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Hydroxysafflor yellow A actives BKCa channels and inhibits L-type Ca channels to induce vascular relaxation
Journal Pre-proof Hydroxysafflor yellow A actives BKCa channels and inhibits L-type Ca channels to induce vascular relaxation Na Wang, Dongmei He, Yuanqun Zhou, Jing Wen, Xiaoqin Liu, Pengyun Li, Yan Yang, Jun Cheng PII:
S0014-2999(19)30825-8
DOI:
https://doi.org/10.1016/j.ejphar.2019.172873
Reference:
EJP 172873
To appear in:
European Journal of Pharmacology
Received Date: 4 July 2019 Revised Date:
10 December 2019
Accepted Date: 16 December 2019
Please cite this article as: Wang, N., He, D., Zhou, Y., Wen, J., Liu, X., Li, P., Yang, Y., Cheng, J., Hydroxysafflor yellow A actives BKCa channels and inhibits L-type Ca channels to induce vascular relaxation, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2019.172873. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Na Wang: Data curation, Investigation, Writing- Original draft preparation, Funding acquisition Jun Cheng: Conceptualization, Methodology, Investigation , Funding acquisition Dongmei He: Data curation, Methodology, Investigation Yuanqun Zhou, Jing Wen, and Xiaoqin Liu: Methodology Pengyun Li: Methodology, Formal analysis Yan Yang: Writing- Reviewing and Editing, Funding acquisition, Project administration
Hydroxysafflor yellow A actives BKCa channels and inhibits L-type Ca channels to induce vascular relaxation Na Wang , Dongmei He , Yuanqun Zhou, Jing Wen, Xiaoqin Liu, Pengyun Li, Yan *
*
Yang, Jun Cheng Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Laboratory of Sichuan Province, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, Sichuan, China. *Equally contributed Corresponding author: Jun Cheng, [email protected] Co- Corresponding author: Yan Yang, [email protected]
Abstract Hydroxy-safflor yellow A (HSYA) can exert a variety of effects upon the vascular system. However, the underlying mechanisms are not clear. The present study is to investigate its vasodilating effect and the mechanisms. Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were enrolled for studying effects of HSYA on blood pressure, vasodilation, intracellular Ca2+ transient and membrane ion channels. Vasodilation and intracellular Ca2+ transient were measured by using vasomotor assay and fluorescence imaging system, respectively. The effect of HSYA on the large conductance Ca2+ activated and voltage-gated potassium channel (BKCa channel) currents in rat mesentery artery and on L-type calcium channel (Ca-L) currents in HEK293cells expressed with Ca-L were investigated using patch clamp techniques. Blood pressure of SHR and WKY rats were concentration dependently reduced by HSYA with a larger effect of HSYA in SHR than that in WKY rats. The tension of mesenteric arteries induced by 3 µM phenylephrine was attenuated by HSYA (IC50=90.8 µΜ). Patch clamp study showed that HSYA could activate BKCa channels and suppress Ca-L channels in a concentration dependent manner. The results of calcium signaling assays indicated that HSYA could reduce the intracellular free Ca2+ level. These findings demonstrate that HSYA could activate BKCa channels and inhibit Ca-L channels and reduce intracellular free Ca2+ level, which are probably important for its vasodilatory effect.
Key Words: Hydroxysafflor yellow A, BKCa, L-type Ca channel, vasodilatation, smooth muscle cells
1. Introduction Hydroxy-safflor yellow A (HSYA), a water soluble monomer and the major bioactive compound extracted from safflower, the extensively used traditional Chinese herbal medicine, can exert a variety of effects upon the vascular system (Yuan et al., 2014). Preliminary studies indicated that HSYA had beneficial effects on hypertensive ventricular remodeling, which may involve mechanisms of inhibiting cell apoptosis and suppressing metalloproteinases expression (Wang et al., 2014). In addition, HSYA had protective effect on bleomycin-induced lung inflammatory response (Wu et al., 2012), the UV-induced skin damage (Kong et al., 2013), and 6-hydroxydopamine-induced Parkinson's disease in rats (Han et al., 2013). In addition to those effects, HSYA displayed vascular relaxation effects on rat pulmonary artery (PA) by activating voltage-gated potassium channels (KV) in pulmonary vascular smooth muscle cells and the effects were endothelium-independent (Bai et al., 2012). Another research indicated that HSYA could significantly reduce blood pressure and heart rate. The author speculated that its effects may be related to activation of the large conductance Ca2+ activated and voltage-gated potassium channel (BKCa) and ATP-sensitive potassium channels (KATP), because BKCa and KATP blocker, but not KV and acetylcholine-activated potassium channel (KAch) blocker, weakened the inhibitory effect of HSYA on heart function and heart rate (Nie et al., 2012). However, the direct effects of HSYA on those ion channel currents have not been assessed. The mechanisms underlying the blood pressure depressing effect of HSYA remain to unveiled. BKCa channel is one of the important potassium channels ubiquitously expressed in blood vessels, especially in endothelial and vascular smooth muscle cells (Contreras et al., 2013). The depolarization of membrane voltage and intracellular local Ca2+ release events through ryanodine receptors (Ca2+ sparks) from the sarcoplasmic reticulum are two key factors to activate BKCa channels (Yang et al., 2013; Yang et al., 2013). When smooth muscle cells contract, BKCa channel will be activated by membrane depolarization and Ca2+-release. Once BKCa channels are activated, the k+ efflux and the cell membrane hyperpolarization lead to the reduced
excitability of the vessels and vasorelaxation (Modgil et al., 2013). Thus BKCa channels exert negative feedback regulation mechanism on the vascular tone and blood flow. L-type calcium channels (Ca-L), the main source of calcium entry pathways, also play a key role in vascular smooth muscle. They are crucial elements in determining vascular tone and other numerous physiological processes. The activation of them causes vasoconstriction. On the contrary, the inhibition of Ca-L can cause vasodilation (Hell et al., 2017; Li et al., 2013). Therefore, both BKCa and Ca-L are attractive targets for discovering drugs clinically relevant to hypertension and other diseases of vascular system (Joseph et al., 2013; Xu et al., 2006). Hence, the present study was designed to reveal the direct vascular effects and the mechanism of HSYA by using vasomotor assay, patch-clamp recordings and calcium signaling assays. The study may help understand vasodilatation effect of HSYA and provide experimental basis for clinical application of HSYA for hypertension and other cardiovascular diseases. 2. Materials and Methods 2.1. Animals and drugs Sixteen-week old spontaneously hypertensive rats (SHR) and their age-matched Wistar-Kyoto (WKY) rats with body weight of 320-380 g were bought from Beijing Weitong Lihua Animal Technology Co. Ltd. (license No. SCXK (Jing) 2016-0011). This study was approved by the Committee on the Ethics of Animal Experiments of Southwest Medical University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996). HSYA was purchased from Bioproduct Research Institute of Chengdu, China (purity>98%) was dissolved in water to make a 10 mM stock solution and added to the bath solution to the desired final concentrations immediately before each experiment. The chemical structure of HSYA (Meselhy et al., 1993) is shown in Fig. 1. Phenylalanine (PE), acetylcholine, tetraethylammonium (TEA), iberiotoxin (IbTX) and nifedipine were purchased from Sigma-Aldrich Inc. (St. Louis, MO, United
States). Fura-2 AM (5-Oxazolecarboxylic acid, 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2-(bis(2((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, (acetyloxy)methyl ester) was purchased from Invitrogen Inc. (San Diego, CA, United States).
Fig.1 The chemical structure of HSYA (C27H32O16). Hydroxysafflor yellow A is a compound with a single chalcone glycoside structure. Molecular weight: 612.53 2.2. Measurement of blood pressure and heart rate of SHR and WKY rats Sixteen-week old adult male rats were enrolled in a blood pressure study under general anesthesia (n=5 each group). Rats were anesthetized with pentobarbital sodium (50 mg/kg). Before blood pressure monitoring, rats underwent surgery for catheterization of the femoral artery. After cutting the skin layer and exposing the femoral artery, a catheter was advanced into the lumen of the femoral artery. Arterial pressure was recorded for 5 min after catheterization. Multi-channel physiological signal acquisition and processing system (RM6240BD) was used (Chengdu Instrument Factory, Sichuan, China) to record changes in blood pressure and heart rate. 2.3. Separation of WKY rat mesenteric artery rings and vasomotor assay Under general anesthesia, the branches of the superior mesenteric arteries were rapidly isolated and placed in solution I containing (in mM): NaCl 127, KCl 5.9, CaCl2 2.4, MgCl2 1.2, glucose 1.2, HEPES 10 (pH adjusted to 7.4 with NaOH). The arteries were sectioned into four sections (2–3 mm width and length) for the tension measurements after cleared of periadvential fat. The endothelial layer was removed mechanically in half of the rings by gently rubbing the intimal surface of the artery lumen with a matchstick. All rings were then mounted in organ baths (AD Instruments,
DMT, Oxford, UK) which were filled with warmed (37°C) and gas-equilibrated (95% O2, 5% CO2) solution I. Removal of the endothelium was confirmed by the absence of relaxation induced by acetylcholine (10 µM). Firstly we applied 60 mM KCl to determine the activity of arteries. Only the arteries with similar contractility after incubation with 60 mM KCl twice can be used in subsequent experiments. After washout of 60 mM KCl by solution I, phenylalanine (1–5 µM) was used as a pre-contract agent. And the relaxing effects of HSYA were measured when contraction effect reached the stable state (Maher et al., 2013; Reis et al., 2013; Sung et al., 2013). 2.4. Intracellular Ca2+ measurement Intracellular Ca2+ transients were measured using the TILLvisION 4.0 imaging system (Till Photonics, Gräfelfing, Germany) at room temperature (21 ± 2℃). As described previously (Grynkiewicz et al.., 1985; Wang et al.., 2003), mesenteric VSMCs were incubated with 5 µM Fura-2/ AM for 30 min. The double-excitation wavelengths Fluorescence excited at 340 nm (20 ms) and 380 nm (10 ms) loaded into the cell, then the emission fluorescence at 510 nm was detected by a photomultiplier tube. The intracellular free Ca2+ concentration ([Ca2+]i) could be calculated using the following equation: [Ca2+]i D Kd* (Sf2/Sb2)* (R – Rmin)/(Rmax – R), where Kd as the dissociation constant for fura-2/calcium complex, performed using a value of 224 nM, Sf2/Sb2 as the ratio of emission fluorescence evoked by 380 nm wavelength excitation in Ca2+-free Tyrode’s solution and saturating [Ca2+] solution, R as the ratio of the emission fluorescence, Rmin as the ratio obtained in the Ca2+-free Tyrode’s solution with 10 mM EGTA, and Rmax as the ratio obtained in the saturating [Ca2+] solution (10 mM [Ca2+] Tyrode’s solution). The cells were incubated with 10 µM Ionomycin for measuring the values of Rmax and Rmin. A drug delivery system (ALA VM4, ALA Scientific Instrument, Farmingdale, NY, United States) was used to add 60 mM high K+ solution for 10 s to stimulate the cells. The peak caused by the intracellular calcium transients was observed. Then the cells were incubated with HSYA for 10 mins to explore the effect of HSYA on high K+-induced Ca2+ transients. Tyrode’s solution was used to wash out the cells during
the 10-min interval. The changes in [Ca2+]i after 10 seconds of high K+ solution treatment showed high K+-induced Ca2+ transients from baseline to peak. Ca2+ transient rise time was defined as the time from the baseline level to the peak of [Ca2+]i. Ca2+ transient decay time was defined as the time from the peak of [Ca2+]i reduction by 90%. 2.5. Whole-cell and single channel recordings Single mesenteric arterial smooth muscle cell of WKY rat were enzymatically isolated. Macroscopic current (MC) of BKCa channels were detected using patch clamp technique in a perforated whole-cell configuration. Amphotericin 2B (250 µg/ml) was added into the patch pipette for perforating cell membrane. Single channel current recordings on single mesenteric arterial smooth muscle cell of rat were conducted under cell-attached and inside-out configuration. Whole-cell and single channel currents were recorded10 min after the recording configuration was established and after a drug application of 5–15 min. Data were low-pass filtered at 1 kHz. The open probability and amplitude of the channel were analyzed with the pCLAMP 10.0 software (Molecular Devices). The data was taken directly from Clamp-fit event statistic window. The channel activity in a patch (the open probability of the channel, NPo) was calculated as the total sum of open times divided by a registration time, where N indicates the maximum number of channels opening simultaneously in a patch. For whole-cell patch experiments, the pipette solution consisted of (in mM): K-aspartate (K-Asp) 130, KCl 10, NaCl 10, MgCl2 1, HEPES-K 10, EGTA 0.05, and the bath solution (in mM): NaCl 134, KCl 6, MgCl2 1, CaCl2 2, HEPES-K 10, glucose 10 (pH 7.4). For single channel patch experiments, the pipette solution consisted of (in mM): K-aspartate (K-Asp) 40, KCl 100, HEPES-K 10 (pH 7.4) and EGTA 2, and the bath solution contained of (in mM): K-Asp 100, KCl 40, HEPES-K 10 (pH 7.4) and EGTA 1. All the current measurements were conducted at a room temperature (25 °C) by using a patch clamp amplifier (HEKE). 2.6. Cell culture and transfection
L-type Ca channel (Cav1.2) cloned from human VSMCs were respectively constructed in the vector pcDNA3.1 and stably expressed in HEK293 cells. The cells were cultured in DMEM (GIBCO) supplemented with 10% FBS (HyClone) and Penicillin-Streptomycin solution at 37°C in 5% CO2. After 24 h of transfection of L-type Ca channel, the cells were dispersed and cultured on the covered glass and then used for patch clamp within 48 h. For whole-cell patch experiments, the pipette solution consisted of (in mM): Cs-aspartate acid 115, CsCl2 20, MgCl2 2.5, Na2ATP 2, HEPES-K 10, EGTA 10, and the bath solution (in mM): NaCl 130, TEA-Cl 4, MgCl2 1.2, CsCl2 1, BaCl2 10, HEPES-K 10, glucose 10 (pH 7.4). 2.7. Statistical analysis Data were expressed as means ± S.E.M. The term n represented the number of the blood vessels or cells tested. Student’s t-tests and analysis of variance (ANOVA) were used for the comparisons between two-group and more than two groups if the evaluations of similar variances were passed. The significance between groups was determined by one-way ANOVA and student-Newman–Keuls test. P < 0.05 was * considered statistically significant (marked as ) and the higher significance level was
set at P < 0.01 (marked as **). 3. Results 3.1. Blood pressure of SHR and WKY rats were significantly reduced by HSYA In order to investigate the effect of HSYA on blood pressure, the mean systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate were measured in SHR and WKY rats with and without intravenous injection of HSYA. Additionally, saline was also injected into the rats as solvent control (Fig. 2 A). HSYA reduced the blood pressure and heart rate in both SHR and WKY rats in a dose-dependent manner (Fig. 2 B-E). The values of SBP, DBP, MAP and heart rate in SHR before and after intravenous injection with 1 mg/Kg HSYA were 208 ± 15 mm Hg, 173 ± 13 mm Hg, 192 ± 14 mm Hg, 394 ± 10 beats/min (before), and 138 ± 14 mm Hg, 117 ± 11 mm Hg, 155 ± 15 mm Hg, 331 beats/min (after), respectively. The values of SBP, DBP, MAP and heart rate in WKY rats before
and after intravenous injection with 1 mg/Kg HSYA were 103 ± 13 mm Hg, 75 ± 11 mm Hg, 84 ± 15 mm Hg, 329 ± 21 beats/min (before), and 93 ± 14 mm Hg, 69 ± 11 mm Hg, 74 ±15 mm Hg, 299 ± 15 beats/min (after), respectively. The reduction of mean blood pressure and heart rate in WKY rats was 4.8% and 8.9%, whereas in SHR rats the reduction was 30.7% and 16.1% after 1 mg/Kg HSYA treatment. In addition, we injected intraperitoneally HSYA (10 mg/kg) into SHR and WKY ratsonce a day for one month and found that the blood pressure of both groups decreased. Taking into account the difference in drug absorption between intraperitoneal injection and intravenous injection, we refer to the intra-abdominal dose of other literature (Pei et al., 2017; Zheng et al., 2019) to increase the dose of intraperitoneal injection. Mean blood pressure and heart rate decreased slightly in WKY rats and significantly decreased in SHR group. After intraperitoneal injection once a day for one month, mean blood pressure of WKY rats decreased from 110 ± 9 mm Hg to 98 ± 14 mm Hg, while the blood pressure of SHR decreased from 189 ± 14 mm Hg to 156 ± 15 mm Hg. And mean heart rate of WKY rats decreased from 329 ± 19 beats/min to 294 ± 14 beats/min, while mean heart rate of SHR decreased from 371 ± 16 beats/min to 296 ± 12 beats/min. All the results of injection intraperitoneally of HSAY were shown in Fig. 3, respectively.
Fig.2 The hypotensive effects of HSYA injection intravenously on SHR and WKY rats. A: The representative recorded traces of changes in blood pressure in rats after administration of vehicle and HSYA. B-E: The dose- response curve showing the dose-dependent reduction in mean systolic blood pressure (mSBP), mean diastolic blood pressure (mDBP), mean pulse pressure difference (mPPD) and heart rate (n=5).
Fig.3 The hypotensive effects of HSYA on SHR and WKY rats after intraperitoneal injection once a day for one month. A and B: The changes of mean blood pressure and heart rate of WKY rats after intraperitoneal injection of vehicle and HSAY once a day for one month (n=5). C and D: The changes of mean blood pressure and heart rate of SHR after intraperitoneal injection of vehicle and HSAY once a day for one month (n=5). 3.2. The vasodilatation effects of HSYA on WKY rats To determine the vasodilatation effect of HSYA on mesenteric arterial, we used the isolated mesenteric arterial of rat for the vasomotor assay. Fig. 4 A showed that 3 µM phenylephrine (PE) caused the blood vessels to contract violently and stabilized for a long time. And Fig. 4 B demonstrated that vehicle did not affect the action of PE. On this basis, HSYA could concentration-dependently reduce the degree of
vasoconstriction induced by PE, with an IC50 of =90.8 µM (Fig. 4 C-D). It is worth noting that IbTX (200 nM), a selective BKCa channel blocker, could obviously reduce and reverse the vasodilatory effect of HSYA (Fig. 4 C), suggesting that potassium channels play a role, especially BKCa channels. Considering that the activation of BKCa channel is an important mechanism of vasorelaxation, IbTX was employed to check the contributions of BKCa channels to the vasodilatory effects of HSYA. Artery rings was incubated with 200 nM IbTX for half an hour and then stimulated with PE. Indeed, IbTX (200 nM) reversed the effect of HSYA (Fig. 4 E). Fig. 4 F showed the relaxation effect of HSYA on vascular tone with and without IbTX-incubation.
Fig.4 The vasodilation effects of HSYA. A: The continuous and stable contraction of
the vascular ring induced by PE. B: The effect of vehicle on the PE-preconstricted artery rings was invalid. C: A typical recording shown that HSYA relaxed the PE-preconstricted artery rings and the effects were reversed by the selective BKCa channel blocker, IbTX. D: The normalized dose- response curve showed that HSYA relaxed blood vessels, in a dose-dependent manner with IC50 of = 90.8 µM (n=5). E: A typical recording shown that after incubated with IbTX, the vasodilating effect of HSYA was significantly reduced. F: The relaxation effect of HSYA on vascular tone with and without IbTX-incubation (n=4).
3.3. Effect on Ca-transient in rat mesenteric arterial smooth muscle cells To further determine the effect of HSYA on intracellular calcium handing in rat mesenteric arterial smooth muscle cells, calcium signaling assays were performed. The Ca2+-transients and intracellular free Ca2+ level were measured in present of 60 mM KCl. We observed that 60 mM KCl significantly increased intracellular calcium level and evoked Ca2+-transients (Fig. 5 A) and 60 µM HSYA attenuated amplitudes of KCl-induced Ca2+-transients without affecting the rising and decay time (Fig. 5 B-D).
Fig.5 Calcium signaling assays in rat mesenteric arterial smooth muscle cell. A: The Ca2+-transients showing changes of intracellular free Ca2+ level were measured in presence of 60 mM KCl. The experiment was repeated three times and intracellular free Ca2+ levels were basically the same. B: KCl-induced Ca2+-transients with and without HSYA-treatment (60 µM for 10 min). HSYA was applied into the extracellular solution. C: KCl-induced changes of intracellular free Ca2+ levels before and after treatment with HSYA. D: The values of rise time and decay time of Ca2+ transients with and without HSYA.
3.4. The effects of HSYA on BKCa channel currents in rat mesenteric arterial smooth muscle cells The above results of the vasodilatation effect of HSYA on mesenteric arterial suggested that BKCa channel was involved. To further confirm the interaction between HSYA and BKCa channel, we tested the effects of HSYA on BKCa channel currents in rat mesenteric arterial smooth muscle cells. We applied step (Fig. 6 A-B) and ramp (Fig. 6 C-D) pulses respectively to record changes in current under the whole-cell
configuration. After HSYA (10, 30, 60 and 120 µM) was applied into the extracellular fluid, we observed that HSYA increased the whole-cell currents in a concentration-dependent manner. IbTX could eliminate the effect of HSYA. As a selective BKCa channel blocker, IbTX sensitive current is considered to be BKCa current.
Fig.6 The effect of HSYA on whole-cell channel currents of BKCa in rat mesenteric arterial smooth muscle cells. A: Typical recordings of the whole-cell currents of BKCa channels before (control) and after application of 60 µM HSYA and 200 nM IbTX. B: Concentration-dependent increased fold of current amplitude induced by a step pulse after incubated with HSYA (at +40 mV membrane potential). The current of control was normalized to 1. C: The recorded traces of BKCa channels induced by ramp pulses before (control) and after applying 60 µM HSYA and 200 nM IbTX. D: Concentration-dependent increased fold of current amplitude induced by a ramp pulse after incubated with HSYA (at +60 mV membrane potential). The current of the control was normalized to 1, and then the relative values of the current values with HSYA were calculated.
In addition, we measured single channel currents in cell-attached and inside-out recording in rat mesenteric arterial smooth muscle cells. In Fig. 7 A and B, the results showed that the BKCa channel current was voltage dependent activation and its conductance was 236.73±3.27 pS. Consistent with the whole-cell current recordings, we observed that HSYA increased the open probability (NPo) of the channels in a concentration-dependent manner (Fig. 7 C-F) in both cell-attached and inside-out recordings.
Fig.7 The effect of HSYA on BKCa channels in rat mesenteric arterial smooth muscle cells in the cell-attached and inside-out recording mode. A: Representative currents of BKCa channels in the inside-out recording mode showing the voltage dependence of BKCa channels. B: I-V curves of BKCa channels in the inside-out recording mode. C
and D: Representative currents of BKCa channels in the cell-attached (C) and inside-out (D) recording mode showing that HSYA activated BKCa channels in a dose-response manner. E and F: The NPo of BKCa currents with and without HSYA in the cell-attached (E) and inside-out (F) mode. *P<0.05, **P<0.01 (n=5).
3.5.The effect of HSYA on L-type calcium channel currents in HEK293 cells In order to check whether HSYA also affect the Ca-L, we used whole-cell patch clamp technique to record the macroscopic currents (MC) in HEK293 cells, in which Ca-Ls were expressed. The application of 1 µM nifedipine confirmed the existence of Ca-L currents in HEK293 cells (Fig. 8 A). Then we applied 50 µM, 100 µM, 150 µM HSYA and matched vehicle in the extracellular fluid and measured the changes of currents (Fig. 8 B). 100 µM HSYA suppressed the current by about 33%, and 150 µM did not lead to further suppression (Fig. 8 C and D).
Fig.8 The effect of HSYA on whole-cell currents of Ca-L expressed in HEK293 cells. A: Identification of Ca-L currents (ICa-L) by nifedipine. B: The recorded currents with HSYA and vehicle treatment. C: Representative I-V curve with and without nifedipine. D: Percentage of inhibition of ICa-L by HSYA (50, 100 and 150 µM) at +20 mV membrane potential.
4. Discussion In the present study, we demonstrate that HSYA activates BKCa and inhibits Ca-L channels and reduces intracellular Ca2+ level, leading to a vasorelaxation. HSYA, one of the most important bioactive monomers and a valuable clinical drug, has been shown to have multiple effects on cardiovascular system (Guo et al., 2017; Pan et al., 2017). Also Han and Li’s research (Han et al., 2016 and Li et al., 2016) shows that HSYA protected against pulmonary arterial hypertension in rats by suppression of inflammation and oxidative stress and inhibition the proliferation and hypertrophy of pulmonary artery smooth muscle cells. In order to make better use of its potential
clinical value, further research is needed in many aspects. Some mechanisms underlying HSYA effects remain to be clarified. The data from this study showed that HSYA had a very good antihypertensive effect in concentration dependent manner when administered intravenously. Simultaneously, it reduced also the heart rate. These results were consistent with previous reports (Nie et al., 2012) that intravenous injection of the HSYA significantly reduced MAP and heart rate in both normotensive rats and SHR in a dose-dependent manner. However, given the possible risk of bradycardia, care should be taken when HSYA is applied in patients, especially in patients with slow heart beatings. The mechanism of action of HSYA to lower blood pressure and vasodilation needs to be further clarified. Through vasomotor assay, we explored the effect of HSYA on vascular tone. The results suggested that HSYA reduced PE-induced vasoconstriction in a concentration-dependent manner. It was worth noting that IbTX (200 nM), a selective BKCa channel blocker, could obviously reduce and reverse the vasodilatory effect of HSYA, confirming the participation of BKCa in the vascular relaxation induced by HSYA. Many other traditional Chinese medicines, like Danshen, Gegen and Scutellaria baicalensis shows similar vasorelaxant effects and mechanisms (Deng et al., 2014; Lin et al., 2010). From a physiological perspective, BKCa channel function plays a key role in the regulation of vascular tone. Previous reports suggested that BKCa channel activated by local increases in intracellular calcium ion concentration (Ca2+ sparks) from the sarcoplasmic reticulum (SR) of smooth muscle
cause vasodilation (Dabertrand et al., 2013; Nelson et al., 1995). Further, our results showed that HSYA decreased the intracellular calcium concentration, but it did not change the values of rise and decay time of Ca2+ transients significantly. Liu et al also reported that HSYA exerted an inhibitory effect on intracellular Ca2+ mobilization (Liu et al., 2018). Here the decrease of the spatially averaged intracellular calcium concentration, which regulates contraction, confirmed the vasorelaxant effect of HSYA. A rise of [Ca2+]i is caused by three pathways which are currently known in vascular smooth muscle cells: (1) transmembrane Ca2+ entry through Ca2+ -permeable ion channels, (2) store Ca2+ release from the sarcoplasmic reticulum, and (3) Ca2+ inward transportation by Na+/Ca2+ exchangers (Bolton et al., 2004; Matchkov, 2010).Change of [Ca2+]i may influence the dynamic equilibrium of transmembrane transport, ER uptake and release of Ca2+. It is well known that Ca2+ transient is important in the process of excitation–contraction (E–C) coupling of SMCs. We still do not know the mechanism of the decrease of the spatially averaged intracellular calcium concentration caused by HSYA and need further research. Interestingly, the activation of BKCa channels induced by Ca2+ sparks was not affected by the decrease of the spatially averaged intracellular calcium, which suggested the intracellular calcium concentration have no direct effect on Ca2+ sparks or HSYA directly open BKCa channels. The reduced Ca2+-transients might result from the combination of IBKCa-enhancing and ICa-L-reducing effects of HSYA. Therefore, the activation of BKCa channels and the inhibition of intracellular calcium, which both caused by HSYA, together leaded to vasodilation. Then we used patch clamp techniques to investigate whether HSYA had direct effect on BKCa. By analyzing the whole-cell current data, we found that HSYA concentration-dependently activated BKCa channels. At the same time, in inside-out recording mode, HSYA increased the probability of opening of BKCa channels in rat mesenteric arterial SMCs. These data indicate that the increase in NPo resulted from the increased frequency of channel opening events. Likewise, NPo was enhanced also in cell-attached recording mode. In agreement with the increased whole-cell currents of BKCa, NPo in single-channel recordings were enhanced by HSYA, a confirmation
HSYA effect on BKCa. The relationship between BKCa channels and vasodilation has been described above, and this part of the results also suggests one of the mechanisms of HSYA vasodilator effect. Another important ion channel that regulates vascular tone is the L-type calcium channel. The activation of Ca-L channel by the depolarization of VSMCs allows Ca2+ influx, increases global Ca2+ level and causes VSMCs contraction (Joseph et al., 2013; Zhang et al., 2018). Conversely, inhibition of Ca-L channel can cause vasodilation. Given the close association between BKCa channel and Ca2+ channel, we chose the cell model to express calcium channels alone to avoid interaction between the two to produce complex results. We found that HSYA had an obviously inhibitory effect on Ca-L channels in whole-cell recording mode, which has not been reported so far. It was linked to the decrease of calcium influx and might further lead to the inactivation of downstream pathways (Dabertrand et al., 2010), which required further research. This indicates that the inhibition of ICa-L might contribute to the vasodilatory action of HSYA. The activation of BKCa channels leads to hyperpolarization and inhibition of Ca-L channels, reducing intracellular Ca2+ level and thus relaxing SMCs (Karlin, 2015; Beleznai et al., 2011). The direct inhibition of ICa-L by HSYA can enhance the SMC relaxation. However, the limitation of our study was that we evaluated the role of HSYA in transfected HEK293 cells and that these cells may not accurately reflect the effect of HSYA on native L-type channels in VSMC. Under physiological conditions, all kinds of factors, like ion channels, blood flow, receptor proteins and so on, are able to influence and regulate vascular tone (Henrion D., 2018). Our research suggests that BKCa channels and Ca-L channels might be the major role of the ion channels which regulate vascular tone by HSYA. And more experiments are needed in the future to prove this. From our data, the vasodilatory effect of HSYA can be explained, at least partially, by its effects on BKCa and Ca-L channels. The reason for its heart beating-slowing effects, however, remains to be revealed. The mechanisms of its effects are still to be further researched (Liu et al., 2017). At present, safflower is clinically used to treat occlusive cerebrovascular disease, coronary heart disease, vasculitis and so on. Safflower yellow pigment is also used to
treat similar diseases. However, HSYA seems to have a long way to go in clinical practice because its complex mechanism of action is not yet clear. The present study provides evidences to support that HSYA is a vasodilator compound. The vasodilatory effect is related to the activation of BKCa channels, the inhibition of Ca-L channels and the reduction of intracellular calcium concentration. Though more detailed clarification on the signal pathways of the effects needs to be further researched (Misárková et al., 2016), HSYA has potential benefits for the treatment of hypertension, a very common disease (Nie et al., 2012; Jin et al., 2013; Goldberg et al., 2017). In the future, we need to further study its antihypertensive mechanism, including the role of the heart, etc. In particular, its inhibition of heart rate indicates that HSYA has a wide range of effects on the cardiovascular system. This is also worthy of special attention.
Acknowledgments The study is supported by grants from the Education Department of Sichuan Province (16ZB0196 to Wang Na), National Natural Science Foundation of China (81173661 to Yang Yan，81600381 to Pengyun Li) and Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease (0903-00021101 to Jun Cheng). The authors would like to thank Prof. Xiao-Bo Zhou and Prof. Shang-Zhong Xu for critical reviews and comments of the manuscript.
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S0014-2999(19)30825-8
DOI:
https://doi.org/10.1016/j.ejphar.2019.172873
Reference:
EJP 172873
To appear in:
European Journal of Pharmacology
Received Date: 4 July 2019 Revised Date:
10 December 2019
Accepted Date: 16 December 2019
Please cite this article as: Wang, N., He, D., Zhou, Y., Wen, J., Liu, X., Li, P., Yang, Y., Cheng, J., Hydroxysafflor yellow A actives BKCa channels and inhibits L-type Ca channels to induce vascular relaxation, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2019.172873. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Na Wang: Data curation, Investigation, Writing- Original draft preparation, Funding acquisition Jun Cheng: Conceptualization, Methodology, Investigation , Funding acquisition Dongmei He: Data curation, Methodology, Investigation Yuanqun Zhou, Jing Wen, and Xiaoqin Liu: Methodology Pengyun Li: Methodology, Formal analysis Yan Yang: Writing- Reviewing and Editing, Funding acquisition, Project administration
Hydroxysafflor yellow A actives BKCa channels and inhibits L-type Ca channels to induce vascular relaxation Na Wang , Dongmei He , Yuanqun Zhou, Jing Wen, Xiaoqin Liu, Pengyun Li, Yan *
*
Yang, Jun Cheng Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Laboratory of Sichuan Province, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, Sichuan, China. *Equally contributed Corresponding author: Jun Cheng, [email protected] Co- Corresponding author: Yan Yang, [email protected]
Abstract Hydroxy-safflor yellow A (HSYA) can exert a variety of effects upon the vascular system. However, the underlying mechanisms are not clear. The present study is to investigate its vasodilating effect and the mechanisms. Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were enrolled for studying effects of HSYA on blood pressure, vasodilation, intracellular Ca2+ transient and membrane ion channels. Vasodilation and intracellular Ca2+ transient were measured by using vasomotor assay and fluorescence imaging system, respectively. The effect of HSYA on the large conductance Ca2+ activated and voltage-gated potassium channel (BKCa channel) currents in rat mesentery artery and on L-type calcium channel (Ca-L) currents in HEK293cells expressed with Ca-L were investigated using patch clamp techniques. Blood pressure of SHR and WKY rats were concentration dependently reduced by HSYA with a larger effect of HSYA in SHR than that in WKY rats. The tension of mesenteric arteries induced by 3 µM phenylephrine was attenuated by HSYA (IC50=90.8 µΜ). Patch clamp study showed that HSYA could activate BKCa channels and suppress Ca-L channels in a concentration dependent manner. The results of calcium signaling assays indicated that HSYA could reduce the intracellular free Ca2+ level. These findings demonstrate that HSYA could activate BKCa channels and inhibit Ca-L channels and reduce intracellular free Ca2+ level, which are probably important for its vasodilatory effect.
Key Words: Hydroxysafflor yellow A, BKCa, L-type Ca channel, vasodilatation, smooth muscle cells
1. Introduction Hydroxy-safflor yellow A (HSYA), a water soluble monomer and the major bioactive compound extracted from safflower, the extensively used traditional Chinese herbal medicine, can exert a variety of effects upon the vascular system (Yuan et al., 2014). Preliminary studies indicated that HSYA had beneficial effects on hypertensive ventricular remodeling, which may involve mechanisms of inhibiting cell apoptosis and suppressing metalloproteinases expression (Wang et al., 2014). In addition, HSYA had protective effect on bleomycin-induced lung inflammatory response (Wu et al., 2012), the UV-induced skin damage (Kong et al., 2013), and 6-hydroxydopamine-induced Parkinson's disease in rats (Han et al., 2013). In addition to those effects, HSYA displayed vascular relaxation effects on rat pulmonary artery (PA) by activating voltage-gated potassium channels (KV) in pulmonary vascular smooth muscle cells and the effects were endothelium-independent (Bai et al., 2012). Another research indicated that HSYA could significantly reduce blood pressure and heart rate. The author speculated that its effects may be related to activation of the large conductance Ca2+ activated and voltage-gated potassium channel (BKCa) and ATP-sensitive potassium channels (KATP), because BKCa and KATP blocker, but not KV and acetylcholine-activated potassium channel (KAch) blocker, weakened the inhibitory effect of HSYA on heart function and heart rate (Nie et al., 2012). However, the direct effects of HSYA on those ion channel currents have not been assessed. The mechanisms underlying the blood pressure depressing effect of HSYA remain to unveiled. BKCa channel is one of the important potassium channels ubiquitously expressed in blood vessels, especially in endothelial and vascular smooth muscle cells (Contreras et al., 2013). The depolarization of membrane voltage and intracellular local Ca2+ release events through ryanodine receptors (Ca2+ sparks) from the sarcoplasmic reticulum are two key factors to activate BKCa channels (Yang et al., 2013; Yang et al., 2013). When smooth muscle cells contract, BKCa channel will be activated by membrane depolarization and Ca2+-release. Once BKCa channels are activated, the k+ efflux and the cell membrane hyperpolarization lead to the reduced
excitability of the vessels and vasorelaxation (Modgil et al., 2013). Thus BKCa channels exert negative feedback regulation mechanism on the vascular tone and blood flow. L-type calcium channels (Ca-L), the main source of calcium entry pathways, also play a key role in vascular smooth muscle. They are crucial elements in determining vascular tone and other numerous physiological processes. The activation of them causes vasoconstriction. On the contrary, the inhibition of Ca-L can cause vasodilation (Hell et al., 2017; Li et al., 2013). Therefore, both BKCa and Ca-L are attractive targets for discovering drugs clinically relevant to hypertension and other diseases of vascular system (Joseph et al., 2013; Xu et al., 2006). Hence, the present study was designed to reveal the direct vascular effects and the mechanism of HSYA by using vasomotor assay, patch-clamp recordings and calcium signaling assays. The study may help understand vasodilatation effect of HSYA and provide experimental basis for clinical application of HSYA for hypertension and other cardiovascular diseases. 2. Materials and Methods 2.1. Animals and drugs Sixteen-week old spontaneously hypertensive rats (SHR) and their age-matched Wistar-Kyoto (WKY) rats with body weight of 320-380 g were bought from Beijing Weitong Lihua Animal Technology Co. Ltd. (license No. SCXK (Jing) 2016-0011). This study was approved by the Committee on the Ethics of Animal Experiments of Southwest Medical University and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996). HSYA was purchased from Bioproduct Research Institute of Chengdu, China (purity>98%) was dissolved in water to make a 10 mM stock solution and added to the bath solution to the desired final concentrations immediately before each experiment. The chemical structure of HSYA (Meselhy et al., 1993) is shown in Fig. 1. Phenylalanine (PE), acetylcholine, tetraethylammonium (TEA), iberiotoxin (IbTX) and nifedipine were purchased from Sigma-Aldrich Inc. (St. Louis, MO, United
States). Fura-2 AM (5-Oxazolecarboxylic acid, 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2-(bis(2((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, (acetyloxy)methyl ester) was purchased from Invitrogen Inc. (San Diego, CA, United States).
Fig.1 The chemical structure of HSYA (C27H32O16). Hydroxysafflor yellow A is a compound with a single chalcone glycoside structure. Molecular weight: 612.53 2.2. Measurement of blood pressure and heart rate of SHR and WKY rats Sixteen-week old adult male rats were enrolled in a blood pressure study under general anesthesia (n=5 each group). Rats were anesthetized with pentobarbital sodium (50 mg/kg). Before blood pressure monitoring, rats underwent surgery for catheterization of the femoral artery. After cutting the skin layer and exposing the femoral artery, a catheter was advanced into the lumen of the femoral artery. Arterial pressure was recorded for 5 min after catheterization. Multi-channel physiological signal acquisition and processing system (RM6240BD) was used (Chengdu Instrument Factory, Sichuan, China) to record changes in blood pressure and heart rate. 2.3. Separation of WKY rat mesenteric artery rings and vasomotor assay Under general anesthesia, the branches of the superior mesenteric arteries were rapidly isolated and placed in solution I containing (in mM): NaCl 127, KCl 5.9, CaCl2 2.4, MgCl2 1.2, glucose 1.2, HEPES 10 (pH adjusted to 7.4 with NaOH). The arteries were sectioned into four sections (2–3 mm width and length) for the tension measurements after cleared of periadvential fat. The endothelial layer was removed mechanically in half of the rings by gently rubbing the intimal surface of the artery lumen with a matchstick. All rings were then mounted in organ baths (AD Instruments,
DMT, Oxford, UK) which were filled with warmed (37°C) and gas-equilibrated (95% O2, 5% CO2) solution I. Removal of the endothelium was confirmed by the absence of relaxation induced by acetylcholine (10 µM). Firstly we applied 60 mM KCl to determine the activity of arteries. Only the arteries with similar contractility after incubation with 60 mM KCl twice can be used in subsequent experiments. After washout of 60 mM KCl by solution I, phenylalanine (1–5 µM) was used as a pre-contract agent. And the relaxing effects of HSYA were measured when contraction effect reached the stable state (Maher et al., 2013; Reis et al., 2013; Sung et al., 2013). 2.4. Intracellular Ca2+ measurement Intracellular Ca2+ transients were measured using the TILLvisION 4.0 imaging system (Till Photonics, Gräfelfing, Germany) at room temperature (21 ± 2℃). As described previously (Grynkiewicz et al.., 1985; Wang et al.., 2003), mesenteric VSMCs were incubated with 5 µM Fura-2/ AM for 30 min. The double-excitation wavelengths Fluorescence excited at 340 nm (20 ms) and 380 nm (10 ms) loaded into the cell, then the emission fluorescence at 510 nm was detected by a photomultiplier tube. The intracellular free Ca2+ concentration ([Ca2+]i) could be calculated using the following equation: [Ca2+]i D Kd* (Sf2/Sb2)* (R – Rmin)/(Rmax – R), where Kd as the dissociation constant for fura-2/calcium complex, performed using a value of 224 nM, Sf2/Sb2 as the ratio of emission fluorescence evoked by 380 nm wavelength excitation in Ca2+-free Tyrode’s solution and saturating [Ca2+] solution, R as the ratio of the emission fluorescence, Rmin as the ratio obtained in the Ca2+-free Tyrode’s solution with 10 mM EGTA, and Rmax as the ratio obtained in the saturating [Ca2+] solution (10 mM [Ca2+] Tyrode’s solution). The cells were incubated with 10 µM Ionomycin for measuring the values of Rmax and Rmin. A drug delivery system (ALA VM4, ALA Scientific Instrument, Farmingdale, NY, United States) was used to add 60 mM high K+ solution for 10 s to stimulate the cells. The peak caused by the intracellular calcium transients was observed. Then the cells were incubated with HSYA for 10 mins to explore the effect of HSYA on high K+-induced Ca2+ transients. Tyrode’s solution was used to wash out the cells during
the 10-min interval. The changes in [Ca2+]i after 10 seconds of high K+ solution treatment showed high K+-induced Ca2+ transients from baseline to peak. Ca2+ transient rise time was defined as the time from the baseline level to the peak of [Ca2+]i. Ca2+ transient decay time was defined as the time from the peak of [Ca2+]i reduction by 90%. 2.5. Whole-cell and single channel recordings Single mesenteric arterial smooth muscle cell of WKY rat were enzymatically isolated. Macroscopic current (MC) of BKCa channels were detected using patch clamp technique in a perforated whole-cell configuration. Amphotericin 2B (250 µg/ml) was added into the patch pipette for perforating cell membrane. Single channel current recordings on single mesenteric arterial smooth muscle cell of rat were conducted under cell-attached and inside-out configuration. Whole-cell and single channel currents were recorded10 min after the recording configuration was established and after a drug application of 5–15 min. Data were low-pass filtered at 1 kHz. The open probability and amplitude of the channel were analyzed with the pCLAMP 10.0 software (Molecular Devices). The data was taken directly from Clamp-fit event statistic window. The channel activity in a patch (the open probability of the channel, NPo) was calculated as the total sum of open times divided by a registration time, where N indicates the maximum number of channels opening simultaneously in a patch. For whole-cell patch experiments, the pipette solution consisted of (in mM): K-aspartate (K-Asp) 130, KCl 10, NaCl 10, MgCl2 1, HEPES-K 10, EGTA 0.05, and the bath solution (in mM): NaCl 134, KCl 6, MgCl2 1, CaCl2 2, HEPES-K 10, glucose 10 (pH 7.4). For single channel patch experiments, the pipette solution consisted of (in mM): K-aspartate (K-Asp) 40, KCl 100, HEPES-K 10 (pH 7.4) and EGTA 2, and the bath solution contained of (in mM): K-Asp 100, KCl 40, HEPES-K 10 (pH 7.4) and EGTA 1. All the current measurements were conducted at a room temperature (25 °C) by using a patch clamp amplifier (HEKE). 2.6. Cell culture and transfection
L-type Ca channel (Cav1.2) cloned from human VSMCs were respectively constructed in the vector pcDNA3.1 and stably expressed in HEK293 cells. The cells were cultured in DMEM (GIBCO) supplemented with 10% FBS (HyClone) and Penicillin-Streptomycin solution at 37°C in 5% CO2. After 24 h of transfection of L-type Ca channel, the cells were dispersed and cultured on the covered glass and then used for patch clamp within 48 h. For whole-cell patch experiments, the pipette solution consisted of (in mM): Cs-aspartate acid 115, CsCl2 20, MgCl2 2.5, Na2ATP 2, HEPES-K 10, EGTA 10, and the bath solution (in mM): NaCl 130, TEA-Cl 4, MgCl2 1.2, CsCl2 1, BaCl2 10, HEPES-K 10, glucose 10 (pH 7.4). 2.7. Statistical analysis Data were expressed as means ± S.E.M. The term n represented the number of the blood vessels or cells tested. Student’s t-tests and analysis of variance (ANOVA) were used for the comparisons between two-group and more than two groups if the evaluations of similar variances were passed. The significance between groups was determined by one-way ANOVA and student-Newman–Keuls test. P < 0.05 was * considered statistically significant (marked as ) and the higher significance level was
set at P < 0.01 (marked as **). 3. Results 3.1. Blood pressure of SHR and WKY rats were significantly reduced by HSYA In order to investigate the effect of HSYA on blood pressure, the mean systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate were measured in SHR and WKY rats with and without intravenous injection of HSYA. Additionally, saline was also injected into the rats as solvent control (Fig. 2 A). HSYA reduced the blood pressure and heart rate in both SHR and WKY rats in a dose-dependent manner (Fig. 2 B-E). The values of SBP, DBP, MAP and heart rate in SHR before and after intravenous injection with 1 mg/Kg HSYA were 208 ± 15 mm Hg, 173 ± 13 mm Hg, 192 ± 14 mm Hg, 394 ± 10 beats/min (before), and 138 ± 14 mm Hg, 117 ± 11 mm Hg, 155 ± 15 mm Hg, 331 beats/min (after), respectively. The values of SBP, DBP, MAP and heart rate in WKY rats before
and after intravenous injection with 1 mg/Kg HSYA were 103 ± 13 mm Hg, 75 ± 11 mm Hg, 84 ± 15 mm Hg, 329 ± 21 beats/min (before), and 93 ± 14 mm Hg, 69 ± 11 mm Hg, 74 ±15 mm Hg, 299 ± 15 beats/min (after), respectively. The reduction of mean blood pressure and heart rate in WKY rats was 4.8% and 8.9%, whereas in SHR rats the reduction was 30.7% and 16.1% after 1 mg/Kg HSYA treatment. In addition, we injected intraperitoneally HSYA (10 mg/kg) into SHR and WKY ratsonce a day for one month and found that the blood pressure of both groups decreased. Taking into account the difference in drug absorption between intraperitoneal injection and intravenous injection, we refer to the intra-abdominal dose of other literature (Pei et al., 2017; Zheng et al., 2019) to increase the dose of intraperitoneal injection. Mean blood pressure and heart rate decreased slightly in WKY rats and significantly decreased in SHR group. After intraperitoneal injection once a day for one month, mean blood pressure of WKY rats decreased from 110 ± 9 mm Hg to 98 ± 14 mm Hg, while the blood pressure of SHR decreased from 189 ± 14 mm Hg to 156 ± 15 mm Hg. And mean heart rate of WKY rats decreased from 329 ± 19 beats/min to 294 ± 14 beats/min, while mean heart rate of SHR decreased from 371 ± 16 beats/min to 296 ± 12 beats/min. All the results of injection intraperitoneally of HSAY were shown in Fig. 3, respectively.
Fig.2 The hypotensive effects of HSYA injection intravenously on SHR and WKY rats. A: The representative recorded traces of changes in blood pressure in rats after administration of vehicle and HSYA. B-E: The dose- response curve showing the dose-dependent reduction in mean systolic blood pressure (mSBP), mean diastolic blood pressure (mDBP), mean pulse pressure difference (mPPD) and heart rate (n=5).
Fig.3 The hypotensive effects of HSYA on SHR and WKY rats after intraperitoneal injection once a day for one month. A and B: The changes of mean blood pressure and heart rate of WKY rats after intraperitoneal injection of vehicle and HSAY once a day for one month (n=5). C and D: The changes of mean blood pressure and heart rate of SHR after intraperitoneal injection of vehicle and HSAY once a day for one month (n=5). 3.2. The vasodilatation effects of HSYA on WKY rats To determine the vasodilatation effect of HSYA on mesenteric arterial, we used the isolated mesenteric arterial of rat for the vasomotor assay. Fig. 4 A showed that 3 µM phenylephrine (PE) caused the blood vessels to contract violently and stabilized for a long time. And Fig. 4 B demonstrated that vehicle did not affect the action of PE. On this basis, HSYA could concentration-dependently reduce the degree of
vasoconstriction induced by PE, with an IC50 of =90.8 µM (Fig. 4 C-D). It is worth noting that IbTX (200 nM), a selective BKCa channel blocker, could obviously reduce and reverse the vasodilatory effect of HSYA (Fig. 4 C), suggesting that potassium channels play a role, especially BKCa channels. Considering that the activation of BKCa channel is an important mechanism of vasorelaxation, IbTX was employed to check the contributions of BKCa channels to the vasodilatory effects of HSYA. Artery rings was incubated with 200 nM IbTX for half an hour and then stimulated with PE. Indeed, IbTX (200 nM) reversed the effect of HSYA (Fig. 4 E). Fig. 4 F showed the relaxation effect of HSYA on vascular tone with and without IbTX-incubation.
Fig.4 The vasodilation effects of HSYA. A: The continuous and stable contraction of
the vascular ring induced by PE. B: The effect of vehicle on the PE-preconstricted artery rings was invalid. C: A typical recording shown that HSYA relaxed the PE-preconstricted artery rings and the effects were reversed by the selective BKCa channel blocker, IbTX. D: The normalized dose- response curve showed that HSYA relaxed blood vessels, in a dose-dependent manner with IC50 of = 90.8 µM (n=5). E: A typical recording shown that after incubated with IbTX, the vasodilating effect of HSYA was significantly reduced. F: The relaxation effect of HSYA on vascular tone with and without IbTX-incubation (n=4).
3.3. Effect on Ca-transient in rat mesenteric arterial smooth muscle cells To further determine the effect of HSYA on intracellular calcium handing in rat mesenteric arterial smooth muscle cells, calcium signaling assays were performed. The Ca2+-transients and intracellular free Ca2+ level were measured in present of 60 mM KCl. We observed that 60 mM KCl significantly increased intracellular calcium level and evoked Ca2+-transients (Fig. 5 A) and 60 µM HSYA attenuated amplitudes of KCl-induced Ca2+-transients without affecting the rising and decay time (Fig. 5 B-D).
Fig.5 Calcium signaling assays in rat mesenteric arterial smooth muscle cell. A: The Ca2+-transients showing changes of intracellular free Ca2+ level were measured in presence of 60 mM KCl. The experiment was repeated three times and intracellular free Ca2+ levels were basically the same. B: KCl-induced Ca2+-transients with and without HSYA-treatment (60 µM for 10 min). HSYA was applied into the extracellular solution. C: KCl-induced changes of intracellular free Ca2+ levels before and after treatment with HSYA. D: The values of rise time and decay time of Ca2+ transients with and without HSYA.
3.4. The effects of HSYA on BKCa channel currents in rat mesenteric arterial smooth muscle cells The above results of the vasodilatation effect of HSYA on mesenteric arterial suggested that BKCa channel was involved. To further confirm the interaction between HSYA and BKCa channel, we tested the effects of HSYA on BKCa channel currents in rat mesenteric arterial smooth muscle cells. We applied step (Fig. 6 A-B) and ramp (Fig. 6 C-D) pulses respectively to record changes in current under the whole-cell
configuration. After HSYA (10, 30, 60 and 120 µM) was applied into the extracellular fluid, we observed that HSYA increased the whole-cell currents in a concentration-dependent manner. IbTX could eliminate the effect of HSYA. As a selective BKCa channel blocker, IbTX sensitive current is considered to be BKCa current.
Fig.6 The effect of HSYA on whole-cell channel currents of BKCa in rat mesenteric arterial smooth muscle cells. A: Typical recordings of the whole-cell currents of BKCa channels before (control) and after application of 60 µM HSYA and 200 nM IbTX. B: Concentration-dependent increased fold of current amplitude induced by a step pulse after incubated with HSYA (at +40 mV membrane potential). The current of control was normalized to 1. C: The recorded traces of BKCa channels induced by ramp pulses before (control) and after applying 60 µM HSYA and 200 nM IbTX. D: Concentration-dependent increased fold of current amplitude induced by a ramp pulse after incubated with HSYA (at +60 mV membrane potential). The current of the control was normalized to 1, and then the relative values of the current values with HSYA were calculated.
In addition, we measured single channel currents in cell-attached and inside-out recording in rat mesenteric arterial smooth muscle cells. In Fig. 7 A and B, the results showed that the BKCa channel current was voltage dependent activation and its conductance was 236.73±3.27 pS. Consistent with the whole-cell current recordings, we observed that HSYA increased the open probability (NPo) of the channels in a concentration-dependent manner (Fig. 7 C-F) in both cell-attached and inside-out recordings.
Fig.7 The effect of HSYA on BKCa channels in rat mesenteric arterial smooth muscle cells in the cell-attached and inside-out recording mode. A: Representative currents of BKCa channels in the inside-out recording mode showing the voltage dependence of BKCa channels. B: I-V curves of BKCa channels in the inside-out recording mode. C
and D: Representative currents of BKCa channels in the cell-attached (C) and inside-out (D) recording mode showing that HSYA activated BKCa channels in a dose-response manner. E and F: The NPo of BKCa currents with and without HSYA in the cell-attached (E) and inside-out (F) mode. *P<0.05, **P<0.01 (n=5).
3.5.The effect of HSYA on L-type calcium channel currents in HEK293 cells In order to check whether HSYA also affect the Ca-L, we used whole-cell patch clamp technique to record the macroscopic currents (MC) in HEK293 cells, in which Ca-Ls were expressed. The application of 1 µM nifedipine confirmed the existence of Ca-L currents in HEK293 cells (Fig. 8 A). Then we applied 50 µM, 100 µM, 150 µM HSYA and matched vehicle in the extracellular fluid and measured the changes of currents (Fig. 8 B). 100 µM HSYA suppressed the current by about 33%, and 150 µM did not lead to further suppression (Fig. 8 C and D).
Fig.8 The effect of HSYA on whole-cell currents of Ca-L expressed in HEK293 cells. A: Identification of Ca-L currents (ICa-L) by nifedipine. B: The recorded currents with HSYA and vehicle treatment. C: Representative I-V curve with and without nifedipine. D: Percentage of inhibition of ICa-L by HSYA (50, 100 and 150 µM) at +20 mV membrane potential.
4. Discussion In the present study, we demonstrate that HSYA activates BKCa and inhibits Ca-L channels and reduces intracellular Ca2+ level, leading to a vasorelaxation. HSYA, one of the most important bioactive monomers and a valuable clinical drug, has been shown to have multiple effects on cardiovascular system (Guo et al., 2017; Pan et al., 2017). Also Han and Li’s research (Han et al., 2016 and Li et al., 2016) shows that HSYA protected against pulmonary arterial hypertension in rats by suppression of inflammation and oxidative stress and inhibition the proliferation and hypertrophy of pulmonary artery smooth muscle cells. In order to make better use of its potential
clinical value, further research is needed in many aspects. Some mechanisms underlying HSYA effects remain to be clarified. The data from this study showed that HSYA had a very good antihypertensive effect in concentration dependent manner when administered intravenously. Simultaneously, it reduced also the heart rate. These results were consistent with previous reports (Nie et al., 2012) that intravenous injection of the HSYA significantly reduced MAP and heart rate in both normotensive rats and SHR in a dose-dependent manner. However, given the possible risk of bradycardia, care should be taken when HSYA is applied in patients, especially in patients with slow heart beatings. The mechanism of action of HSYA to lower blood pressure and vasodilation needs to be further clarified. Through vasomotor assay, we explored the effect of HSYA on vascular tone. The results suggested that HSYA reduced PE-induced vasoconstriction in a concentration-dependent manner. It was worth noting that IbTX (200 nM), a selective BKCa channel blocker, could obviously reduce and reverse the vasodilatory effect of HSYA, confirming the participation of BKCa in the vascular relaxation induced by HSYA. Many other traditional Chinese medicines, like Danshen, Gegen and Scutellaria baicalensis shows similar vasorelaxant effects and mechanisms (Deng et al., 2014; Lin et al., 2010). From a physiological perspective, BKCa channel function plays a key role in the regulation of vascular tone. Previous reports suggested that BKCa channel activated by local increases in intracellular calcium ion concentration (Ca2+ sparks) from the sarcoplasmic reticulum (SR) of smooth muscle
cause vasodilation (Dabertrand et al., 2013; Nelson et al., 1995). Further, our results showed that HSYA decreased the intracellular calcium concentration, but it did not change the values of rise and decay time of Ca2+ transients significantly. Liu et al also reported that HSYA exerted an inhibitory effect on intracellular Ca2+ mobilization (Liu et al., 2018). Here the decrease of the spatially averaged intracellular calcium concentration, which regulates contraction, confirmed the vasorelaxant effect of HSYA. A rise of [Ca2+]i is caused by three pathways which are currently known in vascular smooth muscle cells: (1) transmembrane Ca2+ entry through Ca2+ -permeable ion channels, (2) store Ca2+ release from the sarcoplasmic reticulum, and (3) Ca2+ inward transportation by Na+/Ca2+ exchangers (Bolton et al., 2004; Matchkov, 2010).Change of [Ca2+]i may influence the dynamic equilibrium of transmembrane transport, ER uptake and release of Ca2+. It is well known that Ca2+ transient is important in the process of excitation–contraction (E–C) coupling of SMCs. We still do not know the mechanism of the decrease of the spatially averaged intracellular calcium concentration caused by HSYA and need further research. Interestingly, the activation of BKCa channels induced by Ca2+ sparks was not affected by the decrease of the spatially averaged intracellular calcium, which suggested the intracellular calcium concentration have no direct effect on Ca2+ sparks or HSYA directly open BKCa channels. The reduced Ca2+-transients might result from the combination of IBKCa-enhancing and ICa-L-reducing effects of HSYA. Therefore, the activation of BKCa channels and the inhibition of intracellular calcium, which both caused by HSYA, together leaded to vasodilation. Then we used patch clamp techniques to investigate whether HSYA had direct effect on BKCa. By analyzing the whole-cell current data, we found that HSYA concentration-dependently activated BKCa channels. At the same time, in inside-out recording mode, HSYA increased the probability of opening of BKCa channels in rat mesenteric arterial SMCs. These data indicate that the increase in NPo resulted from the increased frequency of channel opening events. Likewise, NPo was enhanced also in cell-attached recording mode. In agreement with the increased whole-cell currents of BKCa, NPo in single-channel recordings were enhanced by HSYA, a confirmation
HSYA effect on BKCa. The relationship between BKCa channels and vasodilation has been described above, and this part of the results also suggests one of the mechanisms of HSYA vasodilator effect. Another important ion channel that regulates vascular tone is the L-type calcium channel. The activation of Ca-L channel by the depolarization of VSMCs allows Ca2+ influx, increases global Ca2+ level and causes VSMCs contraction (Joseph et al., 2013; Zhang et al., 2018). Conversely, inhibition of Ca-L channel can cause vasodilation. Given the close association between BKCa channel and Ca2+ channel, we chose the cell model to express calcium channels alone to avoid interaction between the two to produce complex results. We found that HSYA had an obviously inhibitory effect on Ca-L channels in whole-cell recording mode, which has not been reported so far. It was linked to the decrease of calcium influx and might further lead to the inactivation of downstream pathways (Dabertrand et al., 2010), which required further research. This indicates that the inhibition of ICa-L might contribute to the vasodilatory action of HSYA. The activation of BKCa channels leads to hyperpolarization and inhibition of Ca-L channels, reducing intracellular Ca2+ level and thus relaxing SMCs (Karlin, 2015; Beleznai et al., 2011). The direct inhibition of ICa-L by HSYA can enhance the SMC relaxation. However, the limitation of our study was that we evaluated the role of HSYA in transfected HEK293 cells and that these cells may not accurately reflect the effect of HSYA on native L-type channels in VSMC. Under physiological conditions, all kinds of factors, like ion channels, blood flow, receptor proteins and so on, are able to influence and regulate vascular tone (Henrion D., 2018). Our research suggests that BKCa channels and Ca-L channels might be the major role of the ion channels which regulate vascular tone by HSYA. And more experiments are needed in the future to prove this. From our data, the vasodilatory effect of HSYA can be explained, at least partially, by its effects on BKCa and Ca-L channels. The reason for its heart beating-slowing effects, however, remains to be revealed. The mechanisms of its effects are still to be further researched (Liu et al., 2017). At present, safflower is clinically used to treat occlusive cerebrovascular disease, coronary heart disease, vasculitis and so on. Safflower yellow pigment is also used to
treat similar diseases. However, HSYA seems to have a long way to go in clinical practice because its complex mechanism of action is not yet clear. The present study provides evidences to support that HSYA is a vasodilator compound. The vasodilatory effect is related to the activation of BKCa channels, the inhibition of Ca-L channels and the reduction of intracellular calcium concentration. Though more detailed clarification on the signal pathways of the effects needs to be further researched (Misárková et al., 2016), HSYA has potential benefits for the treatment of hypertension, a very common disease (Nie et al., 2012; Jin et al., 2013; Goldberg et al., 2017). In the future, we need to further study its antihypertensive mechanism, including the role of the heart, etc. In particular, its inhibition of heart rate indicates that HSYA has a wide range of effects on the cardiovascular system. This is also worthy of special attention.
Acknowledgments The study is supported by grants from the Education Department of Sichuan Province (16ZB0196 to Wang Na), National Natural Science Foundation of China (81173661 to Yang Yan，81600381 to Pengyun Li) and Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease (0903-00021101 to Jun Cheng). The authors would like to thank Prof. Xiao-Bo Zhou and Prof. Shang-Zhong Xu for critical reviews and comments of the manuscript.
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