Eight-week-old male rats (n = 25) were used for this study. All the rats were housed at 22 ± 2℃ with 40-60% humidity in a 12/12-h light/dark cycle, and they were maintained on a diet of animal chow and water ad libitum. All the animal studies were carried out in a semi-pathogen-free barrier zone at the Institute for Laboratory Animal Research in Seoul National University Hospital in accordance with the procedure outlined in the Guide for the Care and Use of Laboratory Animals.

The rats were randomly allocated into either the control group (n = 14) or the diabetic group (n = 11). Diabetes was induced by intravenous administration of a single dose of STZ (60 mg/kg in citrate buffer, pH 4.5) through the tail vein. In the normal group, the same volume of citrate buffer was injected intravenously. Three days after STZ-injection, the blood glucose levels were measured from a drop of blood using a OneTouch Ultra® Blood Glucose Meter (LifeScan, Inc) for determining the successful induction of diabetes. Rats with a blood glucose level > 300 mg/dl were considered diabetic. All the rats were maintained for 4 weeks before intervention.

The rats' blood glucose levels and body weight were measured four weeks after STZ or citrate buffer injection. For each rat, anesthesia was induced with sevoflurane with the rat in a chamber, and the anesthesia was maintained with 2 vol% sevoflurane. Following anesthesia, the rat was placed in a supine position and the four legs and upper front teeth were firmly fixed on a tilt board. The tail artery was cannulated with a 24 G catheter for continuous arterial pressure monitoring. To prevent the effect of postural change on measuring arterial pressure, the transducer was positioned at the level of the heart. After measuring the mean arterial pressure at the supine position (basal MAP), the table was vertically tilted at 45° angle in the head-up direction and the MAPs at 10, 30, 60 and 120 seconds after tilting were recorded. The changes in the MAP related to tilting were compared at each time point between the diabetic and control groups.

After the tilting experiment, the rat was euthanized by cervical dislocation under the deep anesthesia and the third branch of the mesenteric artery and the abdominal aorta were rapidly removed. The arteries were placed on a Petri dish with ice-cold modified Krebs' solution of the following composition (in mM): NaCl: 119, KCl: 4.7, MgSO₄: 1.17, NaHCO₃: 22, CaCl₂: 1.6, HEPES: 8, KH₂PO₄: 1.18, d-glucose: 5, and the arteries were cleaned of fat and connective tissues under a microscope (Zeiss). In addition, the Petri dish was kept cold by surrounding it with crushed ice. Following the cleaning procedure, to exclude the effect of endothelium-derived relaxing factor, the endothelium of the arteries was removed by gently rubbing the lumen with coarse silk and each artery was cut into 3-mm ring segments for the in vitro contractile studies.

Each ring segment was suspended between two triangle-shape tungsten hooks, and this was mounted in a 5 ml water-jacketed temperature-controlled tissue bath (Harvard Apparatus, Holliston, MA, USA) that contained modified Krebs' solution. The lower and upper hooks were attached, respectively, to the bottom of the bath chamber and to a force displacement transducer (NP 72-4493, Harvard Apparatus, Holliston, MA, USA). The tissue bathing solution was gassed with a mixture of 95% O₂ and 5% CO₂ (pH: 7.38-7.42, pCO2: 34-36 mmHg) and maintained at 37℃. Propranolol (1 × 10^-6 M) and yohimbine (1 × 10^-7 M) were always included in the tissue bathing solution to block the β- and α₂-adrenoceptors, respectively. The isometric contractions were recorded using an A/D converter (Powerlab®, ADInstruments, Colorado Springs, CO, USA) and they were analyzed by using Chart for Window Software® (ADInstruments, Colorado Springs, CO, USA).

Following applying an initial tension of 2.0 g, which provided the optimum concentration-response relationships to KCl 60 mM, the arterial strips were allowed to equilibrate for 60 min. When the resting tension reached a stable baseline, no attempt was made to adjust the tissue tension. After the equilibrium, tissues were primed by the addition of phenylephrine (1 × 10^-6 M) to the organ bath. After a steady-state contraction was achieved, the bath contents were replaced with drug-free buffer several times. The tissues were then allowed to reach baseline tension, and the priming procedure was repeated once more before constructing the concentration-response curves. This priming procedure reduces tissue variability in response to repeated agonist administration. In addition, the absence of functional endothelium was also confirmed by the loss of the relaxation response to acethylcholine (1 × 10^-6 M) in the pre-contracted rings with a priming dose of phenylephrine (1 × 10^-6 M) during the priming procedure.

Reproducible cumulative concentration-response curves were constructed by exposing each artery to increasing concentrations (0.5 log unit increments) of phenlyephrine (1 × 10^-9 to 1 × 10^-4) until a sustained maximum response (Emax) was observed. The concentration-response curves to phenylephrine were then obtained (60 min apart each) in the presence of four selective α₁-adrenoceptor antagonists to each artery and the procedure was repeated with a high concentration of antagonists in the same preparation. The incubation time for the selective α₁-adrenoceptor antagonists prazosin (α₁; 1, 3 and 10 × 10^-9 M), 5-methylurapidil (5-MU; α_1A; 3, 10 and 30 × 10^-8 M), and BMY 7378 (α_1D; 3, 10 and 30 × 10^-8 M) was 30 min. In all the experiments, one preparation was run in parallel with the experimental tissues, but it received no antagonist, and it was used to determine any time-dependent changes in agonist sensitivity. Propranolol (1 × 10^-6 M) and yohimbine (1 × 10^-7 M) were always included in the tissue bathing solution to block the β- and α₂-adrenoceptors, respectively.

After removing the vessels and cleaning the extraneous tissues, the arteries were cut into 2-mm rings, embedded in OCT compound (Tissue-Tek® OCT™ Compound, Sakura Finetek) and quickly frozen (-50℃ in isopentane cooled with dry ice). The frozen blocks were cut into 5-µm sections and mounted on the slide. All the sections were stored at -80℃ until use. After warming at room temperature for 30 minutes, the tissue sections were fixed in iced cold acetone for 5 minutes. Following rinsing the sections in PBS, a blocking solution with 10% normal donkey serum was placed on the sections and this was incubated for 30 minutes to block the non-specific binding sites of immunoglobulin. After removal of the blocking solution, the tissue section was incubated with the 1 : 100-diluted primary antibody (α_1D-AR cat. no. s.c.-1475, α_1B-AR cat. no. s.c.-1476, α_1A-AR cat. no. s.c.-1477; Santa Cruz Biotechnologies) for 60 minutes at room temperature. Following washing the primary antibody in PBS, fluoresceinisothiocyanate (FITC)-conjugated donkey anti-goat IgG (1 : 100 dilution, no. s.c.-2024; Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary antibody was placed on the sections for 60 minutes in the dark at room temperature. After removing the secondary antibody by washing with PBS, mounting media was placed and a glass coverslip was applied. For reducing the technical errors, the staining was performed in one slide for same type of artery from two different groups. Three independent investigators observed the expression of the α₁-adrenoceptor subtypes of each artery with using a fluorescence microscope (Olympus VENOX-AHBT3, Tokyo, Japan).

The data is presented as means ± SDs, means ± SEMs, number of rats or number of arterial segments. In each concentration-response curve, the changes in tension related to increasing concentrations of phenylephrine are expressed as the difference of tension (g) between the basal values and the measured value in a certain concentration of phenylephrine. The curves were fitted to all the data by non-linear regression analysis using GraphPad Prism software (GraphPAD software, San Diego, CA, USA) to calculate the negative logarithm of the concentration of agonist that provokes a half-maximal response (pEC₅₀ = agonist potency). The pA₂ values and Schild slopes were calculated by the method of Arunlakshana and Schild [16]. Using three different concentrations of each antagonist, the ratios between the half-maximal concentrations (EC₅₀) of phenylephrine obtained in a specific antagonist concentration and that obtained in its absence (the concentration ratio, R) were respectively calculated when the maximal amplitudes between the concentration-response curves in those two conditions were similar. The data was plotted as negative log-concentrations of the antagonist on the x-axis vs. log (R-1) on the y-axis and the data was fitted as a regression line. For calculation purposes, the slope parameter was set to -1.0 when it was statistically not different from unity. The x-intercept of the fitted regression line was the estimated pA₂. pA₂ is the estimated equilibrium dissociation constant for the specific antagonist and also the dose of antagonist that requires a 2-fold increase in the agonist concentration. Student's t-test was used to determine the statistical differences between the diabetic and control groups. A P value < 0.05 was considered statistically significant.

The initial body weight was 242.8 ± 18.9 g for the diabetic rats and 244.7 ± 14.8 g for the normal rats, respectively (P = 0.79). Four weeks after diabetic induction, the body weight was 242.3 ± 28.0 g and the blood glucose level was 481.7 ± 110.5 mg/dl in the diabetic rats. However, the body weight and blood glucose level in the normal rats were 428.5 ± 21.3 g and 133.1 ± 31.5 mg/dl, respectively. The diabetic rats showed a significantly higher blood glucose level and a lower body weight than that of the normal rats (all P values < 0.001).

After vertical tilting the table at 45° in the head-up direction, the recovery of the mean arterial pressure was significantly slower in the diabetic rats than in that in the normal rats (Fig. 1).

The diabetic rats showed a significantly higher Emax and pEC₅₀ than that of the normal rats on the phenylephrine-induced concentration-response curve of the abdominal aorta (all P values < 0.001, Table 1 and Fig. 2A). However, in the distal mesenteric arteries, the diabetic rats showed a lower Emax and pEC₅₀ than that of the normal rats (all P values < 0.001, Table 1 and Fig. 2B).

Regardless of the arterial type, prazocin, 5-MU and BMY 7378 shifted the concentration-response curve according to phenylephrine to the right direction for both the diabetic and normal rats (Fig. 3).

In addition, the pA₂ values of prazocin, 5-MU and BMY 7378 for inhibiting the contractile response to phenylephrine increased a half order of magnitude in the abdominal aorta from the diabetic rats, but this all decreased by a similar order of magnitude in the distal mesenteric arteries from the diabetic rats, respectively (all P < 0.001, Table 2).

The diabetic rats showed an increased fluorescent intensity of all the α₁-adrenoceptor subtypes in the abdominal aorta (Fig. 4A). However, in the distal mesenteric arteries, the fluorescent intensity of all the α₁-adrenoceptor subtypes was decreased in the diabetic rats (Fig. 4B).