Bay 60 2770

BAY 60-2770 and BAY 41-2272 were provided by Bayer AG (Wuppertal, Germany). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was purchased from Cayman Chemical (Ann Arbor, MI), and hemin was obtained from Frontier Scientific (Newark, NJ). Recombinant human and murine tumor necrosis factor-α (TNF) and human fibronectin were from R&D Systems (Minneapolis, MN). Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin, and streptomycin were from Gibco-Invitrogen (New York, NY). Trizol was from Invitrogen (Carlsbad, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

The following pharmacological agents were used in this study. BAY 60–2770 (a gift from Bayer Pharma AG, Germany); K⁺ channel blocker, 5-HD (5-hydroxydecanoate-sodium salt, Enzo, NY, USA); potent and selective inhibitor of PKG, KT5823 (2,3,9,10,11,12-hexahydro-10R-methoxy-2,9-dimethyl-1-oxo-9S, Cayman Chemical, MI, USA); mitochondria uncoupler, CCCP (carbonyl cyanide 3-chlorophenylhydrazone, Sigma, St. Louis, MO, USA).

Six weeks after 5/6^th nephrectomy or sham procedure, animals were euthanized by inducing hypovolemia via aortic puncture. The carotid arteries were carefully dissected without damaging the endothelium and 2mm long segments were mounted in a wire myograph (DMT, Aarhus, Denmark). During contraction with phenylephrine (10μM) endothelial-dependent relaxation was studied by means of stimulation with acetylcholine (ACh,10⁻¹⁰–10^-5M, both Sigma). Blocking of NOS with L-NAME (10μM, Sigma) was also performed to control for non-NO mediated effects of ACh. The NO-donor sodium nitroprusside (SNP,10⁻¹⁰–10^-5M, Sigma) was used to evaluate the relaxation capacity of SMCs. BAY 41–2272, a stimulator of sGC (Bayer, Leverkusen, Germany) and BAY 60–2770, an activator of oxidized sGC (Bayer) (both kindly provided by Dr. J.P. Stasch, Bayer) were used to evaluate the oxidative state of sGC. 1H-[1 (link),2 (link),4 (link)]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, Sigma) was used to actively oxidize sGC in combination with the BAY compounds.