A. kept under diurnal light conditions and experienced free access to food and water. All animal experiments were authorized by the University or college of Southern California Keck School of Medicine Institutional Animal Care and Use Committee and carried out in accordance with the National Institutes of Health = 5); = 5); and a subset 5-min acute systolic hypertension (= 3). Mean arterial pressure was improved 50C60 mmHg over baseline by constricting the superior mesenteric artery, celiac artery, and abdominal aorta below the renal artery as explained (22). Kidneys were cooled in situ by flushing with chilly PBS and then excised for homogenization and subcellular fractionation as explained below. For immuno-EM analysis, two rats were compared on the same day as explained by Yang et al. (24). In brief, after 20-min acute Rhod-2 AM hypertension or sham operation (= 5 each), kidneys were fixed via perfusion fixation or superfusion of the kidney surface, and blood pressure was monitored continually during fixation. Kidneys were processed as explained below. Series 2: lipid website properties of NCC at basal and elevated blood pressure. In the second series, rats were anesthetized intraperitoneally with Inactin (100 mg/kg) plus a small dose of intramuscular ketamine (100 mg/kg) and sham managed, or blood pressure was elevated for 15 min as explained for (= 5 pairs). Fifteen-minute acute hypertension provoked a Rhod-2 AM significant redistribution of NCC to higher denseness fractions analogous to that seen in Fig. 1(not demonstrated). Kidneys were cooled in situ by flushing with chilly PBS and then excised for homogenization and fractionation on OptiPrep flotation gradients as explained below. Open in a separate windows Fig. 1. hJAL Effects of acutely raising blood pressure (BP) on denseness distribution of NaCl cotransporter (NCC). = 5 in control and 30-min organizations; = 3 in 5-min group. Ideals are means SE. * 0.05 compared with corresponding control, unpaired Student’s (for 20 min); and = 3 in each group. One rat from each group was analyzed on the same day time, and samples were processed and assayed in parallel. Homogenization and subcellular fractionation on sorbitol gradients. The procedure for subcellular fractionation of the renal cortex membranes has been described in detail previously (22) and was identical Rhod-2 AM in all three series. In brief, renal cortices were rapidly dissected and homogenized in isolation buffer (5% sorbitol, 0.5 mM disodium EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 9 g/ml aprotinin, and 5 mM histidine-imidazole buffer, pH 7.5) and centrifuged at 2,000 for 10 min. The pellet was homogenized and centrifuged again, and the low-speed supernatants (So) were pooled, loaded in the interface between two hyperbolic sorbitol gradients (ranging between 35 and 70% sorbitol), and centrifuged inside a swinging bucket rotor (100,000 for 5 h). Twelve fractions were collected from the top, diluted with isolation buffer, pelleted by centrifugation (250,000 for 1.5 h), resuspended in 1 ml of isolation buffer, and stored in aliquots at ?80C, pending assays. Isolation of lipid raft fractions by flotation gradient. So membranes were isolated as explained above and then centrifuged at 200,000 for 75 min and the pellets resuspended in TNE buffer [50 mM TrisHCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and Complete Protease Inhibitor Cocktail (Roche Diagnostics)] through a 25-G needle on snow. Lipid raft fractions were isolated using a changes of methods previously reported (8, 12). Total membranes were incubated in TNE buffer comprising 1% Triton X-100 on snow for 30 min with occasional vortexing. Proteins in ordered lipid domains are resistant to detergent extraction and are more buoyant than proteins in nonordered domains that are extracted from the detergent. These populations can be separated on denseness gradients. The draw out was modified to 50% OptiPrep (Sigma). A volume of 600 l was loaded Rhod-2 AM at the bottom of an ultracentrifuge tube and overlaid with 1 ml each of 40, 30, and 20% and 400 l of 10% OptiPrep in TNE. Following centrifugation at 170,000 at 4C for 4 h, a continuous denseness gradient was created. Six 250-l fractions were collected from the top followed by collection of Rhod-2 AM five 500-l fractions. Immunoblot analysis and antibodies..