, 1984). Full-thickness pieces of the descending colon were excised, shock-frozen in liquid nitrogen and stored at ?70��C until assay. After weighing, the http://www.selleckchem.com/products/azd9291.html frozen tissues were placed, at a ratio of 1 mg: 0.02 mL, in MPO lysis buffer. The composition of this buffer was 200 mmol?L?1 NaCl, 5 mmol?L?1 EDTA, 10 mmol?L?1 Tris, 10% glycine, 0.1 mmol?L?1 phenylmethylsulphonyl fluoride, 1 ��g?mL?1 leupeptide and 28 ��g?mL?1 aprotinin, pH 7.4. The samples were homogenised on ice with an Ultraturrax (IKA, Staufen, Germany) and then subjected to two centrifugations at 6000 ��g and 4��C for 15 min. The MPO (donor: H2O2 oxidoreductase, EC 1.11.1.7) content of the supernatants was measured with an enzyme-linked immunosorbent assay kit specific for the rat and mouse protein (Hycult Biotechnology, Uden, the Netherlands).

The sensitivity of this assay is 1 ng?mL?1 at an intra- and interassay variation of around 10%. Experimental protocols After the completion of surgery, the variables under study were monitored for up to 120 min when the cardiovascular parameters had become stable. Thereafter, baseline values were recorded and averaged during a period of 15 min. In study 1, baseline recordings were taken during the period of 25�C10 min before i.v. injection of vehicle (1 mL?kg?1), alosetron (0.03, 0.1 or 0.3 mg?kg?1), cilansetron (0.1 or 0.3 mg?kg?1), tegaserod (0.3 or 1 mg?kg?1) or L-NAME (0.02 mmol?kg?1). Post-injection recordings were made during the periods of 5�C20 min and 35�C50 min. Study 2 was performed to evaluate the effects of select drug doses in fasted and non-fasted rats over a prolonged period of time.

After the baseline values during the period of 25�C10 min pre injection were recorded, vehicle (1 mL?kg?1), alosetron (0.03 mg?kg?1), tegaserod (1 mg?kg?1) or L-NAME (0.02 mmol?kg?1) was injected i.v.; post-injection recordings were taken for a period of 140 min. In study 3, the drugs were administered i.d. via a soft infant feeding tube (outer diameter 1.5 mm; R��sch, Montevideo, Uruguay), which, during surgery, had been passed down through the oesophagus, stomach and pylorus so that its tip was positioned in the duodenum. After baseline recordings of the parameters under study had been made during the period of 15�C0 min pre injection, vehicle (1 mL?kg?1), alosetron (0.3 mg?kg?1), tegaserod (30 mg?kg?1) or clonidine (0.03 mg?kg?1) was administered i.

d. Post-injection recordings were taken for 90 min. Study 4 was carried out to investigate whether the splanchnic circulation at baseline and after injection of L-NAME is modified by short-term peroral pretreatment with alosetron or tegaserod for 7 days. Each day, the animals were given vehicle (10 mL?kg?1), alosetron (0.3 Batimastat mg?kg?1) or tegaserod (1 mg?kg?1) at 7.30�C8.00 am, 1.00�C1.30 pm and 6.00�C6.30 pm. The solutions were administered intragastrically (IG) through a soft infant feeding tube (outer diameter: 2.