Myography.

Isolated radial MAs were mounted in a small-vessel wire myograph (Multi Wire Myograph System 610M, DMT-USA, Ann Arbor, MI) with two wires (40-µm diameter) threaded through the vessel lumen and connected to either a tension transducer or micrometer in a chamber containing oxygenated (95% O₂-5% CO₂) and warmed (37°C) Krebs buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, and 11 mM d-glucose). MAs were normalized to an internal diameter of 0.9 of L_13.3kPa. After at least 45 min of equilibration, MAs were constricted with 60 mM KCl to establish viability; vessels failing to constrict at least 1 mN (estimated as an appropriate signal-to-noise ratio for tension measurement) were excluded from further study. The effect of AMPK activation on opposing vasoconstriction was assessed by applying increasing concentrations of phenylephrine (PE; 1 nM to 100 µM, Sigma-Aldrich) in the presence or absence of the AMPK agonist A769662 (30 µM, pretreated for 20 min; Cayman Chemical, Ann Arbor, MI) with each PE concentration being added for 3–5 min before the next concentration. MA vasodilation was assessed in vessels preconstricted with 10 µM PE; the vasodilator responses to either A769662 (0.1 nM to 100 µM), acetylcholine (ACh; 0.1–100 µM, Thermo Fisher Scientific), or bradykinin (BK; 0.1–1 µM, Thermo Fisher Scientific) were measured for 3–5 min before addition of the next concentration; and the value after a stable plateau was reached was recorded. To assess the contributions of endothelial factors to the AMPK vasodilator response observed, vessels were incubated with either the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME; 10 µM, BioVision, Inc., Milpitas, CA) or l-NAME (10 µM) plus the cyclooxygenase inhibitor indomethacin (Indo; 10 µM, Sigma-Aldrich) for 20 min before PE-constriction and the application of A769662. In some MAs, the endothelial cell layer was mechanically disrupted by rolling a hair through the vessel lumen; loss of endothelial function was judged as <30% relaxation to 10 µM ACh or 10 nM BK. The force elicited by PE was normalized to that evoked by 60 mM KCl (K_max), whereas A769662 relaxation was calculated as percentage of 10 µM PE contraction. Half-maximal effective concentration (EC₅₀), maximal force, and area under the curve (AUC) were calculated using Graph Pad 8 software (San Diego, CA). Due to the limited number of vessels from each subject, it was not possible to apply all the treatments to the same vessel or vessels from the same subject.