c.), a cannula (PE 50) was inserted retrogradely (1.0 cm) into the portal vein and the vascular mesenteric bed was dissected out at its border with the intestine. The mesenteric venular bed was perfused at a constant rate of 2 mL/min using a peristaltic pump (Miniplus 3, Gilson, France) with Krebs-Henseleit solution, pH 7.4, at 37 °C in the presence of 95% O2 and MK0683 nmr 5% CO2. To confirm the viability of tissues, preparations were exposed to 90 mmol/L KCl for 5 min. After 30 min of washing out the KCl with Krebs solution, Ang II (0.1 nmol) was administered in bolus in a final volume of 100 μL and vascular responses were evaluated as changes
in the perfusion pressure (mmHg) (PowerLab 4S; ADInstruments, Australia). Isolated portal vein ring preparations
were performed according to the method previously described [2]. Rats were anesthetized with chloral hydrate (450 mg/kg, s.c.), the portal vein was excised and connective tissue was removed. Rings of portal veins (3–4 mm length) were mounted under 0.5 g of passive tension in an organ bath (15 mL) containing Krebs-Henseleit solution, pH 7.4, at 37 °C with 95% O2 and 5% CO2. Preparations were allowed to equilibrate for 60 min; during this time, the bath solution was changed every 20 min. To confirm the viability of tissues, the preparations were exposed to 90 mmol/L KCl for 5 min. After 30 min of washing out the KCl with normal Krebs solution, a cumulative-concentration response curve (CCRC) to Ang II (0.1–100 nmol/L) was performed and changes in isometric tension (grams) were recorded (PowerLab 4S, ADInstruments, Australia). CCRC were analyzed by a data analyses JAK inhibitor program (Prism3, GraphPad) Selleck Neratinib to evaluate the EC50 (the concentration of Ang II required to produce 50% maximum response) and maximum response (Emax). Efficacy and sensitivity of portal vein rings preparations in response to Ang II was determined as Emax and pEC50 (−log EC50), respectively. To investigate the mechanisms involved in Ang II-mediated contraction, preparations of mesenteric venous beds
and portal vein rings were incubated with Krebs-Henseleit solution containing losartan (specific AT1R antagonist, 0.1 μmol/L), PD 123319 (specific AT2R antagonist, 0.1 μmol/L), HOE 140 (specific B2R antagonist, 20 nmol/L) [13], indomethacin (COX inhibitor, 10 μmol/L), or L-NAME (inhibitor of NO synthesis, 10 μmol/L) 30 min before Ang II injection. In addition, a group of SHR were treated with celecoxib (specific COX2 inhibitor, 10 mg/kg) [20] administered by gavage 3 h before were killed and the mesenteric venular beds and portal vein rings were prepared. All the concentrations of antagonists/inhibitors used in experiments were based in preliminary studies performed in our laboratory or in the literature, when specified. Total RNA from the portal veins of SHR and Wistar rats was extracted using Trizol reagent (Invitrogen, USA) in accordance with the manufacturer’s protocol.