Five minutes later, dose-response curves to cumulative doses of acetylcholine (ACh, 10−7, 10–6, and 10−5M) were evaluated. The concentration of ACh was increased by one log unit every 1.5 minutes. Response to cumulative doses of ACh was calculated as a percent change in PP. In a different group of rats, a portal perfusion pressure-response curve to Mtx was obtained by adding increasing doses of Mtx (10−6, 10−5, 10−4, 5 − 10−4 mol/L) to the reservoir every 5 minutes. Protein nitrotyrosination (3-NT), this website a marker of peroxynitrite production and oxidative stress due to NO reaction with ROS, was determined by western blotting (see Supporting Information for details). Blots were

probed with a mouse anti–3-NT (1:1,000) monoclonal antibody (Sigma,

Madrid, Spain) and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). X-ray films were exposed, developed, fixed, and scanned. Densitometry of digital images was performed with Melanie version 6 software. GAPDH was used as control of sample loading. Endothelial NO synthase (eNOS) and phosphorylated eNOS (p-eNOS) protein detection was performed with mouse Autophagy inhibitor cost anti-eNOS (1 μg/mL dilution; BD Biosciences, San Jose, CA) and rabbit anti–p-eNOS (1:500 dilution; Cell Signaling Technology) as described for 3-NT. Quantitative densitometric values were compared between eNOS and p-eNOS blots. GAPDH was used as control of sample loading. Measurements of guanosine 3′,5′-cyclic monophosphate, a marker of NO bioavailability, were performed in control and cirrhotic rat liver homogenates from

HC and CIH rats (see Supporting Information for details). The results are expressed as picomoles per milliliter. Mtx and ACh were purchased from Sigma (Madrid, Spain). Statistical analysis was performed using SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL). All data are reported as the mean ± SEM. Comparisons between groups were performed using analysis of variance followed by Student’s t test or the nonparametric test for unpaired data (Mann-Whitney) when appropriate. Differences were considered significant at P < 0.05. MCE 3-NT, nitrotyrosine; ACh, acetylcholine; CBDL, common bile duct ligation; CCl4, carbon tetrachloride; CIH, chronic intermittent hypoxia; eNOS, endothelial nitric oxide synthase; HC, handled controls; MAP, mean arterial pressure; Mtx, methoxamine; NO, nitric oxide; OSAS, obstructive sleep apnea syndrome; p-eNOS, phosphorylated eNOS; PP, portal pressure; ROS, reactive oxygen species. Rats after 12 weeks of CCl4 inhalation and CBDL rats had macroscopic cirrhosis and signs of portal hypertension as shown by the presence of ascites, collateral circulation, or splenomegaly (Table 1). Rats with 8 weeks of CCl4 inhalation showed macroscopic micronodular cirrhosis without ascites. Body weight was recorded to determine whether the exposure protocol altered weight gain.