Nω nitro l arginine methyl ester

Potassium chloride (KCl), PE, N^ω-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), ACh, sodium nitroprusside (SNP), 4-Hydroxy-TEMPO (tempol, a superoxide anion scavenger), indomethacin (a non-selective cyclooxygenase (COX) inhibitor), and methyl-β-cyclodextrin (CD, a cholesterol depleter which disassembles caveolae) were purchased from Sigma-Aldrich (St. Louis, MO, USA); losartan (an Ang II type 1 receptor antagonist) was from Cayman Chemical Company (Ann Arbor, MI, USA). Drug concentrations were expressed as final molar concentrations in the bath chamber. 10× radio-immunoprecipitation assay (RIPA) lysis buffer was from Millipore Corporate Headquarters (Temecula, CA, USA); bicinchoninic acid (BCA) protein assay kit and Pierce enhanced chemiluminescent (ECL) Western Blotting Substrate were from Thermo Fisher Scientific (Rockford, IL, USA); anti-p-eNOS^Thr495 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-eNOS^Ser1177 was from Abcam plc (Cambridge, UK); anti-eNOS, anti-adenosine monophosphate-activated protein kinase (anti-AMPK), anti-Cav-1, anti-β-actin, and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG were from BD Transduction Laboratorie (Lexington, KY, USA); HRP conjugated goat anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).

All the reagents below were of analytical grade, and the stock solutions and buffers were prepared with Milli-Q water (Millipore, Billerica, MA, USA). Ethanol, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), TPTZ (2,4,6-tris (2-pyridyl)-s-triazine), tris(hydroxymethyl)aminomethane, sodium chloride, ascorbic acid, Griess reagent, lansoprazole, ranitidine hydrochloride, 2-thiobarbituric acid, EDTA (ethylene diamine tetraacetic acid), N-acetylcysteine, quercetin, gallic acid, ellagic acid, tannic acid and catechin, IU urease (jack bean urease, type III), Alcian blue, N-ethylmaleimide, N_ω-nitro-L-arginine methyl ester, ranitidine, carbenoxolone were obtained from Sigma-Aldrich (St. Louis, MO, USA). Trolox is obtained from Merck, Düsseldorf, Germany; TNF alpha ELISA kit is from eBioscience, San Diego, CA, USA. Historesin is obtained from Leica Biosystems, Wetzlar, Germany, and acetic acid and glucose were obtained from Vetec, Duque de Caxias, Rio de Janeiro, Brazil. Ethyl ether, formaldehyde, and phenolphthalein are from FMaia, Cotia, São Paulo, Brazil; xylazine and ketamine are obtained from Vetbrands, Paulínia, São Paulo, Brazil. Ki-67 protein (code: sc-23900) and BrdU protein (code: sc-32323) are from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Thoracic aortas were carefully dissected out of the surrounding tissues. The assessment of NO-dependent endothelial function in the isolated rings of rat aortas was determined by dose-dependent relaxation to acetylcholine (Ach, 0.01–10 µM, Sigma-Aldrich, St. Louis, MO, USA) both without and in the presence of the inhibitor of nitric oxide synthase N_ω-Nitro-L-Arginine Methyl Ester (L-NAME, 300 μM, Sigma-Aldrich, St. Louis, MO, USA) as previously described [24 (link)]. The endothelium-independent response was tested by a response evoked by sodium nitroprusside (SNP, 0.001–1 µM, Sigma-Aldrich, St. Louis, MO, USA).

Ranitidine (GlaxoSmithKline laboratory, France) was used as an antiHistamine to highlight the possible effect of the extract on Histamine receptors.
Histamine (Sigma-Aldrich, UK) was used to induce relaxant effects on rat uterine horns by acting on H₂-receptors.
N^ω-nitro-L-arginine methyl ester or L-NAME (Sigma-Aldrich, UK), an inhibitor of nitric oxide synthase, was used to highlight the possible effects of the extract on the relaxation mechanism through the nitric oxide pathway.
Oxytocin was purchased from Ciron Drugs (India). It was used to increase uterus contractions and demonstrate the possible inhibition of extracellular calcium by the extract.

Phenylephrine hydrochloride, acetylcholine chloride, diethylamine NONOate, diethylammonium salt, N^ω-nitro-L-arginine methyl ester, H89, LY 294002, sodium vanadate, SDS, Trizma-Base, and 4-hydroxi-2,2,6,6-tetramethylpiperidinoxyl (Tempol) were purchased from Sigma-Aldrich (Madrid, Spain). U46619 was purchased from Cayman Chemical (Michigan, USA). Stock solutions (10 mmol/L) of drugs were made in distilled water, except for Tempol, H89 and LY 294002, which were dissolved in dimethylsulfoxide (DMSO), and administered so that the maximum DMSO concentration of the medium was less than 0.001%. These solutions were kept at −20 °C and appropriate dilutions were made on the day of the experiment.

The methanolic extract of C. procera was dissolved in 0.9% NaCl (isotonic saline solution). Absolute ethanol (ETOH), N^ω-nitro-L-arginine methyl ester (L-NAME), N-ethylmaleimide (NEM), glibenclamide (GLIB), indomethacin (INDO), and carbenoxolone (CAR) were purchased from Sigma-Aldrich Co. Omeprazole (OME) was obtained from Liomont®. Ketamine and Xylazine were acquired from Pisa®.
All the samples administered by intragastric route, independently of the final concentration used, were always suspended in a volume of 10 ml/kg.