N 2 mercaptopropionyl glycine

Coronary microvascular endothelium-dependent and -independent vasodilator responses were assessed in coronary small arteries (∼300 µm diameter) isolated from the epicardial surface of the left ventricular apex and studied in vitro using a Mulvany wire myograph (Danish Myograph Technology, Aarhus, Denmark) [64 (link), 72 (link)]. Following preconstriction with 10⁻⁶ mol L⁻¹ thromboxane-A2 analogue U46619 (Sigma–Aldrich), concentration–response curves were determined for the endothelium-independent NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 10⁻¹⁰ to 10⁻⁵ mol L⁻¹, Sigma–Aldrich), and for the endothelium-dependent vasodilator bradykinin (BK, 10⁻¹⁰ to 10⁻⁶ mol L⁻¹, Sigma–Aldrich) in the absence and presence of NOS blockade with 10^–4 mol L⁻¹ Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME, Sigma-Aldrich), the highly water soluble prodrug of NLA [48 (link)]. In a subgroup of animals (4 Normal and 5 DM + HFD + CKD swine), the concentration-response curves for bradykinin were also performed in the absence and presence of the ROS scavenger N-2-mercaptopropionylglycine (MPG, 10⁻⁵ mol L⁻¹, Sigma–Aldrich) and the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol 10⁻³ mol L⁻¹, Sigma–Aldrich).

The following commercially available antibodies were used: anti-Rab5 (ab31261, abcam), anti-GABARAP for Drosophila Atg8a (PM037, MBL), anti-Drosophila LAMP1 (ab30687, abcam), anti-human LAMP1 (AB2296838, DSHB), anti-cathepsin D (sc-6487, Santa Cruz), anti-Brp (AB2314866, DSHB), anti-cathepsin L (MAB 22591, R&D systems)^{44 (link),63} , anti-Ref(2)P²⁸, anti-elav (AB528217, DSHB), anti-ATP5α (ab14748, abcam), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (Thermo Fisher Scientific), or near infrared (IR) dye 700/800 (Rockland). The following chemicals were used in the study: N-acetylcysteine amide (A0737, Sigma-Aldrich), N-(2-mercaptopropionyl)glycine (M6635, Sigma-Aldrich), putrescine (51799, Sigma-Aldrich), spermidine (S0266, Sigma-Aldrich), spermine (s4264, Sigma-Aldrich), bis(hexamethylene)triamine (421960, Sigma-Aldrich), DHE (D11347, Thermo Fisher Scientific), LysoTracker^® Red DND-99 (L7528, Thermo Fisher Scientific), LysoSensor™ Yellow/Blue DND-160 (L7545, Thermo Fisher Scientific).