Plant material and extraction
P. yedoensis bark was purchased from Dongwoodang Co., Ltd. (Yeongcheon, Kyungpook, Republic of Korea) in June 2007. Professor Hocheol Kim of Kyung Hee University identified the plants. A voucher specimen P. yedoensis bark (PY001) was deposited at the College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea. Dried P. yedoensis bark (3 kg) was extracted 3 times with 100% MeOH for 3 h in a reflux apparatus. After reflux and filtration, the extract was evaporated using a rotary vacuum evaporator (N-N series, EYELA, Japan) at 60°C and lyophilized to yield 386.8 g of crude extract. MEPY (1 g) was dissolved in dimethyl sulfoxide (DMSO; 10 ml).
Chemicals and drugs
Phenylephrine (PE), acetylcholine (ACh), N
ω-Nitro-L-arginine methyl ester (l-NAME), methylene blue (MB), atropine, indomethacin, ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), tetraethylammonium (TEA), glibenclamide, 4-aminopyridine (4-AP), 1-H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ), and DMSO were purchased from Sigma Aldrich (St. Louis, USA). All other reagents were of analytical purity.
Preparation of rat aortic rings
All procedures involving animals were conducted according to the animal welfare guidelines issued by National Veterinary Research & Quarantine Service and World Organization for Animal Health (OIE); and this study was approved (KHUASP(SE)-10–028) by the Kyung Hee University Institutional Animal Care and Use Committee. The rats were housed under controlled conditions (22 ± 2°C; lighting, 07:00–19:00), and food and water were available ad libitum. Sprague–Dawley rats (weight, 240–260 g; Narabio, Seoul, Korea) were anesthetized by exposure to ether, and the thoracic aorta was removed and immersed in Krebs-Henseleit solution [K-H solution, composition (mM): NaCl, 118.0; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; and glucose, 11.1; pH 7.4], maintained at 37°C, and aerated with a mixture of 95% O2 and 5% CO2. After connective tissue and fat were carefully removed, approximately 2-mm-long aortic rings were cut and suspended in organ chambers containing 10 ml K-H solution at 37°C. The rings in the chambers were aerated with a mixture of 95% O2 and 5% CO2. The aortic rings were placed between 2 tungsten stirrups and connected to an isometric force transducer (Grass Instrument Co., Rhode Island, USA). After incubation under no tension for 30 min, the vessel segments were allowed to equilibrate for 1 h at a resting tension of 1.0 g. The K-H solution was replaced every 15 min during the equilibration period. Changes in tension were recorded via isometric transducers connected to a data acquisition system (PowerLab, ADI instrument Co., New South Wales, Australia). When required, the endothelium was removed by gently rubbing the lumen of the vessel with a thin cotton swab. The presence of functional endothelium was verified by the ability of ACh (10 μM) to induce more than 80% relaxation in rings that were precontracted by PE (1 μM). In endothelium-denuded rings, ACh caused less than 10% relaxation. Ca2+-free K-H solution was prepared by replacing CaCl2 with EGTA (1 mM).
Vasoactivity
We studied the concentration-dependent relaxant effect of MEPY on endothelium-intact and endothelium-denuded aortic rings that were pre-contracted with PE (1 μM) in standard K-H solution. The relaxant effect of MEPY on the aortic rings was calculated as a percentage of the contraction in response to PE.
To study the effect of MEPY on nitric oxide (NO) synthesis pathway, endothelium-intact aortic rings were pre-incubated with l-NAME (10 μM) for 20 min before contraction by PE (1 μM) treatment. The relaxant effect of MEPY on the aortic rings was compared with the control (not treated with l-NAME).
To investigate the effect of MEPY on the NO-cyclic guanosine monophosphate (cGMP) pathway, endothelium-intact aortic rings were pre-incubated with ODQ (10 μM) or MB (10 μM) for 20 min before contraction by PE (1 μM) treatment. The relaxant effect of MEPY on the aortic rings was compared with the control (not treated with ODQ or MB).
To determine whether prostacyclin is involved in MEPY-induced vasorelaxation, endothelium-intact aortic rings were pre-incubated with indomethacin (1 μM) for 20 min before contraction by PE (1 μM) treatment. The relaxant effect of MEPY on the aortic rings was compared with the control (not treated with indomethacin).
To determine whether enhanced NO release by MEPY was associated with the activation of muscarinic receptors, endothelium-intact aortic rings were pre-incubated with atropine (1 μM) for 20 min before contraction by PE (1 μM) treatment. The relaxant effect of MEPY on the aortic rings was compared with the control (not treated with atropine).
To determine whether K+ channels are involved in MEPY-induced vasorelaxation, endothelium-intact aortic rings in standard K-H solution were pre-incubated with various K+ channel blockers, TEA (5 mM), glybenclamide (10 μM), or 4-AP (1 mM) for 20 min before the addition of PE (1 μM). Once a plateau was attained, MEPY (1–100 μg/ml) was cumulatively added. The vasorelaxant effect of MEPY on the aortic rings was calculated as a percentage of the contraction in response to PE.
To investigate the effect of MEPY on extracellular Ca2+-induced contractions, we carried out 2 sets of experiments: (1) evaluation of receptor-operative Ca2+ channels (ROCCs) and (2) evaluation of voltage-dependent Ca2+ channels (VDCCs). In the experiment on ROCCs, we investigated the contractile response induced by CaCl2 (0.3–10 mM) in the endothelium-denuded aortic rings contracted by PE (1 μM) in Ca2+-free K-H solution with and without (control) a 10-min preincubation with MEPY (200 μg/ml). The experiment on VDCCs followed the same procedure, except that the contraction was induced by KCl (60 mM). The contractile responses induced by CaCl2 in the presence and absence (control) of MEPY pre-treatment were compared.
Data analysis
Data are expressed as mean ± standard error of mean (SEM). Statistical comparisons were made using Student’s t-test or one-way analysis of variance (ANOVA) followed by the Tukey’s post-hoc test. All statistical analyses were performed using SPSS v.13.0 statistical analysis software (SPSS Inc., USA). P values less than 0.05 were considered statistically significant.