摘要
研究背景
我们的前期研究发现促胃肠动力药zacopride (5-HT4受体激动剂兼5—HT3受体拮抗剂)对大鼠心室肌内向整流钾电流(inward rectifier potassium current,IK1)表现出选择性增强作用,同时对大鼠心律失常模型表现出抗室性心律失常作用,但是其激动IK1通道的分子机制还远未阐明。已有报道显示,心房肌IK1上调或激动心房5-HT4受体都可能对心房电生理活动产生不利影响,导致心房纤颤等房性心律失常。尽管我们在研究zacopride激动心室肌IK1通道的实验过程未发现对心房电活动的不良影响,但zacopride已被证实是心肌选择性IK1激动剂和5-HT4受体激动剂,因此,不能排除它激动心房IK1通道或激活心房5-HT4受体,并导致潜在的致房性心律失常副作用的可能性。本研究旨在阐明zacopride对心房肌IK1的作用及激动心肌IK1的分子机制,将有助于深入研究IK1通道激动剂的抗心律失常作用及其潜在的临床应用价值。
目前已知,构成IK1通道的亚单位主要来自Kir2亚家族(Kir2.x)中的Kir2.1、Kir2.2和Kir2.3,由它们构成同源及／或异源四聚体通道。编码Kir2.1、 Kir2.2和Kir2.3的基因分别是KCNJ2、KCNJ12和KCNJ4。鉴于不同种属和不同组织部位的IK1电流由不同Kir2.x亚型介导形成,而不同Kir2.x亚型又具有不同的电生理学特性,因此我们推测存在这样一种可能性,即zacopride对Kir2.x亚型的作用是具有选择性的,只针对某类亚型有激动效应,进而使zacoprid对Kir2.x亚型构成不同的大鼠心室肌和心房肌IK1产生不同效应,在抗室性心律失常的同时却不会影响心房的电活动。
一般认为,包括Kir2.1、Kir2.2和Kir2.3在内的Kir2.x通道蛋白的磷酸化是IK1调节的重要机制,并主要涉及PKC和PKA信号转导系统。结合前期动物实验中,zacoprid未引发类似激动5-HT4受体和/或阻断5-HT3受体而导致的异常心电活动,我们推测zacoprid激动IK1是由对其敏感的Kir2.x亚型通道蛋白的磷酸化介导的,而非经5-HT受体途径。
综上所述,本研究拟明确zacopride对大鼠心房肌IK1的作用；分析大鼠心室肌和心房肌IK1通道的Kir2.x亚单位构成和不同亚单位对zacopride激动效应的敏感性,并探讨这种激动效应的信号转导机制,以阐明zacopride激动IK1的分子机制
目的
1.了解zacopride对大鼠心房肌IK1的影响；大鼠心室肌和心房肌的Kir2.x亚型构成；明确zacopride对Kir2.1、Kir2.2和Kir2.3通道的作用。
2.利用干预5-HT受体的工具药观测zacopride激动IK1的作用与细胞膜5-HT4受体和5-HT3受体的关系；利用干预PKA、PKC、PKG信号通路的工具药和基因突变技术分析zacopride激动其敏感Kir2.x通道的信号转导通路。
方法
采用胶原酶法急性分离大鼠心房肌细胞；利用全细胞膜片钳技术观察zacopride对大鼠心房肌细胞IK1电流和细胞跨膜电位的效应。运用Western blots分析大鼠心房肌及心室肌kir2.x通道构成。通过RT-PCR法获得的大鼠心肌Kir2.1、Kir2.2、和Kir2.3基因,并将Kir2.1基因进行点突变获得其磷酸化位点突变基因Kir2.1(S425L);将上述基因插入质粒pEGFP-N1将其转染HEK293细胞,构建大鼠心肌细胞Kir2.x异源表达系统。通过全细胞膜片钳技术了解zacopride对异源表达的大鼠心肌细胞kir2.x通道电流的效应,以及干预5-HT受体和干预PKA、PKC、PKG信号通路的工具药对zacopride调节IK1作用的影响。
结果
1.1μmol/L zacopride对大鼠心房肌细胞IK1电流、静息电位水平和动作电位幅度与时程均无显著作用。
2. Kir2.1、Kir2.2和Kir2.3在大鼠心房及心室均有表达,Kir2.3表达量在大鼠心房及心室无明显差异,而大鼠心房Kir2.1表达量仅为心室相应量的25%,而Kir2.2在心房及心室的含量均较少。Kir2.1和Kir2.3分别是构成大鼠心室肌和心房肌的主要Kir2.x亚型。
3.大约40%-60%瞬时转染后HEK293细胞表达绿色荧光蛋白,同时可记录到IK1电流,表明细胞模型构建成功。100p.mol/L zacopride可使HEK293细胞上表达的同源Kir2.1外向电流增加40.7%±9.7%(在-50mV，P<0.01),对内向电流无显著影响(在-110mV，P>0.05)。100μmol/L zacopride对同源Kir2.2、同源Kir2.3及异源Kir2.1+Kir2.2、Kir2.1+Kir2.3和Kir2.2+Kir2.3通道电流均无明显作用。表明100μmol/L zacopride只对HEK293细胞上表达的同源Kir2.1通道电流的外向部分有激动作用。
4. HEK293细胞上未检测到5-HT3受体,5-HT4受体有少量表达。10μmol/L5-HT4受体拮抗剂RS23597-190不能消除100μmol/L zacopride激动Kir2.1电流的效应(P>0.05)。并且,同为5-HT4受体激动剂和5-HT3受体阻断剂的2-1-(4-Piperonyl) piperazinylbenzothiazole (PBT)(100μmol/L)不能激动反而抑制Kir2.1电流,使-110mV处内向电流减小31.4%±8.6%(P<0.05)。以上结果支持zacopride激动Kir2.1电流的效应与HEK293细胞的5-HT3受体和5-HT4受体的作用无关。PKA抑制剂KT5720能够显著抑制100μmol/L zacopride激动Kir2.1电流的效应(P>0.05),
5.5μmol/L特异性PKC抑制剂GF109203X或5μmol/L特异性PKG抑制剂KT5823不能消除100μmol/L zacopride激动HEK293细胞表达的Kir2.1电流的效应(P>0.05),而5μmol/L特异性PKA抑制剂KT5720能够显著抑制100μmol/L zacopride激动Kir2.1电流的效应(P>0.05)。50μmol/L泉苷酸环化酶激活剂forskolin可使在HEK293细胞上表达的Kir2.1电流外向部分增大24.1%±5.9%(在-50mV, P<0.01);50μmol/L外源性环磷酸腺苷8-bromo-cAMP可使Kir2.1电流外向部分增大22.1%±1.9%(在-50mV,P<0.05)。替换Kir2.1PKA磷酸化位点S425生成的突变体Kir2.1S425L,对zacopride不敏感,100μmol/L zacopride在-50mV可使突变体电流增加13.3%±6.5%,但无统计学意义(P>0.05)。以上结果提示zacopride激动Kir2.1电流的效应是经AC/cAMP/PKA信号通路介导的。
结论
综合上述研究结果,我们得出以下结论：
1. zacopride作为大鼠心室肌IK1选择性激动剂,对大鼠心房肌IK1却没有显著作用,对心房肌静息电位水平和动作电位幅度与时程均无明显影响。
2. zacopride可激动HEK293细胞同源Kir2.1通道,而对同源Kir2.2、Kir2.3以及异源Kir2.1-Kir2.2、Kir2.1-Kir2.3和Kir2.2-Kir2.3通道均无显著作用。
3.构成大鼠心室肌和心房肌IK1通道的亚单位组分不同,心室肌以Kir2.1表达为主,心房肌以Kir2.3为主,这是zacopride对大鼠心室肌和心房肌IK1具有不同作用的分子基础,为zacopride可选择性防治某些室性心律失常的作用提供了理论依据。
4. zacopride增强Kir2.1电流通道的机制主要涉及激活AC/cAMP/PKA信号通路,而与其对5-HT3和5-HT4受体的作用无关,这与前期报道的zacopride增强心室肌IK1通道的作用机制是一致的。
Background
Recently, we reported that zacopride, a known potent5-HT3receptor antagonist and5-HT4receptor agonist which is experimentally used as a gastrointestinal prokinetic agent, is also a selective Ik1channel agonist in rat ventricular myocytes and can suppress triggered ventricular arrhythmia. Upregulation of Ik1in atrial myocytes and activation of5-HT4receptor in atria may lead to potential adverse effect on atrial electrophysiology. It is worth noting that zacopride actually does not induce any atrial arrhythmia when applied to treat triggered ventricular arrhythmia in the rat, although it is the selective agonists both for5-HT4receptor and Ik1channel. The mechanism underlying this "atrial waive" character is poorly understood and is the main focus of the present study.
It is known that Kir2.1(KCNJ2), Kir2.2(KCNJ12) and Kir2.3(KCNJ4) channels which assembled as homotetramers or heterotetramers are the molecular basis of native pore-forming subunit of Ik1channels in the heart. Tissue and species-specific expression profiles of Kir2.x isoforms determine the biophysical and physiological characteristics of Ik1channel in the heart. Based on these understandings, we hypothesize that zacopride exerts differential effects on atrial and ventricular Ik1due to its selective actions on different Kir2.x channels.
It is also known that all of the Kir2.x channels, including Kir2.1, Kir2.2and Kir2.3channels, are the substrates of PKA and PKC. Thus, it is expected that phosphorylations of these Kir2.x channel pore-forming proteins may play important regulatory roles in Ik1function. We surmise that zacopride modulates Ik1channel activities potentially via acting on the phosphorylations of certain Kir2.x proteins, rather than via the5-HT receptor. This signaling pathway may potentially serve as a mechanism by which zacopride suppresses ventricular arrhythmias without inducing atrial arrhythmias.
Objective
In support of this hypothesis, we studied the effects of zacopride on Ik1in rat atrial myocytes, and further observed the effects of zacopride on the homotetrameric or heterotetrameric channels of Kir2.1, Kir2.2, Kir2.3and a mutant Kir2.1channel (Kir2.1S425L) transfected in HEK293cells, aiming to determine which of these Kir2.x channels is the target of zacopride and what is the signaling pathway underlying the tissue-and channel protein-specific actions of the drug.
Methods
Rat atrial myocytes were isolated and routine whole cell recording was performed to measure the Ik1currents. Respective rat cardiac orthologs of Kir2.1, Kir2.2and Kir2.3were cloned by reverse transcriptase-PCR. S425L mutation of KCNJ2gene (encoding Kir2.1) was introduced by site-directed mutagenesis. cDNAs were subcloned into the eukaryotic expression vector pEGFP-Nl. HEK293cells were transfected with genes encoding Kir2.1, Kir2.2, Kir2.3and Kir2.1S425L, and then the respective Ikir2.x were recorded using the same patch clamp technique. Western blots were performed to estimate the relative expression levels of Kir2.x channels in rat ventricles and atria.
Results
1.1.0μmol/L zacopride did not affect the rat atrial IK1and action potential (P>0.05).
2. Kir2.1, Kir2.2and Kir2.3proteins were all expressed in rat ventricles and atria. Similar levels of Kir2.3protein were expressed in atria and ventricles, but atrial Kir2.1protein level was only25%of that measured in the ventricle.
3. Zacopride at100μmol/L significantly increased the outward component of IKir2.1 by40.7%±9.7%at-50mV (P<0.01), but did not affect the inward component of Ikir2.1(changed by9.6%±4.2%at-110mV, P>0.05). Zacopride (100μmol/L) did not affect the currents of both Kir2.2and Kir2.3channels (P>0.05).The heteromeric (Kir2.1plus Kir2.2, Kir2.2plus Kir2.3and Kir2.1plus Kir2.3) channels were insensitive to zacopride (100μmol/L).
4. In HEK293cells which do dot express5-HT3receptor, the enhancement of Ikir2.1by zacopride(100μmol/L) could not be inhibited by a5-HT4receptor antagonist RS23597-190(10μmol/L)(P<0.05).2-1-(4-Piperonyl) piperazinylbenzothiazole (PBT), a known selective5-HT4receptor agonist, did not show an effect on the outward component of IKir2.1at-50mV (P>0.05), observably decreased (rather than increased) the inward IKir2.1by31.4%±8.6%at-110mV (P<0.05).
5. The PKA inhibitor (KT5720,5μmol/L) diminished the Kir2.1-enhancing effect of zacopride alone on the outward component when coapplicated with and zacopride. In contrast, PKC inhibitor (GF109203X,5μmol/L) and the PKG inhibitor (KT5823,5μmol/L) did not significantly alter the Kir2.1activator effect of zacopride. At-50mV,50迷mol/L forskolin (adenylyl cyclase activator) increased the Kir2.1current by24.1%±5.9%(P<0.01) and50μmol/L8-bromo-cAMP (exogenous cAMP) increased the current by22.1%±1.9%(P<0.05). At-50mV, zacopride induced only a mild and statistically insignificant increase of Kir2.1(S425L) current (by13.3%±6.5%,P>0.05).
Conclusion
1. Zacopride failed to impact the Ik1in rat atrial myocytes in which Kir2.3is the predominant isoform of Ik1pore-forming subunits.
2. Zacopride could increase the currents of Kir2.1homomeric channels but not the Kir2.2and Kir2.3homomeric channels and Kir2.1-Kir2.2, Kir2.1-Kir2.3and Kir2.2-Kir2.3heteromeric channels.
3. Zacopride regulates Kir2.1channel via a5-HT receptor-independent pathway
4. Zacopride increases Kir2.1current via a PKA-mediated signaling pathway
In conclusion, the present pharmacological study demonstrates that zacopride selectively activates the homomeic Kir2.1channel via a PKA-mediated signaling pathway. This finding may suggest an important mechanism by which zacopride exerts an Ik1-agonizing effect in the ventricle but not in atria. This unique feature of zacopride suggests that the potential risk of atrial fibrillation induction by this agent may be overestimated when using it as a drug of anti-ventricular arrhythmia. The study may shed light on the development of Ik1channel agonists as anti-arrhythmia drugs.
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