placed into Hepes-Krebs solution (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 4 mM NaHCO3, 1.2 mM KH2PO4, 2 mM CaCl2, 10 mM Hepes, and 6 mM glucose) and then mounted in a pressure arteriograph (Danish MyoTechnology or a custom made device), as previously described (61, 62). The vessels were pressurized to 20 mmHg and then to 60 or 80 mmHg at 37°C and equilibrated for 30 min, brought back to 20 mmHg, and then exposed to incremental increases in pressure to 100 mmHg. Vessels were used only if they displayed robust endothelial cell viability, which was assessed at the end of an agonist-induced constriction using 10 μM acetylcholine (fig. S1C) (61) or the calcium activated intermediate (IK) or small conductance (SK) channel agonist NS309 to induce relaxation of a myogenic constriction (63). Luminal diameters were measured in response to changes in intraluminal pressure or to cumulative concentrations of phenylephrine or inhibitors applied to the circulating bath. Responses to cumulative concentrations of high K+ were achieved by increasing the K+ concentration from 5.9 to 62 mM by substituting equal volumes of Hepes-Krebs solution with depolarizing Hepes-Krebs solution (143.3 mM KCl, 1.2 mM CaCl2*2H2O, 1.2 mM MgCl2*6H2O, 11.6 mM Hepes, and 11.5 mM dextrose) while keeping Ca and Mg concentrations constant. Maximum inner diameter was measured after washing with a Ca2+-free Krebs-Hepes solution supplemented with 1 mM EGTA and 10 μM sodium nitroprusside. Quantification of vessel diameter was performed using the DMT software MyoView. Basal tone and vasoconstriction values were calculated as follows: [maximum diameter − active diameter/maximum diameter] × 100. Acetylcholine and phenylephrine data are expressed as the percentage of the maximum inner diameter.

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