The

volume injected into the LV was 1 or 2 μl. On the day of the experiment rats were anaesthetized with urethane (1.2 g/kg of body weight i.v.) and α-chloralose (60 mg/kg of body weight i.v.) (after the induction with 1% halothane in 100% O2). A femoral artery catheter (PE-10 connected to PE-50) was implanted for the record of pulsatile arterial pressure, mean arterial high throughput screening compounds pressure (MAP) and heart rate (HR). A femoral vein catheter was implanted for administration of anaesthetic. To record pulsatile arterial pressure, MAP and HR, the arterial catheter was connected to a Statham Gould (P23 Db) pressure transducer coupled to a pre-amplifier (model ETH-200 Bridge Bio Amplifier, CB Sciences) and to a Powerlab computer recording system (model Powerlab 16SP, ADInstruments). Recordings began 10 min after the connection of the arterial line to the pressure transducer. selleck compound MAP and HR were continuously recorded during 1 h and were analysed at every 5 min. Baseline values were recorded for 10 min and were analysed immediately before

yohimbine or vehicle injection (first treatment). These values were used as reference to calculate the changes produced by the treatments. Immediately after vein and artery catheterization, an incision was made in ventral midline of the neck to localize the right submandibular/sublingual gland (SSG) complex and the artery that irrigates the SSG complex. The SSG artery, a cervical branch of external maxillary artery is usually larger just above the anterior margin of posterior belly of digastricus.6 and 10 The artery that irrigates the SSG complex was isolated and a miniature pulsed Doppler flow probe (Iowa Doppler Products;

Iowa City, IA) was perfectly adjusted around the artery to record blood flow. A midline laparotomy was also performed and miniature Phosphoprotein phosphatase pulsed Doppler flow probes were placed around the superior mesenteric (SM) artery and the low abdominal aorta for measurement of mesenteric and hindlimb blood flow, respectively. The probes were fixed to the surrounding tissues with suture thread. Data from animals in which the probes moved during the experiment were not considered for analysis. The flow probes were connected to a Doppler flowmeter (Dept of Bioengineering, University of Iowa, Iowa City, IA) coupled to a Powerlab computer record system (model Powerlab 16SP, ADInstruments) for blood flow recording. Details of the Doppler flow recording technique, including the reliability of the method for estimation of flow velocity, have been described previously.18 Relative SSG, mesenteric and hindlimb vascular resistance changes were calculated as the ratio of MAP and Doppler shift. Blood flow was continuously recorded for 1 h. Blood flow and vascular resistance were analysed for every 5 min. Baseline values were recorded for 10 min and were analysed immediately before yohimbine or vehicle injection (first injection). The values were used as reference to calculate the changes produced by the treatments.