Male obese Zucker rats and age-matched male lean Zucker rats were housed in plastic cages and fed normal rodent chow and tap water. Twelve obese Zucker rats (six to eight-weeks old) were randomly divided into two groups: 1) one group consisted of obese rats treated (6 weeks) with oral rosiglitazone maleate (10 mg/kg, suspended in 1% carboxy methyl cellulose in distilled water); and 2) the other group consisted of obese control rats treated with the vehicle (1% carboxy methyl cellulose). Eighteen lean Zucker rats (eight to ten-weeks old) were randomly divided into three groups: 1) control rats fed normal diet and treated with vehicle; 2) rats fed control diet and treated with STZ (single intraperitoneal injection, 65 mg/kg); and 3) rats fed high-fat diet (with 50% fat-derived calories) for one month.
All experiments were approved by the Daping Hospital Animal Use and Care Committee and all procedures were approved by the Experimental Animals Committee of Daping Hospital.
Blood pressure and plasma glucose and insulin measurements
After an overnight fast, obese and lean Zucker rats were anesthetized with pentobarbital (Nembutal) with an initial dose of 60 mg/kg, followed by constant infusion at 40 mg/kg/hr. Following a tracheotomy, the left carotid artery was catheterized with polyethylene-50 tubing for blood pressure monitoring. Blood pressure and heart rate were allowed to stabilize for 10 minutes before accepting the blood pressure and heart rate observed as baseline. The abdomen was opened to expose the abdominal aorta and blood samples were collected from the aorta in EDTA-coated tubes for measurement of plasma glucose and insulin, and then the rats were sacrificed by an overdose of pentobarbital (100 mg/kg body wt). Plasma glucose concentrations were determined by using Accu-Chek Advantage glucose monitoring system. Plasma insulin levels were measured by a rat insulin 96-well plate assay (Millipore Co., St Charles, MO).
Preparation and study of small resistance arteries
Each rat was anesthetized with sodium pentobarbital (60 mg/kg). The entire mesenteric bed was removed carefully and placed in ice-cold physiological salt solution (PSS) containing (mM):NaCl 119, KCl 4.7, CaCl2·H2O 2.5, MgSO4·H2O 1.17, NaH2CO3 25, KH2PO4 1.18, EDTA 0.027, and glucose 5.5, adjusted to pH 7.4. The mesenteric artery was carefully and quickly dissected from the surrounding fat and connective tissues. Third-order branches of the superior mesenteric artery (resting arterial diameter: 200 ± 20 μm) were cut into rings approximately 2 mm in length, and mounted on 40 μm stainless-steel wires in an isometric Mulvany-Halpern small-vessel myograph (model 91 M610, J.P. Trading, Science Park, Aarhus, Denmark). One wire was attached to a force transducer and the other to a micrometer so that wall tension can be measured at a predetermined internal diameter. The rings were maintained in PSS at 37°C and continuously bubbled with oxygen (95%) and carbon dioxide (5%) (Carbogen). The dissecting procedures were performed with extreme care to protect the endothelium from inadvertent damage, proved by a normal response to acetylcholine (Ach) (10-5 Ach-induced relaxation >80% of basal values of arteries preconstricted with phenylephrine HCl [PHE, 10-5M]). In some vessels, the endothelium was removed by pulling a hair along the inside of the vessel; successful denudation of the endothelium was confirmed by the absence of relaxation with Ach (10-5M). Following mounting, the arterial ring was equilibrated in PSS for 1 hr at 37°C at a wall tension of 0.1 mN/mm. Based on preliminary data from >100 vessels, we confirmed that a normalized circumference (L0) = 0.9 L100 resulted in maximal active force development. The vessels were studied at L0 in all subsequent protocols. After determining the response to Ach, as indicated above, the vessels were rinsed three times with fresh PSS and allowed to recover to baseline for 15 minutes. In the first set of experiments, the rings were contracted with phenylephrine HCl (10-5M) and high-potassium PSS (KPSS, 125 mM) to obtain the maximal response. After obtaining the maximal response to PHE (10-5M), the response curves to fenoldopam, a D1 receptor agonist[9, 10], were measured by a cumulative concentration-dependent protocol (10-9 to 10-5M). Response to every single concentration of fenoldopam was observed for 2 minutes. To test the vasorelaxant specific effects of D1 receptors, the arteries were incubated with the D1-like receptor antagonist SCH23390 (10-7M) for 30 minutes before fenoldopam treatment. In order to determine specificity of the vasodilatory effect of D1 receptor stimulation, we studied the vasodilation induced by sodium nitroprusside (SNP, 10-10 to 10-4M) in PHE-preconstricted mesenteric arteries.
Cell and sample preparation
Embryonic thoracic aortic smooth muscle cells from normotensive Berlin–Druckrey IX rats (A10, ATCC, Hercules, CA) were cultured at 37°C in 95% air/5% CO2 atmosphere in Dulbecco’s Modified Eagle’s Medium. A10 cells (80% confluence) and mesenteric arteries from Zucker rats were flash frozen by liquid nitrogen and homogenized in ice-cold lysis buffer (5 ml/gm tissue) (20 mM Tris-HCl, pH 7.4; 2 mM EDTA, pH 8.0; 2 mM EGTA; 100 mM NaCl; 10 μg/ml leupeptin; 10 μg/ml aprotinin; 2 mM phenylmethylsulfonyl fluoride; 1% NP-40), sonicated, kept on ice for 1 hr, and centrifuged at 16,000 g for 30 minutes. All samples were stored at -70°C until use.
After boiling the homogenates in sample buffer (35 mmol/L Tris-HCl, pH 6.8, 4% SDS, 9.3% dithiothreitol, 0.01% bromophenol blue, 30% glycerol) at 95°C for 5 min, 100 μg of protein were separated by SDS-PAGE (10% polyacrylamide), and then electroblotted onto nitrocellulose membranes (Bio-Rad). The blots were blocked overnight with 5% nonfat dry milk in phosphate buffered saline with Tween 20 (PBST) (0.05% Tween 20 in 10 mmol/l phosphate buffered (isotonic) saline) at 4°C with constant shaking, then incubated with polyclonal rabbit anti-rat D1 receptor antibodies (1:400 dilution; Millipore) overnight in the cold-room at 4°C. The membranes were then further incubated with infrared- labeled secondary antibodies (donkey anti-rabbit IRDye 800, Li-Cor Biosciences, Lincoln, NE) added to bind to the primary antibody at room temperature for 1 hr. The membranes were washed three times with PBST. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities.
Determination of basal D1 receptor phosphorylation by co-immunoprecipitation
Equal amounts of lysates (1.0 mg protein/ml supernatant from A10 cells or mesenteric artery) were incubated with polyclonal antiphosphoserine antibody (HKSP, New Territories, HK) (2.5 μg/ml) for 1 hr and protein-G agarose at 4°C for 12 hr. The immunoprecipitates were pelleted and washed four times with lysis buffer. The pellets were suspended in sample buffer, boiled for 10 min, and subjected to immunoblotting with polyclonal affinity-purified rabbit anti-rat D1 receptor antibody. In order to determine the specificity of the bands, normal rabbit IgG (negative control) and D1 receptor antibody (positive control) were used as the immunoprecipitants. The bound complexes were detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities.
Fenoldopam, rosiglitazone maleate, STZ, mitogen-activated protein kinase (MAPK) inhibitor PD98059, Ach, SNP, PHE, and D1 receptor antagonist SCH23390 were obtained from Sigma-Aldrich (St. Louis, MO). Rabbit anti-rat D1 receptor polyclonal antibodies and rat insulin 96 well plate assay kit were obtained from Millipore Corporation (St Charles, MO); and anti-phosphoserine antibodies were obtained from Abcam Ltd (New Territories, Hong Kong, China).
The data are expressed as mean ± SEM. Comparison within groups was made by repeated measures ANOVA (or paired t- test when only 2 groups were compared), and comparison among groups (or t-test when only 2 groups were compared) was made by factorial ANOVA with Holm-Sidak test. A value of P < 0.05 was considered significant.