Male Sprague Dawley rats weighing 250-350 g each were anesthetized by intraperitoneal administration of pentobarbital sodium (50 mg/kg). The aortic rings for tension measurement were prepared according to the method described in the previous studies.18,19 The descending thoracic aorta was dissected free, and surrounding connective tissues and fat were removed under microscopic guidance while aorta was bathed in Krebs solution composed of following components: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 2.4 mM CaCl₂, 25 mM NaHCO₃, 11 mM glucose, and 0.03 mM EDTA. The aorta was then cut into 2.5-mm rings and suspended on Grass isometric transducers (FT-03, Grass Instrument, Quincy, MA, USA) under a 3.0-g resting tension in a 10-mL Krebs bath at 37℃, aerated continuously with 95% O₂ and 5% CO₂ to maintain pH values within the 7.35-7.45 range. The rings were equilibrated at a 3.0-g resting tension for 120 min, during which time bathing solution was changed every 30 min. In all aortic rings, the endothelium was intentionally removed from the aortic rings by inserting a 25-gauge needle into the lumen of the rings and gently rubbing the ring for a few seconds. When the contraction in response to phenylephrine (10^-7 M) had stabilized, endothelial removal was confirmed by the absence of a relaxation response to acetylcholine (10^-5 M). The contractile response evoked by isotonic 30 mM KCl was measured for all aortic rings and used as a reference value. The isotonic 30 mM KCl solution was prepared by replacing the NaCl in the Krebs solution with an equimolar amount of KCl. After washing out the KCl from the organ bath and allowing the isometric tension to return to baseline, a cumulative concentration-response curve to either dexmedetomidine or levomedetomidine was obtained as described below. A single ring was used for each concentration-response curve. The cyclooxygenase inhibitor indomethacin (10^-5 M) and the nitric oxide synthase inhibitor N^W-nitro-L-arginine methyl ester (L-NAME, 10^-5 M) were also included in the Krebs solution to prevent the release of endogenous prostaglandins and nitric oxide, respectively, from any residual endothelium.

The first series of experiments assessed the cumulative concentration-response curves to the medetomidine enantiomers dexmedetomidine or levomedetomidine (10^-9 to 10^-6 M) in the endothelium-denuded rings. A subsequent concentration of dexmedetomidine was added after the previous concentration elicited a sustained and stable contraction for 5 min.

The second series of experiments was designed to determine which isoform of MAPK is functionally important in mediating dexmedetomidine-induced contraction. The effect of MAPK inhibitors (ERK inhibitor: 10^-5 M PD 98059, p38 MAPK inhibitor: 10^-6 and 10^-5 M SB 203580, JNK inhibitor: 10^-6, 3×10^-6, and 10^-5 M SP 600125) on the dexmedetomidine concentration-response curve was assessed by comparing each dexmedetomidine-induced contraction in the presence or absence of each MAPK inhibitor. The incubation period for each MAPK inhibitor was 20 min prior to addition of dexmedetomidine. Inhibitor concentrations were chosen based on the reported 50% inhibitory concentration.12,20

The third series of experiments was designed to examine the effect of dexmedetomidine on the voltage-operated calcium channel-induced contraction. The initial baseline response induced by isotonic 30 mM KCl was recorded in the endothelium-denuded aorta. After washing out KCl from the organ bath and allowing it to return to the baseline resting tension, the second isotonic 30 mM KCl-induced contraction in the same aorta was determined by adding 30 mM KCl solution containing dexmedetomidine (0, 3×10^-8, 10^-7, 3×10^-7, or 10^-6 M) to the organ bath.

Finally, the effects of the L-type calcium channel blocker (10^-5 M verapamil and 5×10^-6 M nifedipine) and the α₂-adrenoceptor antagonist atipamezole (10^-4 M) on the dexmedetomidine concentration-response curve were assessed by comparing cumulative dexmedetomidine concentration-response curves in the presence and absence of each inhibitor. Each inhibitor was added to the organ bath 20 min prior to the addition of dexmedetomidine.

The cells were isolated from the rat thoracic aorta by enzymatic dissociation and grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were subcultured twice a week by harvesting with trypsin/EDTA and seeding in 7.5×10⁵ /mm² flasks. For experiments, the cells between passage numbers 2 and 10 were seeded into dishes (2 cells/mm²), fed every other day, and used at confluence (6-7 days). The cells were deprived of serum overnight prior to treatment.

Western blot analysis was performed according to the method described in the previous study.21 In brief, the cells were lysed in PRO-PREP protein extract solution for total cell lysates, and the lysates were centrifuged at 100,000×g for 20 min at 4℃. Protein concentration was determined using the Bradford method. For preparation of sample loading, equal volumes of 2×sodium dodecyl sulfate (SDS) sample buffer (0.1 mol/L Tris-HCI, 20% glycerol, 4% SDS, and 0.01% bromophenol blue) and supernatant fractions from the lysates were mixed. Proteins (60 microgram) were separated by 10% SDS-polyacrylamide gel electrophoresis for 90 min at 110 V. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes for 2 h at 20 mA using SD Semi-dry Transfer Cells (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the membranes using 5% nonfat milk in Tris-buffed saline (TBS, pH 7.0), the membranes were incubated overnight at 4℃ with specific antibodies at a dilution of 1 : 500 in 5% skim milk in TBS-T. Bound antibody was detected by horseradish peroxidase (HRP)-conjugated anti-goat or anti-rabbit IgG. The membranes were washed and developed using a Western Blotting Luminol Reagent System (iNtRON Biotechnology, Houston, TX, USA) and autoradiography.

All drugs were of the highest purity available commercially: acetylcholine, L-NAME, indomethacin, verapamil, nifedipine, PD 98059, SB 203580, and SP 600125 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dexmedetomidine, levomedetomidine, and atipamezole were kind gifts from Orion Pharma (Turku, Finland). All concentrations are expressed as the final molar concentration in the organ bath. DMEM, FBS, penicillin, streptomycin, trypsin/EDTA, and glutamine were supplied by Gibco BRL (Rockville, MD, USA). PRO-PREP protein extract solution and electrochemiluminescence (ECL) Western blotting detection reagents were supplied by iNtRON Biotechnology (Houston, TX, USA). Anti-phospho-ERK and anti-ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-JNK, anti-JNK, anti-phospho-p38 MAPK, and anti-p38 MAPK antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Nifedipine, PD 98059, SB 203580, and SP 600125 were dissolved in dimethyl sulfoxide (DMSO) (final organ bath concentration: 0.1% DMSO) as was indomethacin. Unless stated otherwise, all other drugs were dissolved and diluted in distilled water.

Values are expressed as the mean±SD of n rats from which descending thoracic aortic rings were obtained. Contractile responses to dexmedetomidine and levomedetomidine are expressed as a percentage of the maximum contraction to 30 mM isotonic KCl. Maximal contraction was defined as the contractile response to 10^-6 M dexmedetomidine. Statistical analysis for the comparison of maximal contractions between the no-drug and inhibitor-treated groups was performed using the Student's t-test for paired samples or a one-way analysis of variance (ANOVA) by Tukey's multiple comparison test. Band intensities from the Western blot analyses were assessed by scanning densitometry, and statistical analysis was performed using the Mann-Whitney U test. Scanning densitometry was performed using an Image Master® VSD (Pharmacia Biotech Inc., San Francisco, CA, USA). The effect of dexmedetomidine on the 30 mM KCl-induced contraction was analyzed by an unpaired Student's t-test. Responses to each concentration of dexmedetomidine were compared using repeated measures ANOVA followed by a Tukey's multiple comparison test. A p value of less than 0.05 was considered statistically significant.