All data are presented as means S.E.M. EET-A reduced kidney ENaC protein expression in AngII hypertension and inhibited the ENaC current and 0.05 compared with SD+vehicle and #0.05 compared with AngII+vehicle; = 6. (SD) rats (225C275 g) and mice lacking cytochrome P450 (Cyp) 2c44 (Cyp2c44?/? mice, 20C25 g). Animal protocols were in accordance with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee. Throughout the experiment, animals were housed under conditions of constant temperature and humidity, with a 12/12 h lightCdark cycle. Animals were allowed to adapt to these conditions for several days before starting any experimental procedures. experiments Telemetry blood pressure measurement In order to measure blood pressure, telemetry transmitters (Data Sciences) were implanted 14 days before the experiment in rats and mice using methods described previously [21,24]. Baseline arterial pressure and heart rate were recorded for 3C5 days before the experimental period. Mean arterial pressure (MAP) or systolic blood pressure and heart rate were recorded continuously throughout the experimental period. Antihypertensive effects of intraperitoneally administered EET analogues in SHRs and AngII-hypertensive rats In the first set of experiments, telemetry transmitters were implanted into male SHRs. After the surgical recovery period, baseline MAP was recorded for 14 days. In this set of experiments, EET analogues (EET-A, EET-X, EET-Y and EET-Z) were administered intraperitoneally (i.p.) continuously using ALZET? osmotic pumps (DURECT) at a dose of 10 mg/kg per day, and blood pressure was monitored constantly. The vehicle-treated rats were administered a solution made up of DMSO, ethanol and PEG-400 (40 %, 15 % and 45 % respectively) for 14 days using ALZET? osmotic pumps. In the second set of experiments, telemetry transmitters were implanted into male SD rats. After 1 week of basal blood pressure recording, ALZET? osmotic pumps were implanted subcutaneously (s.c.) to deliver AngII at a dose of 180 ng/kg per min for 14 days. Around the first day of AngII pump implantation, the EET analogues, EET-A and EET-X were administered (i.p.) using ALZET? osmotic pumps constantly at a dose of 10 mg/kg per day for 14 days and blood pressure was monitored. The vehicle-treated rats were administered either distilled water or a solution made up of DMSO, ethanol and PEG-400 (40 %, 15 % and 45 % respectively) for AZD1981 14 days using AZD1981 ALZET? osmotic pumps. In an additional set of experiments, we implanted osmotic pumps filled with EET-A (10 mg/kg per day) or vehicle-containing DMSO, ethanol and PEG-400 (40 %, 15 % and 45 % respectively) in SD rats to determine the effect of EET-A on their blood pressure. Antihypertensive effects of orally administered EET analogues in AngII-hypertensive rats This experiment was carried out in a set of SD rats implanted with radiotransmitters for continuous monitoring of blood pressure and AngII-filled ALZET? osmotic pumps (s.c.) to deliver AngII at a dosage of 180 ng/kg per minute for 14 days. The rats were treated with EET analogues (10 mg/kg per day), EET-A and EET-X given in drinking water during the 28-day treatment period. Blood pressure was monitored constantly and 24-h urine samples were collected at the end of the treatment period for the measurement of electrolytes and biochemical assays. Biochemical measurements Urinary electrolytes were measured using ion-selective electrode (ISE)-based method (EasyLyte Analyzer, Medica Corporation). sEHi activity was decided using a kit Rabbit Polyclonal to PLAGL1 from Cayman Chemical. experiments Vascular reactivity studies Three sets of vascular experiments were carried out. In the first set, measurements of isometric tone in bovine coronary artery rings were conducted as described previously [18,25]. The arterial rings were slowly stretched to a basal tension of 3.5 g and equilibrated for 1.5 h. KCl (40C60 mM) was repeatedly added and rinsed until reproducible stable contractions were observed. The thromboxane mimetic 9,11-dideoxy-11(U46619; 20 nM) was added to increase basal tension to approximately 50C75 % of maximal KCl contraction. Relaxation responses to cumulative additions of the EET analogues (10?9C10?5 M) were recorded. Basal tension represents tension before the addition of U46619. Results are expressed as the percentage relaxation of the U46619-treated rings; 100 % relaxation represents basal tension. In the second set of vascular experiments, second-order mesenteric arteries were excised from vehicle, EET-A and EET-X-treated AngII-hypertensive rats on day 14 of the experimental protocol to determine the effects of EET analogues around the acetylcholine vasodilator response. The third set of vascular experiments were also carried out with second-order mesenteric arteries of AngII-hypertensive rats treated with EET-A for 14 days, and acetylcholine vascular responses were studied. However, this set of experiments was carried out in the presence and absence of L-is mean total current in a patch. Immunohistochemical analysis The kidney sections were embedded and cut into 4-test (and among groups it was AZD1981 determined by repeated-measure one-way ANOVA followed by.