Arcades of mesentery vessels amenable to pressurization to mimic the myography were developed to have a sufficient amount of vessel material to carry out biochemical experiments (Fig. 1B). Whole mesenteric artery arcades (arteries of second to fourth order) were dissected from mice, and all open ends were closed with suture, incubated for 1 hour at 37°C in Hepes-Krebs buffer, cannulated, and pressurized to 80 mmHg using a sphygmomanometer (Riester Big-Ben Round, Germany). Before and immediately after a pressure step or agonist stimulation, arcades were frozen at 30 s, 90 s, and 5 min, respectively, and analyzed for phosphorylation of MYPT1 at Thr668 and Thr853, RLC20 at Ser19, RSK at Thr577/Ser227, PDK1 at Ser241, and NHE-1 Ser703. Tissues were snap frozen in liquid nitrogen and transferred to a vial with 10% cold trichloroacetic acid/acetone to preserve the phosphorylation state as described previously (25). Agonists were AngII (1 μM) or endothelin 1 (10 nM), phenylephrine (1 μM), and TXA2 receptor mimetic U46619 (1 μM).

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