For this procedure, INK 128 mouse on the

day before the measurement, a catheter that was filled with saline (PE-50) was inserted into the left femoral artery while the subject was under anesthesia (ketamine 70 mg/kg, xylazine 10 mg/kg). The free end of the catheter was exteriorized at the cervical dorsal area. For the BP measurement, the arterial catheter was attached to a 40-cm polyethylene catheter during the 40-min recording period in quiet, conscious rats, allowing the rats’ complete freedom of movement in the cage. The BP was recorded by a pressure transducer coupled to a MP-100 System Guide (model MP100-CE; Biopac Systems, Santa Barbara, CA, USA). The HR was calculated instantaneously from the intervals of pressure pulses. After the measurement of BP and HR, the rats were decapitated and 5 ml of blood was collected in pre-chilled tubes containing heparin sulfate and protease inhibitors: 10−5 mol/l ethylenediaminetetraacetic acid (EDTA), 10−5 mol/l phenylmethylsulphonyl fluoride (PMSF), and 0.5 × 10−5 mol/l pepstatin A. The blood was centrifuged at 4 °C and 2500 rpm (Eppendorf, Hamburg, Germany) for 15 min. The plasma was stored at −80 °C. The right and left atrial appendages, kidneys and mesenteric adipose Dabrafenib tissue were removed, frozen in liquid nitrogen and stored at −80 °C. The dosages of ANP were performed by a double-antibody radioimmunoassay (RIA) as described by Gutkowska et al. [13].

The plasma was thawed, centrifuged for 5 min at 19,400 × g and 4 °C, and the ANP was extracted using Sep-Pak C18 columns (Waters Associates, Milford, MA, USA). The columns were activated with 8 ml of acetonitrile and washed with 8 ml of 0.2% ammonium acetate, pH 4.0. Afterward, 1 ml of plasma was infused into the column

followed by 5 ml of 0.2% ammonium acetate. Finally, the absorbed ANP was eluted with 3 ml of 60% acetonitrile in 0.2% ammonium acetate, evaporated (Speed-Vac, Eppendorf, Hamburg, Germany) and stored at −20 °C for quantification by RIA. To measure the ANP tissue concentrations, each half of the right (RA) and left atria (LA) was thawed and placed in a tube that was filled with 0.1 M acetic Metformin datasheet acid and protease inhibitors (10−5 M EDTA, 10−5 M PMSF and 0.5 × 10−5 M pepstatin A, all purchased from Sigma). The samples were then homogenized and centrifuged at 20,000 × g for 30 min at 4 °C, and the supernatant was diluted (final dilution: 1:2000) in phosphate buffer (0.01 mol/l sodium phosphate, 0.14 mmol/l bovine serum albumin, 0.1% Triton X-100, 0.1 mol/l NaCl and 0.01% sodium azide at pH 7.4) for ANP dosage. The ANP was measured by RIA as was previously described by Gutkowska et al. [13] using a specific antibody that was donated by Jolanta Gutkowska. All of the samples were measured in the same assay, and the intra-assay coefficient of variation was <10%. The protein content of the tissue was determined using the Bradford method [3].