Aortic ring isometric contraction assay

DTAs from wild-type, smLRP1^−/−, and smaLRP1^−/− mice were dissected and placed in a tissue culture dish containing Krebs solution (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 25 mM NaHCO₃, pH 7.4). Aortic rings were prepared and equilibrated as detailed in the online-only Data Supplement. The following stimuli were tested: 120 mM KCl (Sigma-Aldrich), phenylephrine (PE; Sigma-Aldrich), 1 μM U-46619 (Sigma-Aldrich), 1 μM FPL 64176 (Sigma-Aldrich), 0.3 μM calyculin A (Sigma-Aldrich), and 1 mM 4-chloro-m-cresol (4-CmC; Sigma-Aldrich). After each stimulus, aortic rings were washed and re-equilibrated for 15–30 minutes before application of the next stimulus. Force measurements were acquired and recorded using LabChart Pro (ADInstruments). For each stimulus, aortic rings were normalized to their respective baseline force measurement recorded immediately before addition of the stimulus. Aortic ring isometric contraction assays using PE are presented as means ± SEM with 95% confidence intervals indicated.