PART 1 Echinacoside Induces Rat Pulmonary Artery Vasorelaxation By Opening The NO-cGMP-PKG-BKCa Channels And Reducing Intracellular Ca2+ Levels

How does echinacoside induce venous relaxation?

Xiang-Yun GAI1, 2, 3, Yu-hai WEI4, Wei ZHANG2, Ta-na WUREN2, Ya-ping WANG2, Zhan-Qiang LI2, Shou LIU2, Lan MA2, Dian-Xiang LU2, Yi ZHOU1, 2, Ri-li GE1, 2, * 1 Department of Pharmacology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China; 2 Research Center for High Altitude Medicine, Medical College of Qinghai University, Xining 810001, China; 3 School of Pharmacy, Qinghai University for Nationalities, Xining 810007, China; 4 Qinghai Entry-Exit Inspection and Quarantine Bureau, Xining 810001, China

Aim: Sustained pulmonary vasoconstriction as experienced at high altitude can lead to pulmonary hypertension (PH). The main purpose of this study is to investigate the vasorelaxant effect of echinacoside (ECH), a phenylethanoid glycoside from the Tibetan herb Lagotis brevituba Maxim and Cistanche tubulosa, on the pulmonary artery and its potential mechanism.

Methods: Pulmonary arterial rings obtained from male Wistar rats were suspended in organ chambers filled with Krebs-Henseleit solution, and isometric tension was measured using a force transducer. Intracellular Ca2+ levels were measured in cultured rat pulmonary arterial smooth muscle cells (PASMCs) using Fluo 4-AM.

Results: ECH (30–300 μmol/L) relaxed rat pulmonary arteries precontracted by noradrenaline (NE) in a concentration-dependent manner, and this effect could be observed in both intact endothelium and endothelium-denuded rings, but with a significantly lower maximum response and a higher EC50 in endothelium-denuded rings. This effect was significantly blocked by L-NAME, TEA, and BaCl2. However, IMT, 4-AP, and Gli did not inhibit ECH-induced relaxation. Under extracellular Ca2+-free conditions, the maximum contraction was reduced to 24.54%±2.97% and 10.60%±2.07% in rings treated with 100 and 300 μmol/L of ECH, respectively. Under extracellular calcium influx conditions, the maximum contraction was reduced to 112.42%±7.30%, 100.29%±8.66%, and 74.74%±4.95% in rings treated with 30, 100, and 300 μmol/L of ECH, respectively. After cells were loaded with Fluo 4-AM, the mean fluorescence intensity was lower in cells treated with ECH (100 μmol/L) than with NE.

Conclusion: ECH suppresses NE-induced contraction of rat pulmonary artery via reducing intracellular Ca2+ levels, and induces its relaxation through the NO-cGMP pathway and opening of K+ channels (BKCa and KIR).

Keywords: Tibetan herb; echinacoside; pulmonary hypertension; high altitude; vasorelaxation; NO; BKCa; KIR; artery ring; pulmonary arterial smooth muscle cells.

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Introduction

Sustained pulmonary vasoconstriction as experienced at high altitude can lead to pulmonary hypertension (PH) and medial hypertrophy[1], which have been identified as the main cause of right ventricular hypertrophy and heart failure[2]. Individuals living at high altitude, either temporarily or permanently, may present mild to moderate pulmonary hypertension, and those with greater susceptibility to hypoxia may have exaggerated pulmonary vasoconstriction, leading to various high-altitude diseases that are associated with significant morbidity and mortality, such as high-altitude pulmonary edema and heart diseases[3]. Echinacoside (ECH) (Figure 1) is a phenylethanoid glycoside found in a variety of Chinese herbs such as the Tibetan herb Lagotis brevituba Maxim and Cistanche tubulosa[4, 5]. Lagos brevituba Maxim is a species of Lagotis spp belonging to the Scrophulariaceae and grows widely at an altitude over 3000 meters in the Qinghai-Tibet Plateau. ECH has various desirable pharmacological characteristics, such as antioxidative, anti-inflammatory, neuroprotective, hepatoprotective, and nitric oxide (NO) radical-scavenging properties[6]. It can also elicit endothelium-dependent relaxation in rat thoracic aortic rings and cure cardiovascular diseases[7]. However, to the best of our knowledge, there has not been a study investigating its effects on the vascular tone in pulmonary arteries. The purposes of this study are as follows: (1) to explore whether ECH induces in vitro vasorelaxation in rat pulmonary arteries precontracted with noradrenaline (NE), and whether this effect, if any, is endothelium-dependent or acts directly upon vascular smooth muscle; (2) to investigate the effect of ECH on the extracellular calcium influx and intracellular calcium release in rat pulmonary arterial smooth muscle cells (PASMCs); and (3) to identify possible pathways and K+ channels involved in ECH-induced relaxation.

Materials and methods

Reagents

ECH was kindly provided by Dr. Peng-fei TU of Peking University (Beijing, China), with a purity of more than 98% as determined by high-performance liquid chromatography. Dimethyl sulfoxide (DMSO) was purchased from Solar Science & Technology Co, Ltd (Beijing, China); NE, acetylcholine (ACh, ≥99%), indomethacin (IMT), Nω -Nitro-L-arginine methyl ester hydrochloride (L-NAME), tetraethylammonium chloride (TEA), barium chloride (BaCl2), 4-aminopyridine (4-AP), and glibenclamide (Gli) from Sigma Chemical Co (St Louis, MO, USA); high-glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin from Gibco BRL Co, Ltd (Gaithersburg, MD, USA); mouse anti-rat primary α smooth muscle actin (α-SMA) antibody and goat anti-mouse secondary antibody from Boshide Biotech Co, Ltd (Wuhan, China); Fluo Calcium Indicators (Fluo 4-AM) from Invitrogen Corp (Carlsbad, CA, USA); and all inorganic salts from Beijing Chemical Reagent Co, Ltd (Beijing, China). IMT was dissolved in DMSO and 5% NaH2PO4, Gli, Fluo 4-AM, and ECH were dissolved in DMSO, and all other reagents were dissolved in Krebs-Henseleit (KH) or PBS solution. Preliminary experiments showed that DMSO at less than 0.1% (v/v) had no effect on the tension development of isolated pulmonary arterial rings.

Experimental animals

All procedures and protocols were approved by the Animal Care and Use Committee of the Medical College of Qinghai University. Male Wistar rats, 6–8 weeks old, 250–300 g body weight, were purchased from the Animal Center of Lanzhou University (Lanzhou, China) and maintained on a standard laboratory diet and tap water ad libitum at an ambient temperature of 22±2°C and relative humidity of 45%–55% throughout the experiments.

In vitro pulmonary artery perfusion

Preparation of rat pulmonary arterial rings

Rats weighing 250–300 g were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg), and then their hearts and lungs were removed and immersed immediately in ice-cold KH solution containing (in mmol/L): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4·7H2O, 1.2 KH2PO4, 25 NaHCO3, and 11.1 glucose (pH 7.4). The intrapulmonary arteries (0.7–1.5 mm in diameter) were dissected free of surrounding connective tissue and adventitia and then cut into rings of approximately 2–3 mm in length. The rings were suspended in organ chambers filled with 10 mL of KH solution at 37 °C and gassed with 95% O2–5% CO2 [8], and isometric tension was measured using a force transducer (JH-2, Space Medico-Engineering Institute, Beijing, China)

Vessel ring activity assessment and endothelial function assessment

Vessel rings were allowed to equilibrate for 2 h under a basal tension of 400 mg, during which time the KH solution was changed every 15 min; then 1 μmol/L of NE was added into the chamber to assess the activity of each ring by detecting contraction percentage. Before and after the experimental protocol, the contraction percentage was measured (Figure 2A and 2B). Endothelium was removed in some rings by gently rubbing the intimal surface with a fine steel wire, and the integrity was assessed qualitatively by the degree of relaxation caused by ACh (10 μmol/L) in the percentage of contractile tone induced by NE (1 μmol/L). If the relaxation with ACh was greater than 80%, the endothelium remained in good condition; if the relaxation was less than 30%, the endothelium was destroyed (Figure 2A and 2B)[9].

Isolation and culture of rat PASMCs

Distal pulmonary arteries were isolated from pulmonary lobes of rats, and the endothelium and adventitia were removed carefully. After tissues were minced into 2-mm pieces, they were added to the flasks immediately, maintained at 37 °C and 5% CO2 in a humidified incubator for approximately four hours, and then cultured with high-glucose DMEM supplemented with 20% FBS, 100 U/mL of penicillin, and 100 U/mL of streptomycin for approximately five days. Half of the culture media was replaced with fresh media when cells emerged. When cells reached 80% confluence, they were harvested with 0.125% trypsin and seeded into the flasks (1:2 ratio) containing high-glucose DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 U/mL of streptomycin. Cells with three to eight passages were used for all experiments.

Identification of PASMCs by immunohistochemistry

PASMCs were positive for immunochemical staining when typical “hill and valley” features were observed. Cells were seeded in 6-well plates with glass coverslips. When cells reached 80% confluence, they were fixed in 4% paraformaldehyde, blocked with 3% hydrogen peroxide for 15 min, and then incubated with mouse anti-rat primary α-SMA antibody overnight at 4 °C. After being washed three times in PBS for 10 min, cells were incubated with goat anti-mouse secondary antibody at room temperature for 45 min, washed again three times in PBS for 10 min, and visualized with diaminobenzidine.

Detection of the intracellular calcium concentration in rat PASMCs

PASMCs were seeded in 6-well plates with glass coverslips. When cells reached 80% confluence, the culture media were replaced with serum-free DMEM. Twenty-four hours later, cells were loaded with 7 μmol/L Fluo 4-AM at 37 °C for 30 min in a humidified atmosphere of 5% CO2. The residual dye was washed with Hanks’ Balanced Salt Solution (HBSS) solution containing the following (in mmol/L): 1.26 CaCl2, 0.49 MgCl2·6H2O, 0.41 MgSO4·7H2O, 5.33 KCl, 0.44 KH2PO4, 4.17 NaHCO3, 137.93 NaCl, 0.34 Na2HPO4, and 5.56 D-Glucose, with no phenol red. Cells loaded with Fluo 4-AM were exposed to one of three treatment conditions: (1) the control group (con group), in which cells were incubated with serum-free DMEM; (2) the NE group, in which cells were incubated with serum-free DMEM supplemented with 1 μmol/L of NE for 10 min; and (3) the NE+ECH (100 μmol/L) group (NE+ECH100 group), in which cells were incubated with serum-free DMEM supplemented with 1 μmol/L of NE for 10 min and 100 μmol/L of ECH for 20 min. Fluorescence intensity was observed and photographed by fluorescence microscopy (IX71, Olympus, Tokyo, Japan)[11]. Six fields of vision (200×) were collected for each sample. Intracellular calcium concentration was quantified by measuring mean fluorescence intensity using Image Pro-Plus 6.0 software.

Statistical analysis

Data were expressed as the mean±SD. When an appropriate, significant difference was assessed by Dunnett’s test or the Student-Newman-Keuls test for multiple comparisons after one-way analysis of variance (ANOVA). A probability level of P<0.05 was considered significant.

Results Vasorelaxant effects of ECH on isolated pulmonary artery

At the beginning of the experiment, the cumulative addition of ECH from 30 to 300 μmol/L had no significant effect on the basal tension of the pulmonary artery. After the NE-induced vasoconstriction reached a plateau, ECH was added cumulatively (30–300 μmol/L) to the intact endothelium (E+, Figure 2C) or endothelium-denuded rings (E– , Figure 2C). In intact endothelium rings, cumulative addition of ECH dilated the vessels in a concentration-dependent manner, with a maxi-

mum relaxation percentage of 89.22%±0.32% at 300 μmol/L and an EC50 of 51.60±0.57 μmol/L (Figure 2A and 2C). Similarly, in endothelium-denuded rings, ECH also dilated the vessels in a concentration-dependent manner, but with a significantly lower maximum response of 67.72%±1.69% (P<0.01 vs intact endothelium rings) and a higher EC50 of 108.51±2.8 μmol/L (P<0.01 vs intact endothelium rings, Figure 2B and 2C). The sustained plateau of the high-contraction percentage induced by NE (1 μmol/L) after the experimental protocol indicated that the rings pretreated with ECH (30–300 μmol/L) had good activity (Figure 2A and 2B).

Effects of ECH on intracellular calcium release and extracellular calcium influx

The endothelium-denuded rings were exposed to 1 μmol/L of NE until the maximum contraction was attained. Under extracellular Ca2+-free conditions, the rings were equilibrated in Ca2+-free KH solution containing 0.2 mmol/L of EGTA before the initiation of the experiments. Following three washes with Ca2+- and EGTA-free KH solution, the rings were pre-incubated with ECH (30, 100, and 300 μmol/L) for 20 min and then re-exposed to 1 μmol/L of NE. The rings produced

unsustained contractions that rapidly returned to baseline, compared with the initial contractions induced by NE, which served as the reference. The maximum contraction of the control was 33.27%±2.94% (Figure 3A and 3E) but was significantly reduced to 24.54%±2.97% and 10.60%±2.07% in rings pretreated with 100 and 300 μmol/L of ECH, respectively (P<0.01 vs control group; Figure 3C–3E). Under extracellular calcium influx conditions, 2.5 mmol/L CaCl2 was added to the chamber when the curve reached a plateau. The control group produced sustained contractions, with a maximum contraction of 139.89%±7.38% (Figure 3A and 3E); but the maximum contraction was significantly reduced to 112.42%±7.30%, 100.29%±8.66%, and 74.74%±4.95% in rings pretreated with 30, 100, and 300 μmol/L of ECH, respectively (P<0.01 vs control group; Figure 3B–3E). Under these conditions, the maximal effect caused by ECH was more pronounced with 300 μmol/L than with 100 or 30 μmol/L (P<0.01 vs 30 or 100 μmol/L ECH group). Different concentrations of ECH all shortened the platform of sustained contraction, which was induced by extracellular calcium influx at different levels (Figure 3A–3D).

Roles of different inhibitors in ECH-induced rat pulmonary artery vasorelaxation

ECH-induced rat pulmonary artery vasorelaxation was investigated in the presence or absence of the following inhibitors: L-NAME (eNOS inhibitor), IMT (cyclooxygenase inhibitor), TEA (large-conductance Ca2+-activated K+ channel inhibitor), BaCl2 (inward rectifier K+ channel inhibitor), 4-AP (voltage-dependent K+ channel inhibitor), and Gli (ATP-sensitive K+ channel inhibitor). The rings were maximally contracted with 1 μmol/L of NE, and then L-NAME (100 μmol/L), IMT (10 μmol/L), TEA (1 mmol/L), BaCl2 (1 mmol/L), 4-AP (1 mmol/L), or Gli (10 μmol/L) were added, respectively. Once a new plateau was reached, ECH was added in a cumulative manner from 30 to 300 μmol/L. The concentration-response curves in the presence of different inhibitors are shown in Figure 4. The vasorelaxant effect of ECH on pulmonary arterial rings contracted with 1 μmol/L of NE was significantly blocked by L-NAME (100 μmol/L), TEA (1 mmol/L), and BaCl2 (1 mmol/L), with maximum relaxations of 64.41%±3.20%, 84.38%±0.70%, and 75.27%±0.93%, respectively (P<0.01 vs the control group of 89.22%±0.32%), and the values of EC50 were increased to 197.32±2.75 μmol/L, 91.42±2.11 μmol/L, and 115.49±1.75 μmol/L, respectively (P<0.01 vs the control group of 51.60±0.57 μmol/L) (Table 1). However, IMT (10 μmol/L), 4-AP (1 mmol/L), and Gli (10 μmol/L) did not inhibit the ECH-induced relaxation (Figure 4 and Table 1). To validate the effects of these inhibitors on ECH-induced relaxation, the rings precontracted with 1 μmol/L of NE were treated with L-NAME (100 μmol/L), IMT (10 μmol/L), TEA (1 mmol/L), BaCl2 (1 mmol/L), 4-AP (1 mmol/L), and Gli (10 μmol/L). After a new plateau was reached, ECH was added to the chamber at a single concentration of 300 μmol/L rather than in a cumulative manner. The maximum relaxation was reduced to 56.33%±0.90%, 82.21%±0.92%, and 72.16%±0.76% following the addition of L-NAME, TEA, and BaCl2, respected

Identification of rat PASMCs

At low magnification, spindle cells were densely packed, and almost all the cells were stained positively for PASMCs (the PASMC purity was >95%) (Figure 6A). Brown-stained myofilaments were clearly observed in the cytoplasm at high magnification (Figure 6B).

Cistanche echinacoside can resist apoptosis

Effect of ECH on the intracellular calcium concentration in rat PASMCs

NE (1 μmol/L) significantly increased the intracellular calcium concentration (n=6, P<0.01 vs the control group) in rat PASMCs, and ECH could decrease the mean fluorescence intensity (n=6, P<0.01 vs the NE group) (Figure 7)