Measuring Arteriolar Vasoreactivity

Resistance arterioles of ~50 μm in diameter and ~2 mm in length were carefully dissected from the skeletal muscle tissue and cleaned of fat and connective tissue. In an organ perfusion chamber, single vessels were cannulated with glass micropipettes (outer tip diameter ∼40 μm) filled with cold bicarbonate buffer consisting of 123 mmol/L NaCl, 4.4 mmol/L KCl, 2.5 mmol/L CaCl₂, 1.2 mmol/L MgSO₄, 20 mmol/L NaHCO₃, 1.2 mmol/L KH₂PO₄, and 11 mmol/L glucose. Both ends of the vessel were secured with 10‐0 nylon Ethilon monofilament suture, and the vessels were maintained at an intraluminal pressure of 20 mmHg for 30 min. Each preparation was transferred to the stage of an inverted microscope (magnification ×200) attached to a video camera, monitor, and video‐measuring device (model VIA‐100; Boeckeler Instruments, Tucson, AZ). The external bathing medium was continuously superfused with heated buffer solution (pH = 7.4 ± 0.05, Po₂ = 140 ± 10 mmHg) aerated with a gas mixture of 21% O₂–5% CO₂–74% N₂ and maintained at 37°C. The pressure was slowly increased to 100 mmHg and maintained for 30 min. Vessels were preconstricted 30–50% with ET‐1 (100–200 pM). Vessels that did not constrict >30% were excluded from the analysis. Flow was produced by simultaneously changing the heights of the reservoirs in equal and opposite directions to generate an intraluminal pressure gradient of ∆10–∆100 cmH₂O (equivalent to ~7–70 mmHg), which covers the physiological range of arteriolar pressure in the human body (Phillips et al. 2007; Grizelj et al. 2015). In separate experiments, vasoreactivity measures were determined in response to incremental doses of acetylcholine (ACh) (10⁻⁹–10⁻⁴ mol/L). Steady‐state internal arterial diameters were measured before and during intraluminal flow or ACh in the absence or presence of the NO synthase (NOS) inhibitor N^ω‐nitro‐l‐arginine methyl ester (L‐NAME; 10⁻⁴ mol/L). Maximal diameter of every vessel was determined in the presence of papaverine (10⁻⁴ mol/L), and the diameter in response to flow at a gradient of 100 cmH₂O was measured in the presence of papaverine first in one direction, and then with the gradient reversed to verify pipette resistance matching (Phillips et al. 2007).