酸敏感离子通道与异丙酚抗伤害作用及氯喹所致非组胺性瘙痒的关系
摘要
第一部分：异丙酚的抗伤害性作用与酸敏感离子通道的关系
研究背景
异丙酚是一种新型、起效快、作用时间短的静脉麻醉药,并且与其他静脉麻醉药的化学结构有所不同。异丙酚的药理学作用非常复杂,除了具有镇静催眠的作用之外,研究表明其还具有抗伤害性感受的作用：包括降低醋酸引起的内脏痛；抑制炎症性疼痛；降低伤害性信号在脊髓水平的传导等,但其作用机制尚不十分明确。背根神经节(DRG)在机体伤害性感知中具有重要作用,并广泛表达ASIC1a和ASIC3。酸敏感离子通道(ASICs)隶属于上皮钠离子通道家族,广泛表达于中枢和外周神经系统,是由质子(H+)激活的阳离子通道,并可介导酸诱发的伤害性刺激。异丙酚是否对DRG上的ASICs具有调节作用尚不知。
目的
通过测定异丙酚对DRG神经元上的ASICs电流的影响,探讨异丙酚抗伤害性作用可能的机制；通过测定异丙酚对转染于HEK293细胞上的AISC la和ASIC3质粒电流的影响,探讨异丙酚对ASICs调节作用可能的机制。
方法
1、采用膜片钳全细胞技术测定异丙酚对DRG神经元上ASICs电流的作用
分离培养DRG神经元,待细胞贴壁后,选择15～30μM的小直径DRG神经元进行膜片钳全细胞实验,测定异丙酚对ASICs电流的影响。
2、利用膜片钳全细胞技术分别测定异丙酚对转染于人肾上皮细胞株(HEK293)上的ASIC1a和ASIC3电流的影响
采用脂质体2000,分别转染pcDNA3.0-GFP-ASIC1a和pcDNA3.0-GFP-ASIC3质粒于HEK293细胞上。转染后24-48h,选择具有绿色荧光(GFP)的细胞,采用膜片钳全细胞技术分别测定异丙酚对ASIC1a和ASIC3通道电流的影响。
结果
1、异丙酚对大鼠DRG神经元上ASICs电流的影响
(1)pH值6.0的细胞外液可诱发多数DRG神经元产生内向电流,并且内向电流主要有三种类型：①快激活快失活的电流类型；②快激活慢失活并带有小的稳态成分的电流类型；③稳态不失活的电流类型。三种内向电流的快激活相的峰值几乎完全被广谱的ASICs抑制剂(阿米洛利)所抑制,并且三种内向电流的稳态电流被部分抑制。
(2)异丙酚对DRG神经元上的三种类型的ASICs电流峰值幅度的影响
异丙酚(30μM)对三种类型的ASICs电流的峰值幅度均有抑制作用。将电压钳转换为电流钳模式后,pH6.0的溶液可激发部分DRG神经元产生动作电位并且异丙酚(30μM)可降低pH6.0的酸溶液诱发的大鼠DRG神经元的动作电位数目,但对动作电位的动力学没有影响。
2、异丙酚对DRG神经元上ASICs电流峰值幅度抑制作用的剂量-反应关系异丙酚(3、30、100、300μM)剂量依赖性地抑制ASICs电流的峰值幅度。
3、异丙酚对DRG神经元上ASICs电流H+的敏感性的影响异丙酚(30μM)对DRG神经元上ASICs电流H+的敏感性没有影响：细胞外液中加入异丙酚,ASICs电流峰值对H+的剂量-反应曲线没有明显的变化。
4、异丙酚对分别转染于HEK293细胞上的ASIC la和ASIC3电流的峰值幅度的影响
异丙酚(30μM)对HEK293细胞上转染的ASIC1a和ASIC3电流同样具有抑制作用,表明异丙酚对ASIC1a和ASIC3通道具有直接抑制作用。
结论
异丙酚对DRG神经元上ASICs通道电流的峰值幅度具有抑制作用,此作用部分解释了异丙酚抗伤害性的作用机制。
第二部分
酸敏感离子通道3与氯喹非组胺依赖性瘙痒的关系
研究背景
背根神经节(DRG)在伤害性感知中具有重要作用,并广泛表达ASIC1a和ASIC3。与ASIC1a通道的动力学有所不同,ASIC3在微酸环境(pH7.2)下即可被激活,并含有稳态电流成分,从而产生持续的内向电流,在伤害性感知方面具有更重要的作用。除疼痛外,瘙痒也是一种不愉快的伤害性的机体感受,并伴随着皮肤和神经系统的紊乱。痒觉主要通过组胺依赖性和非组胺依赖性两种途径所产生的,其中非组胺依赖性途径占主要地位,但其机制尚不清楚。临床常用抗疟药氯喹是一种非组胺依赖性致痒物质,最近常被当作工具药研究非组胺性瘙痒的细胞及分子机制。近期研究发现DRG神经元上Mas相关G蛋白偶联受体A3(MrgprA3)和瞬时感受器电位香草酸受体1(TRPV1)参与氯喹诱发的瘙痒。以往研究证实ASIC3和TRPV1都可被H+激活,并都与疼痛引起的伤害性感受机制有关,但ASIC3是否也参与氯喹性瘙痒的产生尚不清楚。
目的
通过测定氯喹对转染于中国仓鼠卵母(CHO)细胞上的ASIC3质粒电流的影响,探讨氯喹对ASIC3的调节作用及可能的结合位点；通过动物行为学实验,探讨ASIC3与氯喹性瘙痒的关系。
方法
1、转染ASIC3质粒于CHO细胞上,利用膜片钳全细胞技术测定氯喹对CHO细胞上ASIC3电流的影响
采用脂质体2000,转染pcDNA3.0-GFP-ASIC3质粒于CHO细胞上。转染后24-48h,选择具有绿色荧光的CHO细胞,利用膜片钳全细胞技术测定氯喹对ASIC3电流的影响。
2、采用快速点突变PCR技术,突变ASIC3非质子激活位点,测定氯喹与ASIC3可能的结合位点
采用快速点突变PCR技术,突变ASIC3的非质子激活位点(GLU-79或GLU-423),利用脂质体2000,分别转染突变的ASIC3质粒于CHO细胞上,应用膜片钳全细胞技术测定氯喹对CHO细胞上突变的ASIC3电流的影响。
3、采用膜片钳全细胞技术测定氯喹对DRG神经元上ASIC3样电流的作用分离培养DRG神经元细胞,待细胞贴壁后,选用小直径DRG神经元(15～30μM)进行膜片钳全细胞实验,测定氯喹对ASIC3样电流的影响。
4、采用动物行为学方法,测定ASIC3与氯喹性瘙痒的关系
将含有药物的溶液注射到小鼠面颊部,采用双盲法测定30min内小鼠前爪的擦拭次数(代表痛觉)和后爪的搔抓次数(代表痒觉)。
结果
1、氯喹对转染于CHO细胞上的ASIC3电流动力学的影响
氯喹(300μM)延长了pH7.0激活的ASIC3电流的脱敏时间,并增大了其稳态电流,但对ASIC3电流的峰值幅度没有影响。
2、不同浓度的氯喹对ASIC3稳态电流影响的剂量-效应关系
不同浓度的氯喹(30、100、300μM及1、1.5、2mM)对pH7.0激发的ASIC3的稳态电流具有剂量依赖性的增大作用。为了测定氯喹对持续开放的ASIC3电流的影响,本研究首先采用pH值7.0的细胞外液激活ASIC3,然后连续阶梯性增加氯喹浓度,结果发现ASIC3的稳态电流也相应阶梯型增大。
3、氯喹对ASIC3稳态电流pH敏感性的影响
本实验将细胞外液pH值从7.4阶梯性下降至6.8(以0.1或0.2U为一个阶梯,每阶段持续10s)。结果发现,细胞外液的pH值从7.4降至7.0时,ASIC3的稳态电流逐步增大,之后随着细胞外液pH值的降低,ASIC3的稳态电流反而逐渐变小,从而形成倒钟形曲线。细胞外液中加入氯喹(300μM),再次测定ASIC3的稳态电流对pH阶梯性改变的反应,结果显示,氯喹改变了ASIC3稳态电流对pH的敏感性。
4、氯喹对ASIC3稳态电流的影响与细胞外H+及Ca2+浓度之间的关系
本研究测定了不同pH值(pH7.0、pH6.5和pH6.0)下,氯喹(300μM)对ASIC3稳态电流的作用。结果显示：氯喹增大ASIC3稳态电流的作用随着pH值的降低而减小,然而在无钙(0mM)或高钙(3mM)的细胞外液中,氯喹增强ASIC3稳态电流的作用没有显著性变化。
5、高浓度氯喹可直接激活ASIC3,其可能的结合位点是非质子结合区(Glu-423和Glu-79)
氯喹不仅在酸性环境下增大ASIC3的稳态电流,而且在正常pH的细胞外液中,高浓度氯喹(3mM)也可直接激活ASIC3,从而产生内向稳态电流。
以往研究证实ASIC3上GLU-79和GLU-423位点为非质子结合区域,此区域介导稳态电流的激活,对质子不敏感。为进一步测定高浓度氯喹对ASIC3直接激活作用可能的位点,本研究将ASIC3的GLU-79或GLU-423位点进行突变。结果显示：氯喹(3mM)对CHO细胞上突变GLU-79或GLU-423位点的ASIC3的激活作用显著性降低。
6、氯喹对DRG神经元上ASIC3样电流的影响
ASIC3质粒形成的是同聚体通道,而研究表明AISC3同聚体与异聚体的电流动力学特性有诸多不同。DRG在伤害性感知中具有重要作用,并高表达ASIC3亚基,为此,本研究测定了氯喹(300μM)对DRG神经元细胞上ASIC3样电流的调节作用,结果显示：氯喹(300μM)对DRG神经元细胞上pH6.5激发的ASIC3样电流的稳态成分同样具有增大作用。
7、ASIC3在氯喹诱发的小鼠的搔抓反应中的作用以及ASIC3非质子区激活物质(阿米洛利、GMQ及NPFF)对小鼠搔抓反应的影响结果显示：小鼠面颊部注射氯喹后,产生了明显的搔抓反应,并且此反应可被ASIC3特异性阻断剂(APETx2)所抑制。为进一步证实这个结论,本研究测定了小鼠对皮下注射ASIC3非质子激活物质(阿米洛利、GMQ)的搔抓反应,结果发现两者均可引起小鼠的搔抓反应。以往研究表明,内源性神经肽FF(NPFF)可增强ASIC3的稳态电流。本实验将NPFF注射到小鼠面颊部发现：NPFF同样可诱发大鼠的搔抓反应。
结论
1、氯喹剂量依赖性的增大ASIC3的稳态电流,此作用具有pH值依赖性且与细
胞外液的钙离子浓度无关。
2、氯喹可通过作用于ASIC3的非质子门控区域,直接激活ASIC3.
3、ASIC3特异性阻断剂(APET×2)可阻断氯喹引起的小鼠的搔抓反应,并且ASIC3非质子激活剂(阿米洛利,GMQ和NPFF)也可诱发瘙痒反应,从而表明ASIC3可能是氯喹非组胺性瘙痒新的感受器。
第三部分
氯喹对大鼠回肠粘膜上皮细胞离子转运的影响
研究背景
在临床应用过程中,氯喹除了具有瘙痒的副作用之外,还存在胃肠道方面的副作用,包括恶心或腹泻,但其机制尚不明确。肠道离子转异常可导致水电解质吸收减少或分泌过多进而引起腹泻。研究指出氯喹可激活广泛表达于肠道上皮细胞的苦味受体(TAS2Rs),并且苦味化合物(6-PTU)可诱发大肠粘膜上皮细胞阴离子的分泌。氯喹是否影响肠道粘膜上皮细胞的离子转运及此作用是否与TAS2Rs可能的关系,尚不十分清楚。
目的
采用尤氏灌流(Ussing Chamber)测定氯喹对大鼠回肠粘膜上皮细胞离子转运的影响及可能的机制。
方法
1、采用Ussing Chamber技术,测定氯喹对大鼠回肠粘膜上皮细胞离子转运的影响
急性分离大鼠的回肠粘膜上皮,将其固定于尤氏灌流槽中,测定氯喹对大鼠回肠分泌的影响,加入各种离子通道阻断剂,探讨其可能的机制。
2、利用免疫细胞化学技术,测定参与氯喹对大鼠回肠粘膜上皮细胞离子转运的通道与TAS2Rs的关系
结果
1、氯喹对大鼠回肠粘膜上皮细胞的短路电流(Isc)具有双相作用
在较低浓度(≤5×10-4M)时,氯喹剂量依赖性地增加大鼠回肠粘膜上皮细胞的Isc,在较高浓度(≥10-3M)时,氯喹降低大鼠回肠粘膜上皮细胞的Isc。
2、河豚毒素(TTX)对氯喹诱发的大鼠回肠粘膜上皮细胞Isc变化的影响
肠道神经系统(ENS)在肠上皮细胞离子转运的调节中起着重要的作用。TTX是一种常用的钠离子通道阻断剂,可抑制肠道神经通路对肠上皮分泌的影响。为研究ENS是否参与氯喹对回肠粘膜上皮细胞Isc的影响,研究者将TTX(10-6M)加入到回肠粘膜上皮细胞的浆膜侧的电解液中孵育15min,然后入氯喹,结果显示：TTX对氯喹诱发的回肠粘膜上皮细胞Isc的变化没有影响。
3、多种离子通道阻断剂对氯喹诱发的回肠粘膜上皮细胞Isc变化的影响
为进一步研究氯喹增加回肠粘膜上皮细胞Isc的机制,我们应用多种离子通道阻断剂探讨了氯喹对回肠粘膜上皮细胞浆膜侧Isc影响可能的机制。结果显示：钙激活的氯通道(Calcium-activated chloride channels, CaCCs)参与了氯喹对回肠粘膜上皮细胞Isc的作用。
4, CaCCs-TMEM16A和TAS2Rs在大鼠回肠细胞株(IEC-18)上的关系
研究表明跨膜蛋白16A (TMEM16A)是CaCCs的分子结构。本研究采用免疫细胞化学的方法发现CaCCs-TMEM16A和TAS2Rs在IEC-18上共定位。
5、电解液中的氯离子和钙离子对氯喹诱发的大鼠回肠粘膜上皮细胞Isc变化的影响
本研究采用无氯离子或无钙离子的电解液,进一步研究氯喹对回肠粘膜上皮细胞Isc影响的机制。结果显示：在无氯离子的电解液中,氯喹对回肠粘膜上皮细胞Isc的作用显著性降低。在无钙离子的电解液中,氯喹对回肠粘膜上皮细胞Isc基线的影响几乎完全消失。
6、其他苦味化合物对大鼠回肠粘膜上皮细胞Isc的影响
本研究采用Ussing chamber测量了苦味化合物(苦精,10-4M；奎宁,10-4M)对大鼠回肠粘膜上皮细胞Isc的影响。结果表明：苦精或奎宁同样增加回肠粘膜上皮细胞的Isc。
结论
低浓度的氯喹可通过激活TAS2Rs,进而激活CaCCs导致大鼠回肠粘膜上皮细胞氯离子分泌增加,此作用不依赖于肠道神经系统,氯喹的这一机制为其胃肠道的副作用提供了新的理论依据。
Part I The role of ASICs on the anti-nociceptive effects of propofol Background
Propofol is widely used intravenous anesthetics for the induction and maintenance of general anesthesia. In addition to its anesthetic properties, several studies have shown that propofol has anti-nociceptive effects, including against visceral pain evoked by acetic acid, as well as the capacity to reduce inflammatory pain and depress nociceptive transmission at the spinal level. However, the molecular mechanisms responsible for these anti-nociceptive effects of propofol remain unclear.
Acid-sensing ion channels (ASICs) belong to the epithelial sodium channel/degenerin (ENaC/DEG) family and are activated by extracellular protons. They are widely distributed within both the central and peripheral nervous systems. ASIC activation by protons induces sodium and/or calcium influx, giving rise to depolarization and evoking action potentials in neurons. Dorsal root ganglion (DRG) cells play an important role in the perception of nociception and highly expressed ASIC la and ASIC3. However, the regulation of propofol on ASIC is still unclear.
Objective
The present researched was designed to investigate the effect of propofol on the ASICs in DRG neurons and HEK293cells transfected with either ASIC1a or ASIC3.
Methods
1、Investigated the effect of propofol on ASICs in DRG neuron using whole-cell patch clamping.
DRG neurons were isolated and placed into Petri dishes. DRG neurons in the range of15～30μm diameter were chosen to test the effect of propofol on acid-evoked currents using whole-cell patch clamping.
2、Investigated the effect of propofol on HEK193cells transfected with either ASIC la or ASIC
Cells were transfected with pcDNA3.0constructs encoding ASIC la or ASIC3and green fluorescent protein (GFP) using LipofectamineTM2000. All recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch clamping.
Result
1、Effects of propofol on acid-evoked currents in rat DRG neurons An acid-evoked inward current was observed in the majority of DRG neurons. Three main current types were observed:1) a rapidly inactivating current with a fast transient phase;2) a slow-inactivating transient current followed by a small sustained component;3) a non-inactivating current with a small transient phase.
Amiloride, a broad-spectrum proton-gated channel blocker, completely blocked all three types of transient currents and treatment with propofol (30μM) produced a significant reduction in the peak amplitude of acid-evoked currents (pH6.0) in DRG neurons. Under the current-clamp conditions, propofol decreased the number of action potentials induced by acid stimuli in rat DRG neurons.
2、Dose-response relationship of propofol on acid-evoked currents in rat DRG neurons
The peak amplitudes of the currents were reduced in response to to3,30, and300μM of propofol, indicating that the propofol-induced inhibition was dose-dependent.
3、The pH-dependent activation of ASIC currents in response to propofol in rat DRG neurons
We next investigated whether propofol affects the sensitivity of ASICs to H+. The result showed no apparent shift in the H+dose-response curve.
4、The effects of propofol on acid-evoked currents in ASIC la and ASIC3-transfected HEK293cells
Propofol significantly inhibited both ASIC1a and ASIC3currents transfected in HEK293cells, indicating that propofol directly inhibits ASIC currents.
Conclusion The present results suggested that propofol inhibits proton-gated currents in DRG neurons and this effect could partly explain the anti-nociceptive effects of propofol in primary afferent neurons.
Part Ⅱ The role of ASIC3on the chloroquine-evoked itch
Backgroud
DRG has an important role in the perception of nociception and widely expressed ASIC1a and ASIC3. Different from ASIC la, ASIC3could be activated at slightly acidic environment and has the steady state current which produces sustained inward currents. Therefore, it has a more important role in the perception of nociception. In addition to pain, itch also is an unpleasant sensation that evokes a desire to scratch, which accompanies numerous skin and nervous system disorders. The mechanisms of itch mainly include histamine-dependent and independent pathways. The histamine-independent pathway is more important, but the mechanism is unclear. Chloroquine (CQ) has been used in animal studies and in humans as a tool to study the itch mechanisms. Recent studies have found that Mas-related G protein-coupled receptor A3(MrgprA3) and Trpvl channel involved in CQ-induced itch mechanisms. Previous studies have demonstrated that ASIC3and Trpvl are concerned with the pain caused by nociceptive mechanisms and all can be activated H+. However, whether ASIC3is also involved in CQ-evoked itch is still unclear.
Objective
The present researched was designed to investigate the regulation of CQ on ASIC3currents and the relationship of ASIC3channels with CQ-evoked itch.
Method
1、Investigated the effect of CQ on Chinese Hamster Ovary (CHO) cells transfected with ASIC3.
Cells were transfected with pcDNA3.0constructs encoding ASIC3and green fluorescent protein (GFP) using LipofectamineTM2000. All recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch clamping.
2、Investigated the possible binding sites of CQ on ASIC3by quick point mutation PCR technique.
GLU-79or GLU-423site of ASIC3was mutated using quick point mutation PCR technique. CHO Cells were transfected with mutated ASIC3using LipofectamineTM2000. Recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch
3、Investigated the effect of CQ on ASIC3-like currents in DRG neuron using whole-cell patch clamping.
DRG neurons were isolated and placed into Petri dishes. DRG neurons in the range of15～30μm diameter were chosen to test the effect of CQ on ASIC3-like currents in DRG neuron using whole-cell patch clamping.
4, Investigated the relationship of ASIC3with CQ-evoked itch.
The corresponding drugs were injected in the cheek of mice and counted the number of counted scratching with the hind limb as an indicator of itch and wiping with the forelimb as an indicator of pain.
Result
1、CQ selectively potentiates the sustained currents of proton gated ion channels in ASIC3-transfected CHO cells.
CQ enhanced the sustained current and extended the time of desensitization of ASIC3, which was evoked in CHO cells transfected with ASIC3, however, the peak currents were not. Next, we tested the dose dependence of CQ-mediated enhancement of this sustained current. CQ selectively enhances the sustained current in a dose-dependent manner.
2、CQ reset pH sensitivity of sustained ASIC3current
The pH is dropped from7.4to6.8in steps of0.1or0.2U, with each step held for10seconds. In CHO cells transfected with ASIC3DNA, the activation curve of sustained ASIC3currents was traditional bell-shaped, which peaks at pH7.0. We tested CQ on the sustained current and found that it increases sustained ASIC3current and shifts the bell-shaped activation curve to right. In other words, CQ resets the pH sensitivity of sustained ASIC3current.
3、CQ-induced potentiation of the sustained current was pH dependent and Ca2+ independent.
We found that CQ dramatically enhanced the sustained component without altering the peak component. CQ-induced potentiation of the sustained current was pH dependent. We also investigated the sensitivity of CQ-induced potentiation of the sustained currents to alterations of extracellular Ca2+. The presence of either low extracellular Ca2+(0mM) or high extracellular Ca2+(3raM) has no effect on the CQ-induced potentiation of the ASIC3sustained currents, suggesting that CQ-induced enhancement of the sustained component is independent of extracellular Ca2+.
4, ASIC subunit specificity
To address ASIC subunit specificity, we recorded CQ responses in CHO cells expressing different ASIC subunits. Not like the ASIC3, the homomeric channels ASIC1a was not activated by CQ.
5、Mutation at Glu-79or Glu-423Prevents ASIC3Channel Activation by CQ
Recently, a novel nonproton ligand-sensing domain of ASIC3channels was uncovered, lined by residues around Glu-423and Glu-79of the extracellular "palm" domain of the ASIC3channel. To ascertain that CQ activates ASIC3via interactions with GMQ-sensing domain, we substituted Glu-79and Glu-423with alanine. Mutation at either Glu-79or Glu-423largerly abolished ASIC3channel activation by CQ, which support the view that CQ directly interacts with the non proton ligand.
6、CQ enhances the sustain phase of acid-activated curents in DRG neurons
CQ also selectively enhances the sustained phase of acid-evoked current.
7、 CQ and other compounds such as Ali, GMQ and NPFF, which are known to induce potentiation of the sustain phase of ASIC3current, induce itch in BALB/c mice.
We tested BALB/c mice using the "cheek" assay, which reportedly distinguishes itch and pain responses. CQ injection produces scratching, which was largely alleviated by APETx2, a selective ASIC3channel blocker. To further confirm this result, we test the itch-associated responses of other chemicals in mice, such as Ali and GMQ, which have been reported before as the nonproton ligands to activate ASIC3. Like CQ, those compounds also evoked the scratching response. Another endogenous neuropeptide FF (NPFF), which has been proved that it enhances the sustained current of ASIC3.Unsurprisingly, it also evoked the scratching response in rat.
Conclusion
1、CQ selectively enhances the sustained current in a dose-dependent manner and the potentiating effect of CQ on the sustained current was pH dependent and Ca2+independent.
2、Mutation at Glu-79or Glu-423prevents ASIC3channel activation by CQ.3、CQ and other compounds such as Ali, GMQ and NPFF, which are known to induce potentiating of the sustain phase of ASIC3current, induce itch in BALB/c mice and CQ-induced itch could be blocked by APET×2. These results indicated that ASIC3, a proton-gated ion channel, has the functions as an essential component of itch transduction.
Part Ⅲ The effects of chloriquane on the secretion of rat ileum
Backgroud
Apart from itch, CQ (a bitter tasting drug) is associated gastrointestinal side effects including nausea or diarrhea and the bitter taste receptors (TAS2Rs) are widely expressed in the intestinal tract. The bitter compound could evoke anion secretion in the large intestines of humans and rats. However, whether the side effect of CQ on electrolyte transport in rat ileum has the relation with TAS2Rs is still unclear.
Objective
The present researched was designed to investigate the effect of CQ on electrolyte transport in rat ileum and the relationship with TAS2Rs.
Method
1、Short-circuit current (ISC) was measured in vitro in Ussing chambers.
The tissue preparations were mounted between the2halves of the Ussing chambers and investigate the effect of CQ on electrolyte transport in rat ileum.
2、Investigating of CaCC-TMEM16A and bitter receptor T2R in small intestinal epithelial cells using immunohistochemistry.
Results
1、CQ evoked an increase ISC in rat ileum
CQ dose-dependently increased basal ISC at low concentraons (≤5×101-4M). However, it markedly decreased basal ISC at high concentrations (≥10-3M).
2、The mechanisms of CQ evoked an increase in ISC in rat ileum
CQ-induced increases in ISC were also abolished by CaCCs inhibitors.
Conclusion
Chloroquine could activate TAS2Rs and induces Cl-secretion in rat ileum through CaCCs, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.
研究背景
异丙酚是一种新型、起效快、作用时间短的静脉麻醉药,并且与其他静脉麻醉药的化学结构有所不同。异丙酚的药理学作用非常复杂,除了具有镇静催眠的作用之外,研究表明其还具有抗伤害性感受的作用：包括降低醋酸引起的内脏痛；抑制炎症性疼痛；降低伤害性信号在脊髓水平的传导等,但其作用机制尚不十分明确。背根神经节(DRG)在机体伤害性感知中具有重要作用,并广泛表达ASIC1a和ASIC3。酸敏感离子通道(ASICs)隶属于上皮钠离子通道家族,广泛表达于中枢和外周神经系统,是由质子(H+)激活的阳离子通道,并可介导酸诱发的伤害性刺激。异丙酚是否对DRG上的ASICs具有调节作用尚不知。
目的
通过测定异丙酚对DRG神经元上的ASICs电流的影响,探讨异丙酚抗伤害性作用可能的机制；通过测定异丙酚对转染于HEK293细胞上的AISC la和ASIC3质粒电流的影响,探讨异丙酚对ASICs调节作用可能的机制。
方法
1、采用膜片钳全细胞技术测定异丙酚对DRG神经元上ASICs电流的作用
分离培养DRG神经元,待细胞贴壁后,选择15～30μM的小直径DRG神经元进行膜片钳全细胞实验,测定异丙酚对ASICs电流的影响。
2、利用膜片钳全细胞技术分别测定异丙酚对转染于人肾上皮细胞株(HEK293)上的ASIC1a和ASIC3电流的影响
采用脂质体2000,分别转染pcDNA3.0-GFP-ASIC1a和pcDNA3.0-GFP-ASIC3质粒于HEK293细胞上。转染后24-48h,选择具有绿色荧光(GFP)的细胞,采用膜片钳全细胞技术分别测定异丙酚对ASIC1a和ASIC3通道电流的影响。
结果
1、异丙酚对大鼠DRG神经元上ASICs电流的影响
(1)pH值6.0的细胞外液可诱发多数DRG神经元产生内向电流,并且内向电流主要有三种类型：①快激活快失活的电流类型；②快激活慢失活并带有小的稳态成分的电流类型；③稳态不失活的电流类型。三种内向电流的快激活相的峰值几乎完全被广谱的ASICs抑制剂(阿米洛利)所抑制,并且三种内向电流的稳态电流被部分抑制。
(2)异丙酚对DRG神经元上的三种类型的ASICs电流峰值幅度的影响
异丙酚(30μM)对三种类型的ASICs电流的峰值幅度均有抑制作用。将电压钳转换为电流钳模式后,pH6.0的溶液可激发部分DRG神经元产生动作电位并且异丙酚(30μM)可降低pH6.0的酸溶液诱发的大鼠DRG神经元的动作电位数目,但对动作电位的动力学没有影响。
2、异丙酚对DRG神经元上ASICs电流峰值幅度抑制作用的剂量-反应关系异丙酚(3、30、100、300μM)剂量依赖性地抑制ASICs电流的峰值幅度。
3、异丙酚对DRG神经元上ASICs电流H+的敏感性的影响异丙酚(30μM)对DRG神经元上ASICs电流H+的敏感性没有影响：细胞外液中加入异丙酚,ASICs电流峰值对H+的剂量-反应曲线没有明显的变化。
4、异丙酚对分别转染于HEK293细胞上的ASIC la和ASIC3电流的峰值幅度的影响
异丙酚(30μM)对HEK293细胞上转染的ASIC1a和ASIC3电流同样具有抑制作用,表明异丙酚对ASIC1a和ASIC3通道具有直接抑制作用。
结论
异丙酚对DRG神经元上ASICs通道电流的峰值幅度具有抑制作用,此作用部分解释了异丙酚抗伤害性的作用机制。
第二部分
酸敏感离子通道3与氯喹非组胺依赖性瘙痒的关系
研究背景
背根神经节(DRG)在伤害性感知中具有重要作用,并广泛表达ASIC1a和ASIC3。与ASIC1a通道的动力学有所不同,ASIC3在微酸环境(pH7.2)下即可被激活,并含有稳态电流成分,从而产生持续的内向电流,在伤害性感知方面具有更重要的作用。除疼痛外,瘙痒也是一种不愉快的伤害性的机体感受,并伴随着皮肤和神经系统的紊乱。痒觉主要通过组胺依赖性和非组胺依赖性两种途径所产生的,其中非组胺依赖性途径占主要地位,但其机制尚不清楚。临床常用抗疟药氯喹是一种非组胺依赖性致痒物质,最近常被当作工具药研究非组胺性瘙痒的细胞及分子机制。近期研究发现DRG神经元上Mas相关G蛋白偶联受体A3(MrgprA3)和瞬时感受器电位香草酸受体1(TRPV1)参与氯喹诱发的瘙痒。以往研究证实ASIC3和TRPV1都可被H+激活,并都与疼痛引起的伤害性感受机制有关,但ASIC3是否也参与氯喹性瘙痒的产生尚不清楚。
目的
通过测定氯喹对转染于中国仓鼠卵母(CHO)细胞上的ASIC3质粒电流的影响,探讨氯喹对ASIC3的调节作用及可能的结合位点；通过动物行为学实验,探讨ASIC3与氯喹性瘙痒的关系。
方法
1、转染ASIC3质粒于CHO细胞上,利用膜片钳全细胞技术测定氯喹对CHO细胞上ASIC3电流的影响
采用脂质体2000,转染pcDNA3.0-GFP-ASIC3质粒于CHO细胞上。转染后24-48h,选择具有绿色荧光的CHO细胞,利用膜片钳全细胞技术测定氯喹对ASIC3电流的影响。
2、采用快速点突变PCR技术,突变ASIC3非质子激活位点,测定氯喹与ASIC3可能的结合位点
采用快速点突变PCR技术,突变ASIC3的非质子激活位点(GLU-79或GLU-423),利用脂质体2000,分别转染突变的ASIC3质粒于CHO细胞上,应用膜片钳全细胞技术测定氯喹对CHO细胞上突变的ASIC3电流的影响。
3、采用膜片钳全细胞技术测定氯喹对DRG神经元上ASIC3样电流的作用分离培养DRG神经元细胞,待细胞贴壁后,选用小直径DRG神经元(15～30μM)进行膜片钳全细胞实验,测定氯喹对ASIC3样电流的影响。
4、采用动物行为学方法,测定ASIC3与氯喹性瘙痒的关系
将含有药物的溶液注射到小鼠面颊部,采用双盲法测定30min内小鼠前爪的擦拭次数(代表痛觉)和后爪的搔抓次数(代表痒觉)。
结果
1、氯喹对转染于CHO细胞上的ASIC3电流动力学的影响
氯喹(300μM)延长了pH7.0激活的ASIC3电流的脱敏时间,并增大了其稳态电流,但对ASIC3电流的峰值幅度没有影响。
2、不同浓度的氯喹对ASIC3稳态电流影响的剂量-效应关系
不同浓度的氯喹(30、100、300μM及1、1.5、2mM)对pH7.0激发的ASIC3的稳态电流具有剂量依赖性的增大作用。为了测定氯喹对持续开放的ASIC3电流的影响,本研究首先采用pH值7.0的细胞外液激活ASIC3,然后连续阶梯性增加氯喹浓度,结果发现ASIC3的稳态电流也相应阶梯型增大。
3、氯喹对ASIC3稳态电流pH敏感性的影响
本实验将细胞外液pH值从7.4阶梯性下降至6.8(以0.1或0.2U为一个阶梯,每阶段持续10s)。结果发现,细胞外液的pH值从7.4降至7.0时,ASIC3的稳态电流逐步增大,之后随着细胞外液pH值的降低,ASIC3的稳态电流反而逐渐变小,从而形成倒钟形曲线。细胞外液中加入氯喹(300μM),再次测定ASIC3的稳态电流对pH阶梯性改变的反应,结果显示,氯喹改变了ASIC3稳态电流对pH的敏感性。
4、氯喹对ASIC3稳态电流的影响与细胞外H+及Ca2+浓度之间的关系
本研究测定了不同pH值(pH7.0、pH6.5和pH6.0)下,氯喹(300μM)对ASIC3稳态电流的作用。结果显示：氯喹增大ASIC3稳态电流的作用随着pH值的降低而减小,然而在无钙(0mM)或高钙(3mM)的细胞外液中,氯喹增强ASIC3稳态电流的作用没有显著性变化。
5、高浓度氯喹可直接激活ASIC3,其可能的结合位点是非质子结合区(Glu-423和Glu-79)
氯喹不仅在酸性环境下增大ASIC3的稳态电流,而且在正常pH的细胞外液中,高浓度氯喹(3mM)也可直接激活ASIC3,从而产生内向稳态电流。
以往研究证实ASIC3上GLU-79和GLU-423位点为非质子结合区域,此区域介导稳态电流的激活,对质子不敏感。为进一步测定高浓度氯喹对ASIC3直接激活作用可能的位点,本研究将ASIC3的GLU-79或GLU-423位点进行突变。结果显示：氯喹(3mM)对CHO细胞上突变GLU-79或GLU-423位点的ASIC3的激活作用显著性降低。
6、氯喹对DRG神经元上ASIC3样电流的影响
ASIC3质粒形成的是同聚体通道,而研究表明AISC3同聚体与异聚体的电流动力学特性有诸多不同。DRG在伤害性感知中具有重要作用,并高表达ASIC3亚基,为此,本研究测定了氯喹(300μM)对DRG神经元细胞上ASIC3样电流的调节作用,结果显示：氯喹(300μM)对DRG神经元细胞上pH6.5激发的ASIC3样电流的稳态成分同样具有增大作用。
7、ASIC3在氯喹诱发的小鼠的搔抓反应中的作用以及ASIC3非质子区激活物质(阿米洛利、GMQ及NPFF)对小鼠搔抓反应的影响结果显示：小鼠面颊部注射氯喹后,产生了明显的搔抓反应,并且此反应可被ASIC3特异性阻断剂(APETx2)所抑制。为进一步证实这个结论,本研究测定了小鼠对皮下注射ASIC3非质子激活物质(阿米洛利、GMQ)的搔抓反应,结果发现两者均可引起小鼠的搔抓反应。以往研究表明,内源性神经肽FF(NPFF)可增强ASIC3的稳态电流。本实验将NPFF注射到小鼠面颊部发现：NPFF同样可诱发大鼠的搔抓反应。
结论
1、氯喹剂量依赖性的增大ASIC3的稳态电流,此作用具有pH值依赖性且与细
胞外液的钙离子浓度无关。
2、氯喹可通过作用于ASIC3的非质子门控区域,直接激活ASIC3.
3、ASIC3特异性阻断剂(APET×2)可阻断氯喹引起的小鼠的搔抓反应,并且ASIC3非质子激活剂(阿米洛利,GMQ和NPFF)也可诱发瘙痒反应,从而表明ASIC3可能是氯喹非组胺性瘙痒新的感受器。
第三部分
氯喹对大鼠回肠粘膜上皮细胞离子转运的影响
研究背景
在临床应用过程中,氯喹除了具有瘙痒的副作用之外,还存在胃肠道方面的副作用,包括恶心或腹泻,但其机制尚不明确。肠道离子转异常可导致水电解质吸收减少或分泌过多进而引起腹泻。研究指出氯喹可激活广泛表达于肠道上皮细胞的苦味受体(TAS2Rs),并且苦味化合物(6-PTU)可诱发大肠粘膜上皮细胞阴离子的分泌。氯喹是否影响肠道粘膜上皮细胞的离子转运及此作用是否与TAS2Rs可能的关系,尚不十分清楚。
目的
采用尤氏灌流(Ussing Chamber)测定氯喹对大鼠回肠粘膜上皮细胞离子转运的影响及可能的机制。
方法
1、采用Ussing Chamber技术,测定氯喹对大鼠回肠粘膜上皮细胞离子转运的影响
急性分离大鼠的回肠粘膜上皮,将其固定于尤氏灌流槽中,测定氯喹对大鼠回肠分泌的影响,加入各种离子通道阻断剂,探讨其可能的机制。
2、利用免疫细胞化学技术,测定参与氯喹对大鼠回肠粘膜上皮细胞离子转运的通道与TAS2Rs的关系
结果
1、氯喹对大鼠回肠粘膜上皮细胞的短路电流(Isc)具有双相作用
在较低浓度(≤5×10-4M)时,氯喹剂量依赖性地增加大鼠回肠粘膜上皮细胞的Isc,在较高浓度(≥10-3M)时,氯喹降低大鼠回肠粘膜上皮细胞的Isc。
2、河豚毒素(TTX)对氯喹诱发的大鼠回肠粘膜上皮细胞Isc变化的影响
肠道神经系统(ENS)在肠上皮细胞离子转运的调节中起着重要的作用。TTX是一种常用的钠离子通道阻断剂,可抑制肠道神经通路对肠上皮分泌的影响。为研究ENS是否参与氯喹对回肠粘膜上皮细胞Isc的影响,研究者将TTX(10-6M)加入到回肠粘膜上皮细胞的浆膜侧的电解液中孵育15min,然后入氯喹,结果显示：TTX对氯喹诱发的回肠粘膜上皮细胞Isc的变化没有影响。
3、多种离子通道阻断剂对氯喹诱发的回肠粘膜上皮细胞Isc变化的影响
为进一步研究氯喹增加回肠粘膜上皮细胞Isc的机制,我们应用多种离子通道阻断剂探讨了氯喹对回肠粘膜上皮细胞浆膜侧Isc影响可能的机制。结果显示：钙激活的氯通道(Calcium-activated chloride channels, CaCCs)参与了氯喹对回肠粘膜上皮细胞Isc的作用。
4, CaCCs-TMEM16A和TAS2Rs在大鼠回肠细胞株(IEC-18)上的关系
研究表明跨膜蛋白16A (TMEM16A)是CaCCs的分子结构。本研究采用免疫细胞化学的方法发现CaCCs-TMEM16A和TAS2Rs在IEC-18上共定位。
5、电解液中的氯离子和钙离子对氯喹诱发的大鼠回肠粘膜上皮细胞Isc变化的影响
本研究采用无氯离子或无钙离子的电解液,进一步研究氯喹对回肠粘膜上皮细胞Isc影响的机制。结果显示：在无氯离子的电解液中,氯喹对回肠粘膜上皮细胞Isc的作用显著性降低。在无钙离子的电解液中,氯喹对回肠粘膜上皮细胞Isc基线的影响几乎完全消失。
6、其他苦味化合物对大鼠回肠粘膜上皮细胞Isc的影响
本研究采用Ussing chamber测量了苦味化合物(苦精,10-4M；奎宁,10-4M)对大鼠回肠粘膜上皮细胞Isc的影响。结果表明：苦精或奎宁同样增加回肠粘膜上皮细胞的Isc。
结论
低浓度的氯喹可通过激活TAS2Rs,进而激活CaCCs导致大鼠回肠粘膜上皮细胞氯离子分泌增加,此作用不依赖于肠道神经系统,氯喹的这一机制为其胃肠道的副作用提供了新的理论依据。
Part I The role of ASICs on the anti-nociceptive effects of propofol Background
Propofol is widely used intravenous anesthetics for the induction and maintenance of general anesthesia. In addition to its anesthetic properties, several studies have shown that propofol has anti-nociceptive effects, including against visceral pain evoked by acetic acid, as well as the capacity to reduce inflammatory pain and depress nociceptive transmission at the spinal level. However, the molecular mechanisms responsible for these anti-nociceptive effects of propofol remain unclear.
Acid-sensing ion channels (ASICs) belong to the epithelial sodium channel/degenerin (ENaC/DEG) family and are activated by extracellular protons. They are widely distributed within both the central and peripheral nervous systems. ASIC activation by protons induces sodium and/or calcium influx, giving rise to depolarization and evoking action potentials in neurons. Dorsal root ganglion (DRG) cells play an important role in the perception of nociception and highly expressed ASIC la and ASIC3. However, the regulation of propofol on ASIC is still unclear.
Objective
The present researched was designed to investigate the effect of propofol on the ASICs in DRG neurons and HEK293cells transfected with either ASIC1a or ASIC3.
Methods
1、Investigated the effect of propofol on ASICs in DRG neuron using whole-cell patch clamping.
DRG neurons were isolated and placed into Petri dishes. DRG neurons in the range of15～30μm diameter were chosen to test the effect of propofol on acid-evoked currents using whole-cell patch clamping.
2、Investigated the effect of propofol on HEK193cells transfected with either ASIC la or ASIC
Cells were transfected with pcDNA3.0constructs encoding ASIC la or ASIC3and green fluorescent protein (GFP) using LipofectamineTM2000. All recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch clamping.
Result
1、Effects of propofol on acid-evoked currents in rat DRG neurons An acid-evoked inward current was observed in the majority of DRG neurons. Three main current types were observed:1) a rapidly inactivating current with a fast transient phase;2) a slow-inactivating transient current followed by a small sustained component;3) a non-inactivating current with a small transient phase.
Amiloride, a broad-spectrum proton-gated channel blocker, completely blocked all three types of transient currents and treatment with propofol (30μM) produced a significant reduction in the peak amplitude of acid-evoked currents (pH6.0) in DRG neurons. Under the current-clamp conditions, propofol decreased the number of action potentials induced by acid stimuli in rat DRG neurons.
2、Dose-response relationship of propofol on acid-evoked currents in rat DRG neurons
The peak amplitudes of the currents were reduced in response to to3,30, and300μM of propofol, indicating that the propofol-induced inhibition was dose-dependent.
3、The pH-dependent activation of ASIC currents in response to propofol in rat DRG neurons
We next investigated whether propofol affects the sensitivity of ASICs to H+. The result showed no apparent shift in the H+dose-response curve.
4、The effects of propofol on acid-evoked currents in ASIC la and ASIC3-transfected HEK293cells
Propofol significantly inhibited both ASIC1a and ASIC3currents transfected in HEK293cells, indicating that propofol directly inhibits ASIC currents.
Conclusion The present results suggested that propofol inhibits proton-gated currents in DRG neurons and this effect could partly explain the anti-nociceptive effects of propofol in primary afferent neurons.
Part Ⅱ The role of ASIC3on the chloroquine-evoked itch
Backgroud
DRG has an important role in the perception of nociception and widely expressed ASIC1a and ASIC3. Different from ASIC la, ASIC3could be activated at slightly acidic environment and has the steady state current which produces sustained inward currents. Therefore, it has a more important role in the perception of nociception. In addition to pain, itch also is an unpleasant sensation that evokes a desire to scratch, which accompanies numerous skin and nervous system disorders. The mechanisms of itch mainly include histamine-dependent and independent pathways. The histamine-independent pathway is more important, but the mechanism is unclear. Chloroquine (CQ) has been used in animal studies and in humans as a tool to study the itch mechanisms. Recent studies have found that Mas-related G protein-coupled receptor A3(MrgprA3) and Trpvl channel involved in CQ-induced itch mechanisms. Previous studies have demonstrated that ASIC3and Trpvl are concerned with the pain caused by nociceptive mechanisms and all can be activated H+. However, whether ASIC3is also involved in CQ-evoked itch is still unclear.
Objective
The present researched was designed to investigate the regulation of CQ on ASIC3currents and the relationship of ASIC3channels with CQ-evoked itch.
Method
1、Investigated the effect of CQ on Chinese Hamster Ovary (CHO) cells transfected with ASIC3.
Cells were transfected with pcDNA3.0constructs encoding ASIC3and green fluorescent protein (GFP) using LipofectamineTM2000. All recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch clamping.
2、Investigated the possible binding sites of CQ on ASIC3by quick point mutation PCR technique.
GLU-79or GLU-423site of ASIC3was mutated using quick point mutation PCR technique. CHO Cells were transfected with mutated ASIC3using LipofectamineTM2000. Recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch
3、Investigated the effect of CQ on ASIC3-like currents in DRG neuron using whole-cell patch clamping.
DRG neurons were isolated and placed into Petri dishes. DRG neurons in the range of15～30μm diameter were chosen to test the effect of CQ on ASIC3-like currents in DRG neuron using whole-cell patch clamping.
4, Investigated the relationship of ASIC3with CQ-evoked itch.
The corresponding drugs were injected in the cheek of mice and counted the number of counted scratching with the hind limb as an indicator of itch and wiping with the forelimb as an indicator of pain.
Result
1、CQ selectively potentiates the sustained currents of proton gated ion channels in ASIC3-transfected CHO cells.
CQ enhanced the sustained current and extended the time of desensitization of ASIC3, which was evoked in CHO cells transfected with ASIC3, however, the peak currents were not. Next, we tested the dose dependence of CQ-mediated enhancement of this sustained current. CQ selectively enhances the sustained current in a dose-dependent manner.
2、CQ reset pH sensitivity of sustained ASIC3current
The pH is dropped from7.4to6.8in steps of0.1or0.2U, with each step held for10seconds. In CHO cells transfected with ASIC3DNA, the activation curve of sustained ASIC3currents was traditional bell-shaped, which peaks at pH7.0. We tested CQ on the sustained current and found that it increases sustained ASIC3current and shifts the bell-shaped activation curve to right. In other words, CQ resets the pH sensitivity of sustained ASIC3current.
3、CQ-induced potentiation of the sustained current was pH dependent and Ca2+ independent.
We found that CQ dramatically enhanced the sustained component without altering the peak component. CQ-induced potentiation of the sustained current was pH dependent. We also investigated the sensitivity of CQ-induced potentiation of the sustained currents to alterations of extracellular Ca2+. The presence of either low extracellular Ca2+(0mM) or high extracellular Ca2+(3raM) has no effect on the CQ-induced potentiation of the ASIC3sustained currents, suggesting that CQ-induced enhancement of the sustained component is independent of extracellular Ca2+.
4, ASIC subunit specificity
To address ASIC subunit specificity, we recorded CQ responses in CHO cells expressing different ASIC subunits. Not like the ASIC3, the homomeric channels ASIC1a was not activated by CQ.
5、Mutation at Glu-79or Glu-423Prevents ASIC3Channel Activation by CQ
Recently, a novel nonproton ligand-sensing domain of ASIC3channels was uncovered, lined by residues around Glu-423and Glu-79of the extracellular "palm" domain of the ASIC3channel. To ascertain that CQ activates ASIC3via interactions with GMQ-sensing domain, we substituted Glu-79and Glu-423with alanine. Mutation at either Glu-79or Glu-423largerly abolished ASIC3channel activation by CQ, which support the view that CQ directly interacts with the non proton ligand.
6、CQ enhances the sustain phase of acid-activated curents in DRG neurons
CQ also selectively enhances the sustained phase of acid-evoked current.
7、 CQ and other compounds such as Ali, GMQ and NPFF, which are known to induce potentiation of the sustain phase of ASIC3current, induce itch in BALB/c mice.
We tested BALB/c mice using the "cheek" assay, which reportedly distinguishes itch and pain responses. CQ injection produces scratching, which was largely alleviated by APETx2, a selective ASIC3channel blocker. To further confirm this result, we test the itch-associated responses of other chemicals in mice, such as Ali and GMQ, which have been reported before as the nonproton ligands to activate ASIC3. Like CQ, those compounds also evoked the scratching response. Another endogenous neuropeptide FF (NPFF), which has been proved that it enhances the sustained current of ASIC3.Unsurprisingly, it also evoked the scratching response in rat.
Conclusion
1、CQ selectively enhances the sustained current in a dose-dependent manner and the potentiating effect of CQ on the sustained current was pH dependent and Ca2+independent.
2、Mutation at Glu-79or Glu-423prevents ASIC3channel activation by CQ.3、CQ and other compounds such as Ali, GMQ and NPFF, which are known to induce potentiating of the sustain phase of ASIC3current, induce itch in BALB/c mice and CQ-induced itch could be blocked by APET×2. These results indicated that ASIC3, a proton-gated ion channel, has the functions as an essential component of itch transduction.
Part Ⅲ The effects of chloriquane on the secretion of rat ileum
Backgroud
Apart from itch, CQ (a bitter tasting drug) is associated gastrointestinal side effects including nausea or diarrhea and the bitter taste receptors (TAS2Rs) are widely expressed in the intestinal tract. The bitter compound could evoke anion secretion in the large intestines of humans and rats. However, whether the side effect of CQ on electrolyte transport in rat ileum has the relation with TAS2Rs is still unclear.
Objective
The present researched was designed to investigate the effect of CQ on electrolyte transport in rat ileum and the relationship with TAS2Rs.
Method
1、Short-circuit current (ISC) was measured in vitro in Ussing chambers.
The tissue preparations were mounted between the2halves of the Ussing chambers and investigate the effect of CQ on electrolyte transport in rat ileum.
2、Investigating of CaCC-TMEM16A and bitter receptor T2R in small intestinal epithelial cells using immunohistochemistry.
Results
1、CQ evoked an increase ISC in rat ileum
CQ dose-dependently increased basal ISC at low concentraons (≤5×101-4M). However, it markedly decreased basal ISC at high concentrations (≥10-3M).
2、The mechanisms of CQ evoked an increase in ISC in rat ileum
CQ-induced increases in ISC were also abolished by CaCCs inhibitors.
Conclusion
Chloroquine could activate TAS2Rs and induces Cl-secretion in rat ileum through CaCCs, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.
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