Effects of Intrathecally Administerd NaV1.8 Antisense Oligonucleotide on the Expression of Sodium Channel mRNA in Dorsal Root Ganglion

Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1.8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion （DRG） neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200--260 g were anesthetized with the intraperitoneal injection of 300 mg· kg^-1 choral hydrate. The CCI model was made by loose ligation of sciatic nerve trunk by 4--0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2--4. The animals were decapitated 14 days after the surgery. The L4--L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1.8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1.8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 μg as compared with that in CCI group （P〈0.01）, and it was even greater at the dosage of 90 μg. The intrathecally injected NaV1.8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.

Do age-associated changes of voltage-gated sodium channel isoforms expressed in the mammalian heart predispose the elderly to atrial fibrillation?

Atrial fibrillation(AF)is the most common cardiac arrhythmia worldwide.The prevalence of the disease increases with age,strongly implying an age-related process underlying the pathology.At a time when people are living longer than ever before,an exponential increase in disease prevalence is predicted worldwide.Hence unraveling the underlying mechanics of the disease is paramount for the development of innovative treatment and prevention strategies.The role of voltage-gated sodium channels is fundamental in cardiac electrophysiology and may provide novel insights into the arrhythmogenesis of AF.Na_v1.5 is the predominant cardiac isoform,responsible for the action potential upstroke.Recent studies have demonstrated that Na_v1.8(an isoform predominantly expressed within the peripheral nervous system)is responsible for cellular arrhythmogenesis through the enhancement of pro-arrhythmogenic currents.Animal studies have shown a decline in Na_v1.5 leading to a diminished action potential upstroke during phase 0.Furthermore,the study of human tissue demonstrates an inverse expression of sodium channel isoforms;reduction of Na_v1.5 and increase of Na_v1.8 in both heart failure and ventricular hypertrophy.This strongly suggests that the expression of voltage-gated sodium channels play a crucial role in the development of arrhythmias in the diseased heart.Targeting aberrant sodium currents has led to novel therapeutic approaches in tackling AF and continues to be an area of emerging research.This review will explore how voltage-gated sodium channels may predispose the elderly heart to AF through the examination of laboratory and clinical based evidence.

Voltage- and Use-dependent Effect of 7-chlor-benzylte-trahydropalmatine on Sodium Currents in Guinea Pig Ventricular Myocytes

The whole-cell patch-clamp technique was employed to obtain information about the voltage-dependence and kinetics of interaction of 7-chlor-benzylte-trahydropalmatine (7-Cl-BTHP) with cardiac sodium channels. 7-Cl-BTHP (30 mol/L) significantly decreased the peak sodium current (from 7. 8±1. 8 nA to 5. 3±1. 4 nA, P<0. 01, n=5), without producing a shift of the current-voltage curve. It shifted the inactivation curves of sodium current to hyperpolarized potentials, and the V(0.5) was shifted from - (82. 5±2. 5) mV to - (95±2.4) mV (P <0. 05, n=4). 7-Cl-BTHP produced a significant use-dependent effect that was proportional to the duration of the voltage step. In addition, 7-Cl-BTHP slowed the recovery of sodium channel from inactivation, which could explain its use-dependent effects on sodium current. The characteristics of 7-Cl-BTHP blockage suggest that this agent binds preferentially to inactivated sodium channels.

Modulation of dragon's blood on tetrodotoxin-resistant sodium currents in dorsal root ganglion neurons and identification of its material basis for efficacy

To clarify the modulation of dragon's blood on the tetrodotoxin-resistant (TTX-R) sodium currents in dorsal root ganglion (DRG) neurons and explore its corresponding material basis for the efficacy, using whole-cell patch clamp technique, the effects of dragon's blood and the combined effects of three components (cochinchinenin A, cochinchinenin B, and loureirin B) extracted from dragon's blood on the TTX-R sodium currents in acute-isolated DRG neurons of rats were observed. According to the operational definition of material basis for the efficacy of TCM established, the material basis of the modulation on the TTX-R sodium currents in DRG neurons of dragon's blood was judged from the experimental results. The drug interaction equation of Greco et al. was used to assess the interaction of the three components extracted from dragon's blood. This investigation demonstrated that dragon's blood suppressed the peak TTX-R sodium currents in a dose-dependent way and affected the activations of TTX-R sodium currents. The effects of the combination of cochinchinenin A, cochinchinenin B, and loureirin B were in good agreement with those of dragon's blood. Although the three components used alone could modulate TTX-R sodium currents, the concentrations of the three components used alone were respectively higher than those used in combination when the inhibition rates on the TTX-R sodium currents of them used alone and in combination were the same. The combined effects of the three components were synergistic. These results suggested that the interference with pain messages caused by the modulation of dragon's blood on TTX-R sodium currents in DRG neurons may explain some of the analgesic effect of dragon's blood and the corresponding material basis for the efficacy is the combination of cochinchinenin A, cochinchinenin B, and loureirin B.

Growth Differentiation Factor-15 Produces Analgesia by Inhibiting Tetrodotoxin-Resistant Nav1.8 Sodium Channel Activity in Rat Primary Sensory Neurons

Growth differentiation factor 15(GDF-15)is a member of the transforming growth factor-βsuperfamily.It is widely distributed in the central and peripheral nervous systems.Whether and how GDF-15 modulates nociceptive signaling remains unclear.Behaviorally,we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats.Electrophysiologically,we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia(DRG)neurons.Furthermore,GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents,and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction.GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels,suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel.Immunohistochemistry results showed that activin receptor-like kinase-2(ALK2)was widely expressed in DRG medium-and small-diameter neurons,and some of them were Nav1.8-positive.Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors.Inhibition of PKA and ERK,but not PKC,blocked the inhibitory effect of GDF-15 on Nav1.8 currents.These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK,which mediate the peripheral analgesia of GDF-15.

莲心碱对豚鼠心室肌细胞动作电位及钠与钙电流的影响

为探讨莲心碱 (liensinine,L ien)对心肌离子流的影响及抗心律失常作用机制。采用全细胞膜片钳技术 ,记录了 L ien对单个豚鼠心肌细胞动作电位 (AP)及纳电流 (INa)与 L -型钙电流 (ICa- L)的影响。 L ien 3～ 30μmol/ L 可剂量依赖性地降低 AP幅度 (APA)、静息电位 (RP) ,延长 AP时程。 L ien10 ,30 μm ol/ L 分别使 INa及 ICa- L从给药前的 (8.6± 2 .3) n A和 (75 8± 177) p A降至 (5 .4± 1.7)、(2 .2± 1.6 ) n A和 (335± 12 2 )、(137±10 0 ) p A。L ine10 μmol/ L 抑制 INa和 ICa- L的 I- V曲线并使后者的峰值电流电位略右移。结果表明 L ien有钠、L -型钙通道阻滞作用 。

甲基莲心碱对心肌细胞I_(Na)、I_(Ca-L)及稳态外向K^+电流的影响

目的 为证明甲基莲心碱（ Ｎｅｆｅｒｉｎｅ， Ｎｅｆ） 对单个心肌细胞离子通道电流的影响及抗心律失常作用的离子通道机制。方法 采用全细胞记录膜片钳技术，记录了 Ｎｅｆ 对培养大鼠心室肌细胞钠电流（ Ｉ Ｎａ） 及豚鼠心室肌细胞动作电位、 Ｉ Ｎａ 、 Ｌ 型钙电流（ Ｉ Ｃａ Ｌ） 及稳态外向 Ｋ＋ 电流的影响。结果 Ｎｅｆ ３０ ，１００ μｍｏｌ· Ｌ－ １ 明显抑制培养大鼠心室肌细胞 Ｉ Ｎａ ，分别从对照水平的（３４ ±０７） ｎ Ａ 降至（２１ ±０５） 和（０４ ±０２） ｎ Ａ。 Ｎｅｆ １０ μｍｏｌ· Ｌ－ １ 可降低豚鼠心室肌细胞动作电位振幅、静息电位，延长动作电位时程。 Ｎｅｆ １０ ，３０ μｍｏｌ· Ｌ－ １ 分别使豚鼠心室肌细胞 Ｉ Ｎａ 及 Ｉ Ｃａ Ｌ从给药前的（７９ ±２１） ｎ Ａ 和（６８９ ±２４３） ｐ Ａ 降至（４０ ±１４） 、（１３ ±０６） ｎ Ａ和（３７４ ±１７２） 、（１０９ ±６７） ｐ Ａ。 Ｎｅｆ １０ μｍｏｌ· Ｌ－ １ 还抑制 Ｉ Ｎａ 、稳态外向 Ｋ＋ 电流和 Ｉ Ｃａ Ｌ的 Ｉ Ｖ 曲线并使后者的峰值电流电位略右移。结论 Ｎｅｆ 有钠、 Ｌ 型钙通道阻滞作用并抑制稳态外向 Ｋ＋ 电流。

4-氨基吡啶对豚鼠心室肌钙和钠电流的影响

目的 研究 4 氨基吡啶 ( 4 AP)对心肌细胞L型钙通道和钠通道的影响。方法 用全细胞膜片钳技术考察 4 AP对豚鼠心室肌细胞L型钙电流和钠电流的作用。结果 4 AP 0 1,0 5 ,1 0mmol·L-1浓度依赖性地抑制L型钙电流 (ICa,L)和钠电流 (INa) ,抑制率分别为 ( 11 6± 1 7) % ,( 37 5± 8 3) %和 ( 5 4 5± 6 9) %以及 ( 2 2 1±14 3) % ,( 39 4± 8 8) %和 ( 6 2 3± 6 8) %。 0 5mmol·L-14 AP使ICa ,L和INaI -V曲线均上移。结论 4

卡维地洛对大鼠心室肌细胞钠通道的影响

目的研究卡维地洛对大鼠心室肌细胞膜钠通道的影响,在离子通道水平探讨卡维地洛的抗心律失常作用机制。方法用急性酶解法获得单个大鼠心室肌细胞,标准的全细胞膜片钳技术记录钠通道电流。结果①卡维地洛呈浓度依赖性抑制钠通道电流,IC50=(6.35±0.40)μmol·L-1。②10μmol·L-1卡维地洛能使心肌细胞钠通道电流-电压关系曲线明显上移,峰电流从(17.31±1.68)pA/pF减少至(6.58±1.35)pA/pF(n=8,P<0.05),激活电位、峰电位和翻转电位无明显改变。③卡维地洛能使钠通道电流失活曲线明显左移。④卡维地洛对钠通道电流的激活和复活曲线无明显影响。⑤卡维地洛呈频率依赖性地抑制钠通道电流。⑥1μmol·L-1卡维地洛能明显阻断10μmol·L-1异丙肾上腺素增加钠通道电流效应。结论卡维地洛能够抑制心肌细胞钠通道电流,呈浓度依赖性和频率依赖性。

绞股蓝总皂甙对单个豚鼠心室肌细胞的钙、钠、钾电流及动作电位的影响

目的研究绞股蓝总皂甙（ＧＰ）对成年豚鼠单个心室肌细胞的动作电位（ＡＰ）、Ｌ－型钙通道电流（ＩＣａ）、快钠通道电流（ＩＮａ）以及内向整流钾通道电流（ＩＫ１）的影响。方法膜片钳全细胞记录。结果５０ｍｇＬ－１ＧＰ能使动作电位时程（ＡＰＤ９０）由给药前的（７０８±２４）ｍｓ缩短到给药后的（３９６±１６）ｍｓ（Ｐ＜０．０１），抑制率５８．１％，动作电位幅值（ＡＰＡ）由给药前的（１２１±７．２）ｍＶ降到给药后的（８６±８．６）ｍＶ（Ｐ＜０．０１），抑制率２４．１％，静息膜电位无明显影响；５～５０ｍｇＬ－１的ＧＰ能浓度依赖性阻滞ＩＣａ和ＩＮａ，但对ＩＫ１无明显影响。结论ＧＰ对缺血心肌的保护作用在于减少“钙超载”和抑制异常兴奋性的产生。

雷诺嗪对兔心房肌细胞快钠电流的影响及使用依赖性阻滞作用

目的研究雷诺嗪对兔心房肌细胞快钠电流的影响及其是否存在使用依赖性阻滞作用。方法应用全细胞膜片嵌记录方法,了解雷诺嗪对兔单个心房肌细胞快钠电流的作用,及在不同刺激频率(1 Hz、3.3 Hz、5 Hz)时对快钠电流的影响。结果30μmmol/L雷诺嗪对兔心房肌细胞快钠电流有明显的抑制作用,其IC50为(25.6±1.8)μmmol/L,并且随着刺激频率的增加其作用加强,存在使用依赖性。结论雷诺嗪对兔心房肌细胞快钠电流有一定的抑制作用,其存在使用依赖性阻滞作用。

葛根素对大鼠心室肌细胞钠通道的影响

目的研究葛根素对大鼠心室肌细胞膜钠通道电流的影响,探讨葛根素在离子通道水平的作用机制。方法用急性酶解法获得单个大鼠心肌细胞,用标准全细胞膜片钳技术记录钠通道电流。结果0.3,1,3g/L葛根素能剂量依赖性增加钠通道电流,增加值分别(3.4±0.5)%、(34.1±2.7)%和(55.9±2.5)%,n=5。1g/L葛根素能使心肌细胞钠通道电流鄄电压曲线显著下移,最大峰电流密度从(17.4±1.6)pA/pF增加至(23.3±1.8)pA/pF,n=5,P<0.05,但原有的电流鄄电压依赖特征不变;葛根素对钠通道电流失活和复活曲线也无明显影响。1μmol/L普萘洛尔能明显阻断1g/L葛根素增加钠通道电流作用。结论葛根素能浓度依赖性地增加心肌钠大鼠通道电流。

卡维地洛对抗乌头碱诱发的大鼠心律失常作用的研究

评价卡维地洛抗大鼠实验性心律失常的作用 ,探讨其抗心律失常作用的离子通道机理 ,为临床用药提供理论依据。采用乌头碱诱发大鼠心律失常模型 ;采用膜片钳技术 ,观察乌头碱、卡维地洛对急性分离的大鼠心室肌细胞钠通道电流 (INa)的影响。结果 :诱发大鼠出现室性早搏、室性心动过速、心室颤动及心脏停搏时 ,对照组及卡维地洛组所用乌头碱的量 (μg)分别为 :室性早搏 :2 1.75± 3.4 7vs 31.81± 2 .0 4 ;室性心动过速 :2 3.5 2± 4 .13vs 36 .0 6± 3.79;心室颤动 :37.6 3± 7.94vs 6 4 .13± 1.4 0 ;心脏停搏 :6 9.31± 1.85vs 84 .6 5± 5 .2 5。利用膜片钳技术观察到 :对照组INa密度为 :5 8.6 3± 11.6 5 pA/pF ;卡维地洛组加入乌头碱后以及再加入卡维地洛后的INa密度分别为73.35± 12 .80 (pA/pF) ;10 .19± 0 .0 2 (pA/pF) ,与自身加药前相比P <0 .0 5。乌头碱及卡维地洛使INaI V曲线分别向下方及向上方移位。结论 :卡维地洛具有抗乌头碱诱发的大鼠心律失常的作用 ,其抗心律失常作用机制之一可能与Ⅰ类抗心律失常药类似 ,即抑制INa。

尼氟灭酸对大鼠心室肌细胞钠通道及动作电位的影响

目的:观察尼氟灭酸(niflumicacid,NFA)对大鼠心室肌细胞钠电流及动作电位的影响。方法:分别用全细胞膜片钳及电流钳技术记录单个心室肌细胞电压门控钠电流(INa)和动作电位(AP)。结果:尼氟灭酸(100μmol/L)能可逆性地抑制INa和AP,与对照组比较,抑制的程度分别为:60.1%±12.1%(-30mV,n=6,P<0.01)及78.2%±10.3%(n=5,P<0.01)。天冬氨酸根离子(Asp-)替代细胞外液的氯离子可引起类似尼氟灭酸的作用。结论:尼氟灭酸对钠通道有抑制作用并影响动作电位。

电压门控钠离子通道在大鼠肠易激综合征模型中的变化

目的:内脏高敏感性是肠易激综合征(IBS)的重要病理生理学机制之一,本文研究电压门控钠离子通道与内脏高敏感性之间的关系以进一步了解IBS的发病机制。方法:以新生大鼠直肠内气囊扩张制作动物模型,取成年后大鼠L6～S2脊髓背根神经节,急性酶解分离制成单细胞,采用膜片钳全细胞模式研究电压门控钠离子通道电流的峰值以及激活和失活情况。结果:造模组河豚毒敏感电流与河豚毒不敏感电流峰值之比显著高于对照组,而两组间河豚毒敏感电流和河豚毒不敏感电流激活和失活情况差异无显著性。结论:新生大鼠直肠内气囊扩张使电压门控钠离子通道发生了改变,后者可能参与了模型大鼠内脏高敏感性的发生和发展。

莲心碱对大鼠心室肌细胞I_(Na)、I_(Ca-L)及稳态外向K^+电流的影响

采用膜片钳技术，研究了莲心碱（Liensinine，Lien）对大鼠心室肌细胞动作电位及钠电流（INa）、L-型钙电流（ICa－L）及稳态外向K＋电流的影响，Lien30μmol／L可显著地降低动作电位幅度、静息膜电位，延长动作电位时程。Lien10，30μmol／L分别使INa及ICa－L从给药前的（7．9±2．2）nA和（699±175）pA降至（4．3±1．7）、（1．8±1．6）nA和（203±132）、（147±121）pA。Lien10μmol／L还抑制INa、稳态外向K＋电流和ICa－L的I－V曲线并使后者的峰值电流电位略右移。提示Lien有钠、L型钙通道阻滞作用并抑制稳态外向K＋电流，这些可解释Lien广泛的抗心律失常作用机制。

罗比卡因对大鼠背根神经节神经元河豚毒素不敏感钠电流的影响

目的 探讨酰胺类局麻药罗比卡因硬膜外麻醉对钠电流 (INa)影响。方法 以全细胞膜片钳技术记录罗比卡因对急性分离大鼠背根神经节神经元河豚毒素不敏感钠电流 (TTX RINa)的影响。结果 在电压钳的状态下 (钳制电压Vh 90mV ,刺激电压Vt0mV) ,罗比卡因 (10～10 0 0 μmol/L) 对TTX RINa均有抑制作用 ,且呈浓度依赖性 ,与其他局麻药不同的是罗比卡因使TTX R激活曲线向去极化方向移动。结论 罗比卡因对大鼠背根神经节神经元TTX RINa有抑制作用 ,且使TTX R激活曲线向去极化方向移动 。

早钠通道电流增大对心肌电生理的影响

目的应用海葵毒素(ATXⅡ)改变钠通道关闭的动力过程,研究钠通道电流变大对心肌电活动的影响。方法在酶解分离的豚鼠心肌细胞、离体心室肌标本和整体动物上,分别应用膜片钳技术、细胞内微电极技术以及心电图Ⅱ导联,记录豚鼠心肌细胞早钠通道电流、动作电位和整体心电图。结果当ATXⅡ特异性地与钠通道结合后,早钠通道电流增大。表现为早钠通道开放的出现频率增加,开放时间常数显著延长,此变化与ATXⅡ的浓度成正相关。与其相对应,心肌细胞动作电位的电位幅值明显增加,0期最大除极速率Vm ax显著增大,而静息电位基本不变;整体动物心电图出现QRS波波宽减小。结论早钠通道电流的增大,可使心肌除极速度和幅度显著增加,使兴奋波在心室内传导速度加快,导致QRS波变窄,亦可导致心律失常中早期后除极的出现。

一个新发现的SCN5A突变A1180V初步功能分析

目的我们曾报道在一个伴进行性心脏传导障碍的扩张型心肌病家系中发现了SCN5A基因的新突变A1180V,家系所有患病成员均存在A1180V突变,而年长未发病的成员及200例无血缘关系的正常人群未见该突变。为了明确该突变的致病机制,该文对这个新发现的突变A1180V进行初步的功能分析。方法将野生型和突变型cDNA分别转染至可以稳定表达SCN5Aβ亚基的HEK293细胞中,转染后48～64h进行电生理试验,膜片钳记录室温下两种钠通道的动作电位,计算激活期和稳态失活期的依赖电压,得出失活期及恢复期的时间曲线。将电生理的数据进行统计分析。结果电生理结果显示,与野生型钠通道相比,A1180V突变型通道没有明显改变激活期的电生理特点;但突变型通道改变了稳态失活期的电流-电压曲线,有-4.5mV的位移;突变型通道在去极化脉冲250ms末存在轻度增加的迟发电流;失活后的恢复期也有差异,突变型通道恢复更慢,而且更慢的恢复期导致突变型通道在受到重复去极化脉冲的刺激下出现的钠电流减少,减少的电流取决于重复刺激的速率,速率越大电流减少越多。结论A1180V突变型钠通道与野生型钠通道相比有轻度的表型变化,A1180V突变本身通过干扰钠内流影响细胞内钠稳态从而引起心脏的结构改变。

布比卡因对人肺上皮细胞短路电流影响的研究

目的布比卡因是长效酰胺类局麻药,本研究旨在观察布比卡因对人肺上皮细胞短路电流的影响,并探讨可能的机制。方法应用尤斯灌流室装置测定H441单层细胞的短路电流。由总电流减去阿米洛利抑制后的电流算得阿米洛利敏感性电流,以用药前H441单层细胞的阿米洛利敏感性电流初始值为100%对照。用100μmol·L^(-1)布比卡因处理H441细胞,在0、15、30、60 min 4个时间点提取蛋白用于Western blot,研究布比卡因对ERK1/2蛋白磷酸化的影响。结果布比卡因能够剂量依赖性抑制H441单层细胞的短路电流,此电流可被阿米洛利抑制;Western blot结果显示,布比卡因能够促进ERK1/2蛋白磷酸化。结论布比卡因通过抑制人肺上皮细胞阿米洛利敏感性电流而降低肺泡上皮离子转运,其机制可能与其促进ERK1/2蛋白磷酸化有关。临床上对伴有肺部疾患的病人应用布比卡因时应考虑其可能对肺泡上皮液体清除的影响。