Hypoxic pulmonary hypertension and novel ATP-sensitive potassium channel opener: the new hope on the horizon

Hypoxic pulmonary hypertension(HPH) is a syndrome characterized by the increase of pulmonary vascular tone and the structural remodeling of peripheral pulmonary arteries.The aim of specific therapies for hypoxic pulmonary hypertension is to reduce pulmonary vascular resistance,reverse pulmonary vascular remodeling,and thereby improving right ventricular function.Iptakalim,a lipophilic para-amino compound with a low molecular weight,has been demonstrated to be a new selective ATP-sensitive potassium(K ATP) channel opener via pharmacological,electrophysiological,biochemical studies,and receptor binding tests.In hypoxia-induced animal models,iptakalim decreases the elevated mean pressure in pulmonary arteries,and attenuates remodeling in the right ventricle,pulmonary arteries and airways.Furthermore,iptakalim has selective antihypertensive effects,selective vasorelaxation effects on smaller arteries,and protective effects on endothelial cells,but no effects on the central nervous,respiratory,digestive or endocrine systems at therapeutic dose.Our previous studies demonstrated that iptakalim inhibited the effects of endothelin-1,reduced the intracellular calcium concentration and inhibited the proliferation of pulmonary artery smooth muscle cells.Since iptakalim has been shown safe and effective in both experimental animal models and phase I clinical trials,it can be a potential candidate of HPH in the future.

Iptakalim,an ATP-sensitive potassium channel opener,confers neuroprotection against cerebral ischemia/reperfusion injury in rats by protecting neurovascular unit cells

Objective:To investigate the role of iptakalim,an ATP-sensitive potassium channel opener,in transient cerebral ischemia/reperfusion (I/R) injury and its involved mechanisms.Methods:Intraluminal occlusion of middle cerebral artery (MCAO) in a rat model was used to investigate the effect of iptakalim at different time points.Infarct volume was measured by staining with 2,3,5-triphenyltetrazolium chloride,and immunohistochemistry was used to evaluate the expressions of Bcl-2 and Bax.In vitro,neurovascular unit (NVU) cells,including rat primary cortical neurons,astrocytes,and cerebral microvascular endothelial cells,were cultured and underwent oxygen-glucose deprivation (OGD).The protective effect of iptakalim on NVU cells was investigated by cell viability and injury assessments,which were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and release of lactate dehydrogenase.Caspase-3,Bcl-2 and Bax mRNA expressions were evaluated by real-time polymerase chain reaction (PCR).Results:Administration of iptakalim 0 or 1 h after reperfusion significantly reduced infarct volumes,improved neurological scores,and attenuated brain edema after cerebral I/R injury.Iptakalim treatment (0 h after reperfusion) also reduced caspase-3 expression and increased the ratio of Bcl-2 to Bax by immunohistochemistry.Iptakalim inhibited OGD-induced cell death in cultured neurons and astrocytes,and lactate dehydrogenase release from cerebral microvascular endothelial cells.Iptakalim reduced mRNA expression of caspase-3 and increased the ratio of Bcl-2 to Bax in NVU cells.Conclusions:Iptakalim confers neuroprotection against cerebral I/R injury by protecting NVU cells via inhibiting of apoptosis.

ATP-sensitive potassium channels:novel potential roles in Parkinson's disease

The ATP-sensitive potassium（KATP）channels which extensively distribute in diverse tissues（e.g.vascular smooth muscle,cardiac cells,and pancreas）are well-established for characteristics like vasodilatation,myocardial protection against ischemia,and insulin secretion.The aim of this review is to get insight into the novel roles of KATPchannels in Parkinson＇s disease（PD）,with consideration of the specificities KATPchannels in the central nervous system（CNS）, such as the control of neuronal excitability,action potential,mitochondrial function and neurotransmitter release.

Adenosine triphosphate-sensitive potassium channel opener protects PC12 cells against hypoxia-induced apoptosis through PI3K/Akt and Bcl-2 signaling pathways

Although previous studies have shown the neuroprotective effects of the adenosine triphosphate （ATP）-sensitive potassium （KATP） channel opener against ischemic neuronal damage, little is known about the mechanisms involved. Phosphatidylinositol-3 kinase （PI3K）/v-akt murine thy-moma viral oncogene homolog （Akt） and Bcl-2 are thought to be important factors that mediate neuroprotection. The present study investigated the effects of KATP openers on hypoxia-induced PC12 cell apoptosis, as well as mRNA and protein expression of Akt and Bcl-2. Results demon-strated that pretreatment of PC12 cells with pinacidil, a KATP opener, resulted in decreased PC12 cell apoptosis following hypoxia, as detected by Annexin-V fluorescein isothiocyanate/ propidium iodide double staining flow cytometry. In addition, mRNA and protein expression of phosphorylated Akt （p-Akt） and Bcl-2 increased, as detected by immunofluorescence, Western blot analysis, and reverse-transcription polymerase chain reaction. The protective effect of this preconditioning was attenuated by glipizide, a selective KATP blocker. These results demonstrate for the first time that the protective mechanisms of KATP openers on PC12 cell apoptosis following hypoxia could result from activation of the PI3K/Akt signaling pathway, which further activates expression of the downstream Bcl-2 gene.

Effect of G_(αq/11) Protein and ATP-sensitive Potassium Channels on Ischemic Preconditioning in Rat Hearts

Objectives To investigate the effect of Gαq/11 signaling pathway and ATP-sensitive potassium channel ( KATP channel ) on ischemic preconditioning (IPC) protection in rat hearts. Methods Two series of experiments were performed in Wistar rat hearts. In the first series of experiment, ischemic preconditioning was induced by left anterior descending occlusion (three, 5 min episodes separated by 5 min of reperfusion), ischemia-reperfusion injury was induced by 30 min coronary artery occlusion followed by 90 min reperfusion. Hemodynamics, infarct size and scores of ventricular arrhythmias were measured. The expression of Gαq/11 protein in the heart was measured by Western blot analysis in the second series. Results Ischemic preconditioning rats showed decreased infarct size and scores of ventricular arrhythmia vs non-IP control rats. The effect of IPC was significantly attenuated by glibenclamide (1 mg/kg, ip), a nonselective KATP channel inhibitor. IPC caused a significant increase in the expression of Gαq/11 protein. Conclusions Activations of Gαq/11 signal pathway and KATP channel played significant roles in the classical cardioprotection of ischemic precon-ditioning rat heart and might be an important mechanism of signal transduction pathway during the ischemic preconditioning.

Beneficial effects of adenosine triphosphate-sensitive K^+ channel opener on liver ischemia/reperfusion injury

AIM: To investigate the effect of diazoxide administration on liver ischemia/reperfusion injury.

K-ATP channel openers facilitate glutamate uptake by GluTs in rat primary cultured Astrocytes

Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels(K-ATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus K-ATP channel openers(KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that K-ATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters(GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium(MPP+). These potentiated effects were completely abolished by mitochondrial K-ATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C(PKC). These findings are the first to demonstrate that activation of K-ATP channel, especially mitochondrial K-ATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.

钾离子通道在抑郁症治疗中的研究进展

抑郁症是一种由多种因素引起的情感障碍性疾病,其发病率呈现逐年上升趋势,严重危害人类的身心健康。当前抗抑郁药的治疗机制主要是以增加脑内5-羟色胺、去甲肾上腺素和多巴胺等单胺递质的浓度为主,但其存在抗抑郁响应率低、不良反应严重等缺点。因此,抑郁症治疗仍未满足临床需求。近年来越来越多的临床前研究和临床实验表明,钾离子通道开放剂或拮抗剂在抑郁症治疗中具有巨大潜力。该文重点综述了近年来钾离子通道中电压门控钾离子通道、双孔钾离子通道与抑郁症的相关研究进展,为新型抗抑郁药物的研发提供新的思路。

Activation of SUR2B/Kir6.1-type K ATP channels protects glomerular endothelial,mesangial and tubular epithelial cells against oleic acid renal damage

Cumulative evidence suggests that renal vascular endothelial injury play an important role in initiating and extending tubular epithelial injury and contribute to the development of ischemic acute renal failure.Our previous studies have demonstrated that iptakalim's endothelium protection is related to activation of SUR2B/Kir6.1 subtype of ATP sensitive potassium channel(K ATP) in the endothelium.It has been reported that SUR2B/Kir6.1 channels are widely distributed in the tubular epithelium,glomerular mesangium,and the endothelium and the smooth muscle of blood vessels.Herein,we hypothesized that activating renal K ATP channels with iptakalim might have directly neroprotective effects.In this study,glomerular endothelial,mesangial and tubular epithelial cells which are the main cell types to form nephron were exposed to oleic acid(OA) at various concentrations for 24 h.0.25 μl/ml OA could cause cellular damage of glomerular endothelium and mesangium,while 1.25μl/ml OA could lead to the injury of three types of renal cells.It was observed that pretreatment with iptakalim at concentrations of 0.1,1,10 or 100 μmol/L prevented cellular damage of glomerular endothelium and tubular epithelium,whereas iptakalim from 1 to 100 μmol/L prevented the injury of mesangial cells.Our data showed iptakalim significantly increased survived cell rates in a concentration-dependent manner,significantly antagonized by glibenclamide,a K ATP blocker.Iptakalim played a protective role in the main cell types of kidney,which was consistent with natakalim,a highly selective SUR2B/Kir6.1 channel opener.Iptakalim exerted protective effects through activating SUR2B/Kir6.1 channels,suggesting a new strategy for renal injury by its endothelial and renal cell protection.

Structure of an ATP-sensitive potassium channel(K_(ATP))

Subject Code:C05 With the support by the National Natural Science Foundation of China,the collaborative research team led by Prof.Chen Lei(陈雷)at the State Key Laboratory of Membrane Biology,Institute of Molecular Medicine,Peking-Tsinghua Center for Life Sciences,Beijing Key Laboratory of Cardiometabolic

线粒体ATP敏感性钾离子通道开放剂调控冠心病大鼠心肌凋亡的功能及机制

目的:探讨线粒体ATP敏感性钾离子通道开放剂对冠心病大鼠心肌细胞凋亡的影响及机制。方法:将50只SD大鼠随机分为对照组、冠心病组及二氮嗪低、中、高剂量组,除对照组外,其余各组大鼠均用高脂饮食联合垂体后叶素构建冠心病大鼠模型,造模后二氮嗪低、中、高剂量组大鼠分别灌胃3,5,7 mg/kg的二氮嗪,每日给药1次,共14 d,对照组和冠心病组大鼠灌胃等体积的生理盐水。治疗14 d后,取各组大鼠心肌组织,苏木精-伊红(HE)染色检测心肌损伤,原位缺口末端转移酶标记法(TUNEL)检测心肌细胞凋亡,酶联免疫吸附法(ELISA)检测血清炎性细胞因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)浓度,蛋白免疫印迹法(Western Blot)检测心肌组织中Cleaved-Caspase 3、Bcl-2、Bax、磷酸化蛋白激酶B(p-AKT)、蛋白激酶B(AKT)、磷酸化磷脂酰肌醇-3-激酶(p-PI3K)、磷脂酰肌醇-3-激酶(PI3K)表达。结果:相比于对照组,冠心病组大鼠心肌损伤严重,血清TNF-α、IL-1β、IL-6显著增加,心肌细胞凋亡指数增加,Cleaved-Caspase 3和Bax表达增加,Bcl-2表达、PI3K和AKT磷酸化水平降低(P<0.05)。相比于冠心病组,二氮嗪低、中、高剂量组大鼠心肌损伤均有缓解,TNF-α、IL-1β、IL-6降低,心肌细胞凋亡指数降低,Cleaved-Caspase 3和Bax表达下调,Bcl-2表达、PI3K和AKT磷酸化水平增加(P<0.05)。结论:线粒体ATP敏感性钾离子通道开放剂二氮嗪可缓解冠心病大鼠心肌细胞损伤及凋亡,其机制为激活抗凋亡的PI3K/AKT信号通路。

钾通道启开剂研究(Ⅰ)─—反式-4-氨基-3-羟基-3,4-二氢-2,2-二甲基-2-氢-1-苯并吡喃类化合物的合成及其心血管活性

通过溴醇两步环氧化法并采用三本基膦脱氧合成了一系列新型的反式－4－氨基－3－羟基-3，4－二氢－2，2－二甲基－2－氢－1－苯并吡喃类化合物，通过IR、NMR、MS和元素分析等手段对其结构进行了确证．研究了其中13个化合物对KCI刺激大鼠胸主动脉条收缩抑制作用，考察了其体外血管扩张活性；还对其中11个化合物进行了S．D．大鼠体内降血压实验．结果表明，所测化合物对低钾刺激引起的收缩均有不同程度的抑制作用，其中化合物Ⅰ1、Ⅰ2、Ⅱ5、Ⅱ6效果较好，部分化合物显示较好的降血压活性．

3,4-二酰氧基苯骈吡喃类化合物的合成及生物活性

苯骈吡喃类钾通道启开剂为研究抗高血压药物开辟了新途径。前胡丙素具有类似结构，为寻找活性高毒副作用小的降压药物，结合二者结构特征，设计合成了２４个３，４二酰氧基苯骈吡喃类化合物，药理初筛表明某些化合物有一定程度的降压活性。

钾通道调节剂的高通量筛选模型

目的 建立钾通道调节剂高通量筛选的细胞模型。方法 96孔板上细胞负载荧光染料DiBAC4(3 ) ,测定不同化合物对荧光强度的影响 ,反映细胞膜电位的变化 ,间接反映化合物对钾通道的作用。结果 高钾去极化和钾通道阻断剂 4 AP ,TEA ,E 40 3 1,glibenclamide ,quinidine和nifedipine均能增强细胞的荧光强度 ,钾通道开放剂cromakalim能减弱细胞的荧光强度 ,上述化合物在一定剂量范围内均有剂量效应关系。利用该模型筛选 76个化合物 ,发现 9个化合物的荧光强度变化值有较好的剂量效应关系 ,有待膜片钳技术的进一步验证和筛选。结论 此方法简单 ,易于操作 ,重现性好 。

苯并吡喃-4-腙类化合物的合成及其血管舒张活性

目的 寻找高效低毒并具有组织选择性的苯并吡喃类钾通道开放剂。方法 以对氰基苯酚为原料 ,经酰化、Fries重排、环合、成腙和取代等反应合成了 3个系列 2 0个苯并吡喃 4 腙类新化合物 ,所有目标化合物结构均经IR ,1 HNMR ,MS和元素分析确证 ,并测定其对低钾 (30mmol·L- 1 KCl)和高钾 (80mmol·L- 1 KCl)诱导的大鼠主动脉条收缩抑制作用。结果 合成了 2 0个新化合物 (I1～ 9,II1～ 4 和III1～ 7)。离体扩血管活性实验表明 ,大部分化合物具有一定的血管舒张活性。结论 化合物I9,III2 和III5对低钾诱导的血管收缩抑制活性在 1× 10 - 6 mol·L- 1 浓度下略低于对照药emakalim ,但对高钾诱导的血管收缩抑制活性在浓度为 1× 10 - 5mol·L- 1 下强于对照药emakalim 。

ATP敏感性钾通道开放剂埃他卡林对大鼠低氧性肺动脉高压的影响

目的 :探讨新型ATP敏感性钾通道开放剂 (KATPCO)埃他卡林 (iptkalim ,Ipt)对低氧性肺动脉高压 (HPH)大鼠肺血管重构的影响。方法 :将大鼠置于常压低氧舱内 (O2 1 0 %± 0 .5 % ) ,8h/d ,每周 6d ,4周后测定平均肺动脉压(mPAP)、RV/ (LV +S) ;用图象分析仪测量与呼吸性细支气管伴行的肺小动脉外径 (ED)、动脉中层壁厚 (MT)、动脉管壁中层面积 (MA)、动脉管腔面积 (VA)和血管总面积 (TAA)。结果 :慢性低氧组大鼠的mPAP和RV/ (LV +S)显著高于正常对照组 (P <0 .0 1 ) ;图象分析显示低氧组大鼠肺小动脉中层壁厚与动脉外径百分比 (MT % )、动脉壁中层面积与血管总面积百分比 (MA % )均显著高于对照组 (P <0 .0 1 ) ;慢性低氧组大鼠肺小动脉管腔面积 (VA)与血管总面积 (TAA)百分比显著低于正常组 (P <0 .0 1 )。Ipt 0 .75mg·kg- 1 ·d- 1 和 1 .50mg·kg- 1 ·d- 1 均可显著抑制低氧性肺血管壁重构 ,降低肺动脉压 ,减少右心室肥厚 ,1 .50mg·kg- 1 ·d- 1 则可逆转持续低氧所致的所有病理性变化。结论

新型抗高血压药物盐酸埃他卡林对小动脉的选择性扩张作用及其药理学机制

目的 :研究新型抗高血压药物盐酸埃他卡林(Ipt)对小动脉的作用特性及其药理学机制。方法 :采用大鼠尾动脉螺旋状血管条和主动脉离体血管环两种组织 ,对比观察盐酸埃他卡林对大、小动脉扩张作用的药理学特性 ,并且利用膜片钳技术观察盐酸埃他卡林对大鼠尾动脉平滑肌细胞钾电流的影响。结果 :Ipt在 1 0 - 7～ 1 0 - 3 mol·L- 1范围内对KCl预致收缩的大鼠尾动脉血管条产生剂量依赖性舒张反应 ,且具有部分内皮依赖性 ,但对主动脉离体血管环无明显的舒张反应 ,该作用在高血压状态时显著增强 ,能被ATP敏感性钾通道特异性阻断剂格列苯脲阻断 ,并且对大鼠尾动脉平滑肌细胞的钾电流具有显著增强作用。结论 :盐酸埃他卡林具有选择性舒张小动脉作用 。

细胞外Ba^(2+)对双基因内向整流钾通道的阻断作用

目的 :研究 Ba2 +对非洲爪蟾卵母细胞表达的双基因内向整流钾通道 (IRK1- T)的阻断作用。方法 :采用双微电极电压钳 (TEV)法。结果 :细胞外 Ba2 +浓度分别为 1,3,10和 10 0μmol/ L ,K+浓度为 90 mm ol/ L ,可见 Ba2 +的阻断作用对 IRK1- T的瞬间电流 (施加电压后 1m s)具有浓度依赖性、时间依赖性和电压依赖性 ;快速开通道阻断剂 Ba2 +对 IRK1- T的通道开关特性几乎无影响作用 ,IRK1- T对之不通透。三级指数拟合表明 :细胞外 Ba2 +低浓度(1和 3μm ol/ L )时 ,Ba2 +与 K+相互竞争同一结合位点 ,随着 Ba2 +浓度的增加 ,时间常数不增加但拟合的权数却浓度依赖性增加 ,所以失活过程随 Ba2 +浓度的增加越来越快 ;细胞外 Ba2 +高浓度 (10和 10 0μm ol/ L )时 ,时间常数随Ba2 +浓度的增加而减少 ,拟合的权数却浓度依赖性减少 ,失活过程也越来越快 ,说明 Ba2 +作用位点由通道的表面部位进入通道更深的地方。结论 :Ba2 +对 IRK1-

大黄素对大鼠近端结肠平滑肌细胞电压依赖性钾通道的影响

目的研究大黄素对大鼠近端结肠电压依赖性钾离子通道的影响,以探讨其增强结肠运动的机制。方法采用全细胞膜片钳技术测定电压依赖性钾离子通道快速激活型钾电流及延迟整流型钾电流。结果大黄素(1～30μmol·L-1)浓度依赖性地阻断延迟整流性钾通道,加快电流失活,其阻断作用不需要钾通道的开放。30μmol·L-1大黄素可抑制快速激活型钾电流。5μmol·L-1大黄素对钾通道的激活动力学及失活动力学没有影响,但30μmol·L-1大黄素使其激活动力学曲线明显右移,斜率常数由(13.0±0.6)上升至(19.6±2.5)mV,同时也使失活动力学曲线明显右移。结论大黄素可阻断延迟整流型钾通道及快速激活型钾通道,其阻断作用不是开放阻断。

埃他卡林等三种结构类型的钾通道开放剂对非血管平滑肌舒张作用的选择性

目的:研究新结构类型的ATP敏感性钾通道(KATP)开放剂(KCO)、抗高血压新药埃他卡林(iptakalim,Ipt)对胃、回肠和膀胱等非血管平滑肌舒张作用的影响,并比较Ipt与其结构完全不同的KCO、氰胍类的吡那地尔(pinacidil,Pin)和苯并噻二嗪类的二氮嗪(diazoxide,Dia)舒张作用的差异,为进一步明确Ipt舒张作用的选择性特征提供一定的依据。方法:采用大鼠离体胃底、回肠和膀胱肌条3种组织,以10-5mol·L-1乙酰胆碱预收缩,观察不同浓度的药物对它们的舒张作用。结果:Ipt在10-8～10-4mol·L-1范围内对3种组织均无显著的舒张作用;Pin在10-8～10-4mol·L-1范围内对胃底条和膀胱平滑肌等均无显著的舒张作用,但在10-5和10-4mol·L-1时对回肠的舒张率分为28.8%和51.9%,诱发显著的舒张作用;Dia对3种组织的作用与Ipt相似,均不引起明显的舒张作用。结论:Ipt不影响胃、回肠和膀胱的舒张作用,选择性优于Pin,与Dia相似;化学结构类型不同的KCO对回肠、胃和膀胱的作用既相似也有不同。