Box-Behnken设计优化金雀花中总黄酮纯化工艺研究

目的 研究大孔吸附树脂纯化金雀花中总黄酮的工艺条件。方法 以乌鲁木齐石人沟采集的金雀花为原料,以超声提取的方法得到总黄酮粗提物,对比7种大孔吸附树脂对金雀花中总黄酮的吸附-解吸效果,采用Box-Behnken设计优化总黄酮纯化工艺,以树脂的吸附率和解吸率为指标,考察进样液浓度、进样体积、进样流速、洗脱液、洗脱体积、洗脱流速等因素对纯化效果的影响。结果 筛选得到AB-8型大孔吸附树脂为最适树脂,并获得最佳吸附条件和解吸条件,吸附条件：进样浓度为1.14 mg·mL^-1、进样体积为2.8 BV、进样流速为2.5 mL·min^-1;解吸条件：洗脱液浓度为70%、洗脱体积为2.0 BV、洗脱流速为3.0 mL·min^-1。在此条件下的总黄酮百分含量从1.72%提高到了22.95%,纯度升高了13.3倍。结论 本纯化工艺操作简便、可行,为金雀花中总黄酮的进一步研究提供了实验依据。     

Disruption of Cell Volume Regulation by Mercuric Chloride Is Mediated by an Increase in Sodium Permeability and Inhibition of an Osmolyte Channel in Skate Hepatocytes

The mechanism by which mercury leads to cell swelling and impairs the normal regulatory volume decrease (RVD) in cells swollen in hypotonic media was examined in hepatocytes isolated from the little skate,Raja erinacea,an osmoconforming marine elasmobranch. Skate hepatocytes treated with 50 μ HgCl₂in isotonic medium swelled to volumes double those of control cells, and this was associated with an increase in Na⁺and K⁺permeability. The gain in intracellular Na⁺exceeded the K⁺loss by 0.27 μEq/mg protein, accounting in large part for the observed cell swelling. The effects of mercury were blunted when hepatocytes were incubated in medium in which the Na⁺was replaced with K⁺, and were essentially absent when Na⁺was replaced with choline⁺, indicating an important role of Na⁺influx in mediating mercury's effects on cell volume regulation. The inhibition of RVD by mercury was prevented if the metal was administered as a mercaptide with dithiothreitol or glutathione. However, when these chelating agents were added after the mercury, only the membrane permeant dithiothreitol was able to reverse the inhibition of RVD, suggesting an intracellular site of action. Mercuric chloride also produced a concentration-dependent inhibition of the ATP-sensitive volume-regulatory osmolyte channel in skate hepatocytes, as assessed by inhibition of swelling-activated [¹⁴C]taurine efflux. [¹⁴C]Taurine efflux was inhibited at mercury concentrations (20–40 μ ) that had no effect on intracellular ATP levels or ATP/ADP ratios, consistent with a direct interaction with the channel. These findings indicate that mercury impairs cell volume regulation in skate hepatocytes at multiple sites, including the volume-regulatory osmolyte channels, and Na⁺and K⁺permeability pathways. The combined effects of increased Na⁺influx and the inability to extrude organic osmolytes may account for the inhibition of RVD.     

UPLC-MS/MS快速鉴定乔松素在人肝微粒体中的代谢产物

目的以超高效液相色谱质谱联用法(UPLC-MS/MS)快速鉴定乔松素在人肝微粒体中的代谢产物。方法以人肝微粒体体外孵育体系对乔松素进行代谢研究,以UPLC-MS/MS鉴定乔松素的体外代谢产物。结果建立了乔松素的体外代谢孵育体系,UPLC-MS/MS在6 min内检测到乔松素的4个代谢产物,分别鉴定为柚皮素、5,6,7-三羟基二氢黄酮、5,7,8-三羟基二氢黄酮和乔松素-7-葡萄糖醛酸苷,这4个代谢产物均为首次发现的乔松素人肝微粒体体外代谢产物。结论乔松素在人肝微粒体中的主要代谢途径为羟基化和葡萄糖醛酸化,5,7,8-三羟基二氢黄酮是其主要羟基化产物,乔松素-7-葡萄糖醛酸苷可能为乔松素的主要代谢失活形式。     

Suppression of ClC-3 channel expression reduces migration of nasopharyngeal carcinoma cells

Recent studies suggest that chloride (Cl-) channels regulate tumor cell migration. In this report, we have used antisense oligonucleotides specific for ClC-3, the most likely molecular candidate for the volume-activated Cl- channel, to investigate the role of ClC-3 in the migration of nasopharyngeal carcinoma cells (CNE-2Z) in vitro. We found that suppression of ClC-3 expression inhibited the migration of CNE-2Z cells in a concentration-dependent manner. Whole-cell patch-clamp recordings and image analysis further demonstrated that ClC-3 suppression inhibited the volume-activated Cl- current (I(Cl,vol)) and regulatory volume decrease (RVD) of CNE-2Z cells. The expression of ClC-3 positively correlated with cell migration, I(Cl,vol) and RVD. These results strongly suggest that ClC-3 is a component or regulator of the volume-activated Cl- channel. ClC-3 may regulate CNE-2Z cell migration by modulating cell volume. ClC-3 may be a new target for cancer therapies.     

Honey flavonoids inhibit hOATP2B1 and hOATP1A2 transporters and hOATP-mediated rosuvastatin cell uptake in vitro

1. Some flavonoids contained in the common diet have been shown to interact with important membrane uptake transporters, including organic anion transporting polypeptides (OATPs). OATP2B1 and OATP1A2 expressed in the apical membrane of human enterocytes may significantly contribute to the intestinal absorption of drugs, e.g. statins. This study is aimed at an evaluation of the inhibitory potency of selected food honey flavonoids (namely galangin, myricetin, pinocembrin, pinobanksin, chrysin and fisetin) toward hOATP2B1 and hOATP1A2 as well as at examining their effect on the cellular uptake of the known OATP substrate rosuvastatin.

2. Cell lines overexpressing the hOATP2B1 or hOATP1A2 transporter were employed as in vitro model to determine the inhibitory potency of the flavonoids toward the OATPs.

3. Chrysin, galangin and pinocembrin were found to inhibit both hOATP2B1 and hOATP1A2 in lower or comparable concentrations as the known flavonoid OATP inhibitor quercetin. Galangin, chrysin and pinocembrin effectively inhibited rosuvastatin uptake by hOATP2B1 with IC₅₀ ～1–10?μM. The inhibition of the hOATP1A2-mediated transport of rosuvastatin by these flavonoids was weaker.

4. The found data indicate that several of the tested natural compounds could potentially affect drug cellular uptake by hOATP2B1 and/or hOATP1A2 at relative low concentrations, a finding which suggests their potential for food–drug interactions.     

豨莶草总黄酮对大鼠脑缺血损伤的保护作用及其作用机制研究

目的 探讨豨莶草总黄酮对大鼠局灶性脑缺血损伤的影响及其作用机制。方法 将SD大鼠随机分为6组:假手术组、模型组、银杏叶片(50 mg/kg)组及豨莶草总黄酮低、中、高剂量(5、10、20 mg/kg)组。采用线栓法建立大鼠局灶性脑缺血模型。观察豨莶草总黄酮对大鼠神经行为、脑梗死面积和脑组织含水量的影响。测定大鼠脑组织中丙二醛(MDA)含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性。结果 豨莶草总黄酮可明显改善大鼠神经功能缺陷,减轻脑水肿,减少脑梗死面积,降低脑组织MDA含量,提高脑组织SOD及GSH-Px的活性。结论 豨莶草总黄酮有抗脑缺血损伤作用,作用机制可能与其抗氧化作用有关。     

The membrane transport of flavonoids from Crossostephium chinense across the Caco-2 monolayer

The permeation and efflux of six polysubstituted flavonoids isolated from Crossostephium chinense, a Chinese traditional and herbal drug for the treatment of diabetes, were investigated using the Caco‐2 cell monolayer. The six flavonoids (selagin, apometzgerin, tricetin‐3′,4′,5′‐trimethylether, quercetagetin‐3,6,7‐trimethylether, hispidulin and quercetagetin) sharing a similar parent skeleton structure with varied substituents in the heterocyclic ring B were selected for the study. Quercetagetin exhibited a low bi‐directional permeability comparable to that of atenolol, suggesting a paracellular diffusion mechanism. The remaining compounds exhibited time‐ and concentration‐ dependent permeation with apparent permeability coefficient (P_app) values in the range 10^?6–10^?5 cm/s, suggesting transcellular diffusion pathways. Selagin exhibited significantly larger basolateral to apical P_app than that of the reverse direction, suggesting the existence of efflux mechanisms. ATP‐depletion and probenecid pretreatment led to a significant inhibition of the efflux of selagin, whereas verapamil had no effect on the basolateral to apical transport of selagin, suggesting that multidrug resistance proteins (MRPs) and not P‐glycoprotein play a role in the intestinal efflux of selagin. Based on present and previous results, the structure–permeation relationship and the role of MRPs in mediating the efflux of flavonoids are discussed. Experimental results in this study provide useful information for pharmacological applications of the flavonoids from C. chinense. Copyright © 2010 John Wiley & Sons, Ltd.     

Copper effects on ion transport across lamprey erythrocyte membrane: Cl(-)/OH(-) exchange induced by cuprous ions.

We studied the effects of prelytic copper concentrations on cell volume, intracellular pH, and ion transport in lamprey erythrocytes. Ion fluxes and pH were measured by radioactive tracer technique, patch clamp, and flame photometry. Prelytic CuSO(4) concentration of 100 microM caused anion-dependent intracellular acidification and increase in Cl(-) influx after 2 min lag-phase. In the presence of ascorbate copper effect was amplified and lag-phase was skipped. Pretreatment of the cells with N-phenyl maleimide abolished copper-induced changes completely. Copper treatment caused an increase in Na(+) fluxes in both directions and a net Na(+) uptake. Copper-induced Na(+) transport was partially amiloride(MIA)-sensitive representing Na(+)/H(+) exchange. The nature of the amiloride-insensitive fraction of copper-activated Na(+) influx remains unknown. Cell swelling after 15 min of copper exposure induced regulatory volume decrease response involving KCl extrusion via K(+) and Cl(-) volume-sensitive channels. We suggest that the effects of copper on ion transport fit the following sequence of events: (i) cupric ions are reduced to cuprous state on the membrane surface, (ii) electroneutral pairs CuCl and CuOH mediate chloride/hydroxyl exchange, as shown before for trialkyltin, dissipating transmembrane pH gradient, and (iii) changes in intracellular pH result in the activation of the Na(+)/H(+) exchange and consecutive volume changes cause the RVD response.     

骨疏康胶囊联合利塞膦酸钠治疗绝经后骨质疏松症的临床研究

目的 比较黄芩酒炙前后化学成分变化,为建立全面的黄芩饮片质量评价方法提供参考。方法 HPLC法建立黄芩片、酒黄芩的特征图谱,并生成对照特征图谱,标定共有峰并进行相似度评价。采用UPLC-Q-TOF/MS定性分析黄芩酒炙前后化学成分,利用UPLC-TQMS对2种饮片中11种黄酮类成分（黄芩苷、黄芩素、汉黄芩苷、汉黄芩素、千层纸素A、千层纸苷、野黄芩苷、芹菜素、高车前素、木犀草苷、白杨素）进行定量分析,并以含量为变量进行多元统计分析。结果 建立了黄芩片、酒黄芩的特征图谱,标定9个共有峰,2种饮片相似度均在0.947以上。在黄芩饮片中,共发现50种成分,通过多级质谱数据分析,保留时间匹配,并结合对照品及数据库检索,鉴定了其中44种成分。UPLC-TQMS定量结果显示酒炙后黄芩中黄芩苷、汉黄芩苷等黄酮苷类成分含量略有下降,黄芩素、汉黄芩素等黄酮苷元类成分含量稍有增加。经多元统计学分析,2种饮片有明显的分离趋势,载荷图结果表明黄芩苷、汉黄芩苷、千层纸苷的含量差异可能是引起黄芩酒炙前后化学成分变化的主要因素。结论 黄芩酒炙后没有新增或消失成分,但成分含量有所变化。所建立黄芩饮片的定性、定量方法重现性好,分析快速、准确,可用于黄芩饮片酒炙前后的质量评价。     

K+通道在Jurkat细胞调节性体积减小中的作用

目的：研究K^ 通道在Jurkat细胞调节性体积减小(RVD)中的作用。方法：调节细胞外不同离子浓度，用细胞成像系统测定Jurkat细胞(人T淋巴细胞肿瘤细胞株)在不同渗透压时细胞容积的变化。结果：细胞培养液中缺少Ca2^ ，或细胞培养液中的K^ 浓度升高，或使用Ca^2 敏感的K^ 通道(KCa)阻断剂均可抑制Jurkat细胞的RVD；应激免疫抑制蛋白(ISPS)具有抑制Jurkat细胞RVD的作用。结论：Jurkat细胞中KCa通道是RVD不可缺少的因素。ISPS对免疫功能抑制作用的机理之一，可能是抑制了T淋巴细胞上的K^ 通道。     

Cardiac-specific overexpression of the human short CLC-3 chloride channel isoform in mice

1. ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2. Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3. Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4. The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I(-) > Cl(-) > Asp(-)) and inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5. Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6. In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.     

Mechanisms of Food Effects of Structurally Related Antiarrhythmic Drugs,Disopyramide and Bidisomide in the Rat

Purpose. To determine whether the rat is a good animal model for the food effects observed with bidisomide but not with the structurally similar antiarrhythmic drug, disopyramide in man and to explore a reason for the differences in the food effects of these compounds. Methods. The following effects on the absorption of bidisomide and/ or disopyramide were examined in the rat: Food effects, gastrointestinal transit time under fasting and nonfasting conditions, pH effects, hypertonic solution effect of NaCl and glucose, bile effects, permeability, inhibitory effects by Gly, Gly-Gly, Gly-Pro, glucose and mannitol and drug binding to food. Results. Remarkable food effects were observed with bidisomide but not with disopyramide. There was no difference in the GI transit time with and without food. The pH effect with and without food was similar. Effect of salt concentrations on bidisomide and disopyramide was similar. There was no bile effect on absorption of both compounds. Binding of bidisomide and disopyramide to food was similarly low. The apparent permeability of bidisomide was much lower than disopyramide especially in the ileum and its absorption was more inhibited by Gly, Gly-Gly and Gly-Pro. Conclusions. In the rat, as previously seen in humans, the food effect was observed with bidisomide but not with disopyramide. This difference was in part due to both lower intestinal permeability of bidisomide compared to disopyramide and greater inhibition of absorption by the amino acid, Gly and the dipeptides, Gly-Gly and Gly-Pro.     

Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca^2 + ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca^2 + concentration ([Ca^2 +]_C), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na⁺/Ca^2 + exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca^2 + entry and ERK/synapsin I signaling pathway.     

Effects of long-term Ailanthus altissima extract supplementation on fear,cognition and brain antioxidant levels

IntroductionAilanthus altissima is an indigenous plant known for various remedial properties. The present study aimed to evaluate the neuroprotective potential of methanolic extract Ailanthus altissima (AA) bark as current scientific trend is searching plant for neurodegenerative diseases, worldwide.MethodologyIn in-vitro experiments, the AA was analyzed for phenols, flavonoids, antioxidative and cholinesterase inhibitory properties with subsequent detailed characterization for secondary metabolites. The in-vivo neurological effects were evaluated in rats through behavioral assessment for anxiety and memory after chronic administration (28 days) of 50–200 mg/kg of AA. At the end of behavior studies, isolated brains were biochemically tested to determine antioxidant enzyme activity.ResultsAA was found rich in phenols/flavonoids and active in radical scavenging with the presence of 13 secondary metabolites in UHPLC-MS analysis. The AA yielded anxiolytic effects dose-dependently in the open field, light/dark and elevated-plus maze tests as animals significantly (P < 0.05 vs control group) preferred open arena, illuminated zone and exposed arms of maze. Similarly, the animals treated with AA showed significant (P < 0.05 vs amnesic group) increase in spontaneous alternation, discrimination index in y-maze, novel object recognition tests. Further, AA.Cr treated rats showed noticeably shorter escape latencies in Morris water maze tests.In biochemical analysis, the dissected brains AA treated rats showed reduced levels of AChE and malondialdehyde with increased levels of first-line antioxidant enzymes i.e. glutathione peroxidase and superoxide dismutase. These observed biological effects might be attributed to phenols and flavonoids constituents owned by AA. -The in-silico studies showed thatconessine and lophirone J phytocompounds have good blood–brain barrier permeability and interaction with AChE.ConclusionThe outcomes of this study validate that bark of Ailanthus altissima might work as a source of bioactive phytochemicals of neuroprotective potential.     

半枝莲和白花蛇舌草药对中总黄酮的提取及纯化工艺研究

目的 筛选出半枝莲和白花蛇舌草药对中总黄酮提取及纯化的最佳工艺条件。方法 以总黄酮提取率和浸膏量为评价指标,选择两者配比、乙醇体积分数、料液比、提取时间4个因素作为考察对象,L₉（3⁴）正交试验优选总黄酮提取工艺。然后结合静态和动态实验方法,以吸附率、解吸率为指标,确定聚酰胺树脂纯化的工艺条件及参数。结果 确定药对中总黄酮提取的工艺条件：半枝莲与白花蛇舌草药对的配比为4：1,25倍量体积分数为70%的乙醇,提取3次,每次2 h。聚酰胺树脂纯化药对中总黄酮的工艺条件：上样液中总黄酮浓度为63.14 mg/ml,pH为4.0,以1.5 ml/min的流速上样,先用流速为3 ml/min的3.1床容积（BV）的水洗脱,再用流速为3 ml/min的9.3 BV体积分数为65%的乙醇洗脱,收集洗脱液,测得总黄酮纯度为40.39%,转移率为81.57%。结论 优化的提取工艺条件稳定、可行,黄酮提取率较高。聚酰胺树脂纯化的工艺条件简便,效果良好。     

FKBP12 Depletion Leads to Loss of Sarcoplasmic Reticulum Ca2+ Stores in Rat Vas Deferens

The immunophilin 12-kDa FK506 binding protein (FKBP12) stabilizes intracellular Ca²⁺ release channel (CRC) activity in different tissues. In this work, the presence of FKBP12 in rat vas deferens (RVD) and its possible contribution to RVD function was investigated. Treatment under appropriate pH, temperature, and ionic conditions was used to strip FKBP12 from CRC binding sites; Western blotting revealed FKBP12 in control but not in treated homogenates. Disruption of the FKBP12-CRC complex in RVD decreased the Ca²⁺ content of sarcoplasmic reticulum (SR) by increasing Ca²⁺ leakage through the ryanodine receptor (RyR3 isoform) but not through 1,4,5-iNOSitol trisphosphate receptors (IP₃R1 and IP₃R3 isoforms). The decrease of SR Ca²⁺ content was not related to inhibition of SERCA ATPase. It seems that dissociation of FKBP12-RyR leads to conformational changes in RyR that make it difficult for ryanodine to access its binding site. Rapamycin, which is commonly used as a pharmacological tool to disrupt the FKBP12-RyR complex, decreased phenylephrine-induced contractions in RVD epididymal halves. The data suggest that FKBP12 is expressed in RVD in a labile association with RyR3. Disruption of the FKBP12-RyR3 complex may lead to modifications of RVD physiology and in consequence may compromise male fertility.     

金花葵粗黄酮提取物的免疫调节作用研究

目的:研究金花葵粗黄酮提取物对小鼠的免疫调节作用.方法:通过碳廓清实验、NK细胞实验、血清半数溶血值实验、抗体生成细胞实验、小鼠淋巴细胞转化实验、迟发性变态反应实验、免疫器官/脏体比实验考察金花葵粗黄酮提取物的免疫调节作用.结果:金花葵粗黄酮提取物可以增强小鼠巨噬细胞的吞噬百分率、单核吞噬细胞的吞噬指数、NK细胞活性和脾淋巴细胞增殖能力,增加脾抗体形成细胞和血清半数溶血值,但对小鼠足跖厚度及免疫器官脏体比值没有影响.结论:金花葵粗黄酮提取物具有一定的调节免疫的作用.     

Impact of the whole-cell patch-clamp configuration on the pharmacological assessment of the hERG channel: Trazodone as a case example

IntroductionVoltage- and state-dependent blocks are important mechanisms by which drugs affect voltage-gated ionic channels. However, spontaneous (i.e. drug-free) time-dependent changes in the activation and inactivation of hERG and Na⁺ channels have been reported when using conventional whole-cell patch-clamp in HEK-293 cells.MethodshERG channels were heterologously expressed in HEK-293 cells and in Xenopus laevis oocytes. hERG current (I_hERG) was recorded using both conventional and perforated whole-cell patch-clamp (HEK-293 cells), and two microelectrode voltage-clamp (Xenopus oocytes) in drug-free solution, and in the presence of the drug trazodone.ResultsIn conventional whole-cell setup, we observed a spontaneous time-dependent hyperpolarizing shift in the activation curve of I_hERG. Conversely, in perforated patch whole-cell (HEK-293 cells) or in two microelectrode voltage-clamp (Xenopus oocytes) activation curves of I_hERG were very stable for periods ~50 min. Voltage-dependent inactivation of I_hERG was not significantly altered in the three voltage clamp configurations tested. When comparing voltage- and state-dependent effects of the antidepressant drug trazodone on I_hERG, similar changes between the three voltage clamp configurations were observed as under drug-free conditions.DiscussionThe comparative analysis performed in this work showed that only under conventional whole-cell voltage-clamp conditions, a leftward shift in the activation curve of I_hERG occurred, both in the presence and absence of drugs. These spontaneous time-dependent changes in the voltage activation gate of I_hERG are a potential confounder in pharmacological studies on hERG channels expressed in HEK-293 cells.     

��1-Adrenoceptor-mediated depletion of phosphatidylinositol 4, 5-bisphosphate inhibits activation of volume-regulated anion channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE

Volume-regulated anion channels (VRACs) play an important role in cell-volume regulation. α₁-Adrenoceptor stimulation by phenylephrine (PE) suppressed the hypotonic activation of VRAC current in mouse ventricular cells and regulatory volume decrease (RVD) was also absent in PE-treated cells. We examined whether the effects of α₁-adrenoceptor stimuli on VRAC current were modulated by phosphatidylinositol signalling.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp method was used to record the hypotonicity-induced VRAC current in mouse ventricular cells. RVD was analyzed by videomicroscopic measurement of cell images.

KEY RESULTS

The attenuation of VRAC current by PE was suppressed by α_1A-adrenoceptor antagonists (prazosin and WB-4101), anti-G_q protein antibody and a specific phosphoinositide-specific phospholipase C (PLC) inhibitor (U-73122), but not by antagonists for α_1B-, α_1D- or β-adrenoceptor, or protein kinase C inhibitors. The inhibition of VRAC by PE was antagonized by intracellular excess phosphatidylinositol 4,5-bisphosphate (PIP₂), while intracellular anti-PIP₂ antibody (PIP₂ Ab) inhibited the activation of VRAC currents. When cells were loaded with phosphatidylinositol 3,4,5-trisphosphate (PIP₃) with or without PIP₂ Ab, PE little affected the VRAC current. Extracellular m-3M3FBS (an activator of PLC) suppressed VRAC in the absence of PE, and this effect was reversed by intracellular excess PIP₂.

CONCLUSIONS AND IMPLICATIONS

Our results indicate that the stimulation of α_1A-adrenoceptors by PE inhibited the activation of cardiac VRAC current via PIP₃ depletion brought about by PLC-dependent reduction of membrane PIP₂ level.