血管外膜源性舒张因子的作用机制

目的： 验证血管外周脂肪组织释放血管外膜源性舒张因子(ADRF)，并对ADRF的作用机制做初步探讨。方法：检测WKY大鼠带完整血管外周脂肪组织的血管环和不带血管外周脂肪组织的裸血管环的收缩力；并用液体转移方法鉴证血管外周脂肪组织释放ADRF，使用工具药对ADRF作用途径进行观察。结果：与配对裸血管相比，带完整血管外周脂肪组织的胸主动脉在苯肾上腺素(PHE,10-6 mol/L)诱发的血管收缩力下降20.5%；把孵育带完整血管外周脂肪组织的胸主动脉的生理池液体转移到配对裸血管，引起裸血管舒张20.8%；在无钙液中，有无血管外周脂肪组织的血管之间PHE诱发的收缩力无区别；钙激活的钾通道 (K_Ca)阻断剂四乙胺和ATP敏感性钾通道（K_ATP）阻断剂格列苯脲均有效阻断ADRF的血管舒张作用。结论：血管外周脂肪组织释放一种引起血管舒张的ADRF，ADRF通过激活K_Ca、K_ATP发挥作用，且需钙离子参与。     

线粒体KATP通道开放剂与缺氧预适应对乳鼠窦房结细胞起搏离子流作用的比较

目的： 通过比较线粒体K_ATP通道开放剂及缺氧预适应（HP）对缺氧/复氧（H/R）乳鼠窦房结细胞起搏离子流（I_f）的作用，探讨HP对H/R时窦房结细胞电生理活动的保护机制。 方法： 取培养 2 d 的乳鼠窦房结细胞，随机分为①对照组；②H/R组；③HP组；④ diazoxide（线粒体KATP通道开放剂）+H/R组；⑤5-HD（线粒体K_ATP通道阻断剂）+HP组。以全细胞膜片钳技术检测I_f。 结果： ①H/R组各指令电压下的If密度显著高于对照组，激活曲线右移，半数最大激活电压由(-98.9±2.4)mV变为(-85.1±2.5)mV(P＜0.01)；②HP及diazoxide预处理能显著抑制I/R后升高的I_f密度，使激活曲线左移，半数最大激活电压分别为(-90.7±5.0)mV(P＜0.01)及(-92.2±1.9)mV(P＜0.01)；③5-HD预处理阻断HP效应，使I_f密度增加并使激活曲线右移，半数最大激活电压为(-86.3±2.7)mV(P＜0.01)。 结论： 线粒体K_ATP通道开放剂预处理能模拟HP效应，对抗H/R对乳鼠窦房结细胞I_f 的影响，有助于维护H/R时窦房结细胞电生理活动的相对稳定性。     

链脲佐菌素诱导的糖尿病早期小鼠胸主动脉钾通道变化

目的:探讨链脲佐菌素诱导的糖尿病早期小鼠胸主动脉钾通道的变化。方法:实验采用离体血管的方法测定糖尿病鼠和正常鼠胸主动脉环对血管收缩剂:60mmol/LKCl和苯肾上腺素(PE)、内皮非依赖性舒张剂:硝普钠(SNP)以及电压依赖性钾通道(K_V通道),钙激活型钾通道(K_Ca通道),ATP敏感钾通道(K_ATP通道)阻断剂的反应。结果:糖尿病鼠胸主动脉环对60mmol/LKCl、PE和SNP的效应都显著大于对照组;K_Ca通道阻断剂四乙铵(TEA)显著降低糖尿病小鼠胸主动脉环在PE的激动下SNP的舒张效应,而且其-logIC₅₀的差值较对照组显著增大;K_V通道阻断剂4-氨基吡啶(4-AP)显著降低糖尿病和正常小鼠胸主动脉环对SNP的舒张效应,但是-logIC₅₀差值无显著差异;K_ATP通道阻断剂格列苯脲(Glibenclamide)显著降低糖尿病小鼠胸主动脉环对SNP的舒张效应,而对照组无显著阻断作用,-logIC₅₀的差值也无显著差异。结论:糖尿病早期小鼠胸主动脉平滑肌细胞K_Ca通道的开放或表达显著增强,也证实了K_ATP通道开放增强。     

磷酯酶A2抑制剂DTNB对大鼠心肌 缺血再灌注心律失常的影响

目的:研究非钙依赖性的磷脂酶A₂ (PLA₂)和ATP敏感性钾通道(K_ATP)在大鼠心肌缺血再灌注(I/R)心律失常中的作用。方法:结扎大鼠左冠状动脉前降支造成缺血10 min,然后放开再灌注10 min。在心肌缺血前5 min分别给予PLA₂抑制剂5,5'-dithio-bis(2-nitrobenzoic acid)(DTNB)(16 mg/kg),K_ATP开放剂pinacidil(0.2 mg/kg),K_ATP阻断剂glibenclamide(0.3 mg/kg)。结果:与对照组相比,DTNB可降低心律失常评分、磷酸肌酸激酶(CPK)和乳酸脱氢酶 (LDH)值(P＜0.05);而glibenclamide对I/R心律失常无影响,但升高CPK,LDH值(P＜0.05);与glibenclamide组比,在glibenclamide阻断K_ATP后,DTNB可引起致死性心律失常,并可升高LDH值(P＜0.05);pinacidil可完全抑制I/R引起的室颤和室速。结论:PLA₂抑制剂DTNB可减轻I/R损伤,表明PLA₂在I/R心律失常中起着重要作用,同时也表明激活的K_ATP具有抗I/R心律失常的作用。但DTNB对抗I/R心律失常的作用与K_ATP的关系仍有待进一步研究。     

雌激素通过KATP通道影响大鼠肠系膜动脉的反应性

目的: 观察雌激素对大鼠肠系膜动脉平滑肌ATP敏感性钾离子通道 (K_ATP 通道)mRNA表达的影响,探讨K_ATP通道在雌激素调节大鼠肠系膜血管反应性中的作用。方法: 雌性SD大鼠48只,体重(100±10) g,随机分为假手术组(sham)、卵巢切除组(Ovx)和卵巢切除后补充雌激素组(Ovx +E)。采取实时荧光定量PCR检测大鼠肠系膜动脉中K_ATP通道mRNA的表达;观察各组大鼠肠系膜动脉对去甲肾上腺素(NE)升压效应的反应性。结果: 与sham组相比,Ovx组大鼠肠系膜动脉K_ATP 通道的Kir6.1及SUR2B亚单位mRNA表达减少(P<0.05),而Ovx+E组则表达增加(P<0.05)。与sham组相比,Ovx组大鼠的血管反应性明显增加(P<0.05),Ovx+E组无明显差异。给予K_ATP通道阻滞剂格列本脲后,sham组和Ovx+E组的动脉反应性增加(P<0.05),Ovx组无明显变化(P>0.05),此时3组间比较无明显差异(P>0.05)。结论: 雌激素可能通过上调肠系膜动脉平滑肌细胞K_ATP通道的表达来降低动脉对去甲肾上腺素升压效应的反应性。     

缺血预处理延缓大鼠心肌缺血引起的细胞间电脱耦联

目的:研究缺血预处理(IPC)延缓心肌细胞间电脱耦联现象及其可能的机制,尤其是线粒体膜ATP敏感性钾通道(mitoK_ATP)在其中的作用。方法:大鼠心脏Langendorff离体灌流,用四电极法测量心肌整体阻抗(R_t),监测R_t在心肌缺血后的变化来判断心肌细胞发生电脱耦联的时间。结果:(1)对照组心肌缺血40 min后复灌30 min,心肌细胞间电脱耦联发生平均时间为(13.29±0.95) min;(2)IPC可以明显延迟电脱耦联的发生时间、促进心肌缺血复灌后收缩功能的恢复;(3)IPC前给予mitoK_ATP特异阻断剂5-hydroxydecanoate(5-HD,100 μmol/L)取消了IPC的心脏作用;(4)MitoK_ATP特异开放剂diazoxide(60 μmol/L)预处理可以模拟IPC延迟电脱耦联、促进心肌收缩功能恢复;(5)Diazoxide的IPC模拟作用能被5-HD取消,也能被L型钙通道特异阻断剂verapamil(2.0 μmol/L)和自由基清除剂N-(2-mercaptopropionyl)glycine(300 μmol/L)取消。结论:IPC可以通过激活mitoK_ATP延缓大鼠心肌缺血造成的细胞间电脱耦联和改善心肌收缩功能。     

微元素粉末对大鼠血管内皮和平滑肌细胞膜电位的影响

目的:探讨微元素粉末(由铝、钛、硅等元素组成的超细粉末,粉末直径在0.5μm以下,microelementpowder,MP)对大鼠血管内皮和平滑肌膜电位的影响,探讨其改善微循环的机理。方法:培养大鼠肺血管内皮细胞和主动脉平滑肌细胞,用电位敏感的荧光探针和激光共聚焦显微镜测定细胞膜电位动态变化。结果:MP使平滑肌细胞显著超极化。ATP敏感钾通道(ATPsensitiveK⁺channel,K_ATP)阻滞剂优降糖(2μmol/L)对平滑肌膜电位无明显影响,但能完全逆转MP对平滑肌的超极化作用;MP使血管内皮细胞轻度超极化,其作用不受优降糖影响。结论:MP激活血管平滑肌K_ATP通道,使细胞膜超极化,从而扩张微血管和改善微循环。     

模拟缺血-再灌注对窦房结细胞自发性动作电位的影响及吡那地尔的干预作用

目的:探讨模拟缺血-再灌注对窦房结细胞动作电位的影响及K_ATP通道开放剂的干预效果。方法:取培养2d的乳鼠窦房结细胞进行实验,随机分为对照组、模拟缺血-再灌注组(I/R)、K_ATP通道开放剂pinacidil干预组(P+I/R)及K_ATP通道阻断剂5-HD干预组(5-HD+P+I/R及5-HD+I/R)。采用全细胞膜片钳技术记录窦房结细胞动作电位,测定最大舒张电位(MDP)、0期去极化速度(UV)、超射值(APO)、动作电位周期(IBI)及50%复极时程(APD₅₀)的变化。结果:①I/R组MDP(-55.2±4.5)mV明显低于对照组(P＜0.05),APD₅₀(64.3±3.8)ms明显短于对照组(P＜0.01),而UV(3.8±0.5)V/s及APO(14.2±2.0)mV则明显小于对照组(P＜0.01),但IBI无明显改变。②P+I/R组的MDP及APO明显大于I/R组,IBI明显长于、UV显著快于I/R组,但APD₅₀进一步缩短。③5-HD不能阻断pinacidil对模拟缺血-再灌注窦房结细胞MDP、IBI和APD₅₀的影响,但能使pinacidil引起的UV及APO改变发生逆转。结论:Pinacidil可能通过开放不同种类K_ATP通道对模拟缺血-再灌注窦房结细胞动作电位产生影响。     

K+通道在正常与慢性低氧大鼠低氧性肺血管收缩反应中的作用

目的:探讨K⁺通道在慢性低氧致低氧性肺血管收缩反应降低中的作用。方法:采用离体肺灌流实验,研究4-AP(4-aminopyridine,电压依赖性K⁺通道-Kv阻滞剂)、TEA(tetraethylamonium,Ca²⁺激活性K⁺通道-K_Ca阻滞剂)、GLIB(glibenclamide,ATP敏感性K⁺通道-K_ATP阻滞剂)对正常与慢性低氧大鼠肺血管低氧反应的影响。结果:4-AP、TEA均可使正常大鼠肺动脉基础压上升,且使其肺血管低氧反应明显增强;对于慢性低氧大鼠,其肺血管对低氧反应明显低下,4-AP、TEA升肺动脉基础压的作用明显低于对照鼠肺,GLIB也呈现升高肺动脉基础压力作用,4-AP、TEA、GLIB均可使肺血管低氧反应大大增强,增强的比例明显大于正常对照组。结论:在离体灌流鼠肺HPV中,Kv、K_Ca的开放起调节作用,大鼠经慢性低氧后,肺血管反应性明显降低,可能与Kv、K_Ca、K_ATP在HPV中的调节作用相对增强有关。     

钾离子通道在大鼠慢性低氧所致肺血管低反应中的作用

目的与方法：采用阻断剂及离体肺内动脉血管环方法,研究不同类型钾离子通道对慢性低压低氧的大鼠模型的低氧性肺血管收缩反应（HPV）的作用,旨在探讨钾离子通道在慢性低氧肺血管低反应机制中的作用。结果：①慢性低氧15 d、30 d可降低肺血管对急性低氧的收缩反应。②分别阻断正常对照组、慢性低氧组大鼠肺血管钙激活性钾通道（K_Ca）、ATP敏感的钾通道（K_ATP）,均使其HPV反应明显增强,其中慢性低氧组增强幅度显著高于正常对照组（P<0.01）。③阻断正常对照组、慢性低氧组大鼠肺血管延迟整流性钾通道（K_DR）对其HPV均无明显影响。结论：K_Ca、K_ATP在HPV反应起着重要的调节作用,慢性低氧可使此调节作用显著加强,这可能是导致肺血管低反应一个重要机制。     

Functional effects of KCNJ11 mutations causing neonatal diabetes: enhanced activation by MgATP

Recent studies have shown that heterozygous mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, cause permanent neonatal diabetes either alone (R201C, R201H) or in association with developmental delay, muscle weakness and epilepsy (V59G,V59M). Functional analysis in the absence of Mg2+, to isolate the inhibitory effects of ATP on Kir6.2, showed that both types of mutation reduce channel inhibition by ATP. However, in pancreatic beta-cells, K(ATP) channel activity is governed by the balance between ATP inhibition via Kir6.2 and Mg-nucleotide stimulation mediated by an auxiliary subunit, the sulphonylurea receptor SUR1. We therefore studied the MgATP sensitivity of KCNJ11 mutant K(ATP) channels expressed in Xenopus oocytes. In contrast to wild-type channels, Mg2+ dramatically reduced the ATP sensitivity of heterozygous R201C, R201H, V59M and V59G channels. This effect was predominantly mediated via the nucleotide-binding domains of SUR1 and resulted from an enhanced stimulatory action of MgATP. Our results therefore demonstrate that KCNJ11 mutations increase the current magnitude of heterozygous K(ATP) channels in two ways: by increasing MgATP activation and by decreasing ATP inhibition. They further show that the fraction of unblocked K(ATP) current at physiological MgATP concentrations correlates with the severity of the clinical phenotype.     

成年大鼠脑内ATP敏感性钾通道亚型mRNA表达的研究

为探讨ATP敏感性钾通道(KATP)亚型在大鼠脑组织中的表达,本研究采用半定量逆转录聚合酶链反应 (RT PCR)检测了大鼠小脑、大脑皮质、海马、纹状体、黑质的KATP亚型mRNA的表达。结果显示Kir6. 1、Kir6. 2、Sur1、Sur2B在小脑、大脑皮质、海马、纹状体、黑质中均有表达,Sur2A在脑组织中未见表达。Kir6. 1mRNA在海马和黑质的相对表达水平明显高于小脑、大脑皮质和纹状体(P<0.01);Kir6. 2和Sur1mRNA在黑质的相对表达水平明显高于小脑、大脑皮质、海马和纹状体 (P<0.01 );Sur2BmRNA在黑质、海马和纹状体的相对表达水平明显高于小脑和大脑皮质(P<0.01 )。以上结果提示KATP在脑内具有广泛表达,其表达水平在不同部位存在着差异性。     

Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome

Activating mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 and SUR1 are a common cause of neonatal diabetes. Here, we analyse the molecular mechanism of action of the heterozygous mutation F132L, which lies in the first set of transmembrane helices (TMD0) of SUR1. This mutation causes severe developmental delay, epilepsy and permanent neonatal diabetes (DEND syndrome). We show that the F132L mutation reduces the ATP sensitivity of K(ATP) channels indirectly, by altering the intrinsic gating of the channel. Thus, the open probability is markedly increased when Kir6.2 is co-expressed with mutant TMD0 alone or with mutant SUR1. The F132L mutation disrupts the physical interaction between Kir6.2 and TMD0, but does not alter the plasmalemma channel density. Our results explain how a mutation in an accessory subunit can produce enhanced activity of the K(ATP) channel pore (formed by Kir6.2). They also provide further evidence that interactions between TMD0 of SUR1 and Kir6.2 are critical for K(ATP) channel gating and identify a residue crucial for this interaction at both physical and functional levels.     

A heterozygous activating mutation in the sulphonylurea receptor SUR1 (ABCC8) causes neonatal diabetes

Neonatal diabetes is a genetically heterogeneous disorder with nine different genetic aetiologies reported to date. Heterozygous activating mutations in the KCNJ11 gene encoding Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, are the most common cause of permanent neonatal diabetes. The sulphonylurea receptor (SUR) SUR1 serves as the regulatory subunit of the K(ATP) channel in pancreatic beta cells. We therefore hypothesized that activating mutations in the ABCC8 gene, which encodes SUR1, might cause neonatal diabetes. We identified a novel heterozygous mutation, F132L, in the ABCC8 gene of a patient with severe developmental delay, epilepsy and neonatal diabetes (DEND syndrome). This mutation had arisen de novo and was not present in 150 control chromosomes. Residue F132 shows evolutionary conservation across species and is located in the first set of transmembrane helices (TMD0) of SUR1, which is proposed to interact with Kir6.2. Functional studies of recombinant K(ATP) channels demonstrated that F132L markedly reduces the sensitivity of the K(ATP) channel to inhibition by MgATP and this increases the whole-cell K(ATP) current. The functional consequence of this ABCC8 mutation mirrors that of KCNJ11 mutations causing neonatal diabetes and provides new insights into the interaction of Kir6.2 and SUR1. As SUR1 is expressed in neurones as well as in beta cells, this mutation can account for both neonatal diabetes and the neurological phenotype. Our results demonstrate that SUR1 mutations constitute a new genetic aetiology for neonatal diabetes and that they act by reducing the K(ATP) channel's ATP sensitivity.     

Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes

ATP-sensitive potassium (K_ATP) channels, composed of pore-forming Kir6.2 and regulatory sulphonylurea receptor (SUR) subunits, play an essential role in insulin secretion from pancreatic beta cells. Binding of ATP to Kir6.2 inhibits, whereas interaction of Mg-nucleotides with SUR, activates the channel. Heterozygous activating mutations in Kir6.2 (KCNJ11) are a common cause of neonatal diabetes (ND). We assessed the functional effects of six novel Kir6.2 mutations associated with ND: H46Y, N48D, E227K, E229K, E292G, and V252A. K_ATP channels were expressed in Xenopus oocytes and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the beta cell type of SUR). All mutations reduced the sensitivity of the K_ATP channel to inhibition by MgATP, and enhanced whole-cell K_ATP currents. Two mutations (E227K, E229K) also enhanced the intrinsic open probability of the channel, thereby indirectly reducing the channel ATP sensitivity. The other four mutations lie close to the predicted ATP-binding site and thus may affect ATP binding. In pancreatic beta cells, an increase in the K_ATP current is expected to reduce insulin secretion and thereby cause diabetes. None of the mutations substantially affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting patients carrying these mutations may respond to these drugs.     

COPD合并慢性缺氧患者肺血管对急性缺氧反应变化中肺动脉平滑肌细胞钾通道的作用

目的：以人离体肺动脉环缺氧张力变化为对象研究钾通道在缺氧性肺血管收缩（HPV）发生中的作用。 方法： 从手术室切取人肺动脉环，进行人肺动脉环张力试验，分正常对照组，单纯慢性阻塞性肺病（chronic obstructive pulmonary disease ，COPD）组和COPD合并慢性缺氧组，分别利用相应的特异性阻断剂观察电压门控性钾通道（KV），钙离子激活的钾通道（KCa）， ATP敏感的钾通道（KATP），在缺氧性肺血管收缩（HPV）的作用。 结果： （1）3组肺动脉环在急性缺氧时血管收缩张力均增加，同时COPD＋慢性缺氧组增加百分比显著低于对照组；COPD组与对照组无显著差异。（2）4-AP阻断Kv后较阻断前，3组肺血管环缺氧张力增加幅度均显著降低（P<0.05），同时正常对照组和COPD组降低幅度明显大于慢性缺氧组。TEA阻断KCa后以及格列苯脲（glybenclamide）阻断KATP后二者较阻断前，3组肺血管环缺氧张力均显著增加（P<0.05），同时COPD＋慢性缺氧组增加的幅度明显大于正常组（分别是P<0.01和P<0.05）。 结论： （1）慢性缺氧可降低肺血管的收缩性及肺血管对急性缺氧的收缩反应；（2）Kv在3组人离体肺动脉环HPV反应中均起介导作用，慢性缺氧可使此介导作用加强；KCa和KATP在3组人离体肺动脉环HPV反应中均起调节作用，慢性缺氧可使此调节作用加强。     

Functional modulation of the ATP-sensitive potassium channel by extracellular signal-regulated kinase-mediated phosphorylation

ATP-sensitive potassium (K(ATP)) channels play an important role in controlling insulin secretion and vascular tone as well as protecting neurons under metabolic stress. We have previously demonstrated that stimulation of the K(ATP) channel by nitric oxide (NO) requires activation of Ras- and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. However, the mechanistic link between ERK and the K(atp) channel remained unknown. To investigate how ERK modulates the function of K(ATP) channels, we performed single-channel recordings in combination with site-directed mutagenesis. The Kir6.2/SUR1 channel, a neuronal K(ATP) channel isoform, was expressed in human embryonic kidney (HEK) 293 cells by transient transfection. Direct application of the activated ERK2 to the cytoplasmic surface of excised, inside-out patches markedly enhanced the single-channel activity of Kir6.2/SUR1 channels. The normalized open probability (NPo) and opening frequency were significantly increased, whereas the mean closed duration was reduced. The single-channel conductance level was not affected. The ERK2-induced stimulation of Kir6.2/SUR1 channels was prevented by heat-inactivation of the enzyme. Furthermore, alanine substitutions of T341 and S385 to disrupt the potential ERK phosphorylation sites present in the Kir6.2 subunit significantly abrogated the stimulatory effects of ERK2, while aspartate substitutions of T341 and S385 to mimic the (negative) charge effect of phosphorylation rendered a small yet significant reduction in the ATP sensitivity of the channel. Taken together, here we report for the first time that ERK2/MAPK activates neuronal-type K(ATP) channels, and this stimulation requires ERK phosphorylation of the Kir6.2 subunit at T341 and S385 residues. The ERK2-induced K(ATP) channel stimulation can be accounted for by changes in channel gating that destabilize the closed states and by reduction in the ATP sensitivity. As Kir6.2 is the pore-forming subunit of K(ATP) channels, ERK2-mediated phosphorylation may represent a common mechanism for K(ATP) channel regulation in different tissues.     

The regulation of rotenone-induced inflammatory factor production by ATP-sensitive potassium channel expressed in BV-2 cells

Our previous studies have demonstrated that activating ATP-sensitive potassium channel (K(ATP) channel), not only improved Parkinsonian behavior and neurochemical symptoms, but also reduced iNOS activity and mRNA levels in striatum and nigra of rotenone rat model of Parkinson's disease (PD). In this study, it was first shown that the subunits of K(ATP) channels are expressed in BV-2 cells, and then it was investigated whether K(ATP) channel was involved in regulating inflammatory factor production from BV-2 cells activated by rotenone. It was found that K(ATP) channel was expressed in BV-2 cell and formed by the combination of Kir 6.1 and SUR 2A/2B. K(ATP) channel openers (KCOs) including pinacidil, diazoxide and iptakalim (Ipt) exerted beneficial effects on rotenone-induced morphological alterations of BV-2 cells, decreased tumor necrosis factor alpha (TNF-alpha) production and the expression and activity of inducible isoform of nitric oxide synthase (iNOS). Either glibenclamide or 5-hydroxydecanoate acid (a selective mitochondrial K(ATP) channel blocker) could abolish the effects of KCOs, suggesting that K(ATP) channels, especially mitochondrial ATP-sensitive potassium channels (mitoK(ATP) channels), played a crucial role in preventing the activation of BV-2 cells, and subsequently the production of a variety of proinflammatory factors. Therefore, activation of K(ATP) channel might be a new therapeutic strategy for treating neuroinflammatory and neurodegenerative disorders.     

Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain

Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.     

Growth inhibitory effects of ATP and its derivatives on human fibroblasts immortalized with 60Co-gamma rays

In our previous study (Katayama B et al, Int J Mol Med 2: 603-606, 1998), cell growth inhibition caused by ATP added to cultures was found to be greater in immortalized human fibroblasts than in the normal human fibroblasts. Since it has been reported that ATP affects cells via P2-purinergic receptors, growth inhibitory effects of ATP and its derivatives on immortalized human fibroblasts were investigated in the present study in order to learn what type of receptors are involved in ATP cytotoxicity. The ATP derivatives used in this study were: ATP, ADP, beta, gamma-methyleneadenosine 5'-triphosphate (MeATP), 2' & 3'-o-(4-benzoylbenzoyl) adenosine, triethylammonium salt (BzATP), adenosine 5'-o-(3-thiotriphosphate) (ATPgammaS), 2-methylthioadenosine 5'-triphosphate (2-MeSATP) and UTP. The extent of cytotoxicity induced by these drugs was found to be in the order of: ATP=ADP>ATPgammaS>MeATP=BzATP. On the other hand, neither 2-MeSATP nor UTP showed any cytotoxicity. These findings indicate that ATP may exert the cell growth inhibition by certain kinds of signal transduction via P2x or P2y purinergic receptors which affect intrinsic channels/pores of cell membrane and/or G protein activation. As a result, intracellular elevation in the concentrations of ions such as calcium and potassium, membrane depolarization, loss of endogenous ions/metabolites, and activation of inositol phospholipid-specific phospholipase C may occur. Actually, a dihydropyridine calcium channel blocker, nifedipine, and an ATP-sensitive K+-channel blocker, glybenclamide, reduced the growth inhibitory effects of ATP on the cells to some extent. The growth inhibition caused by ATP was not due to apoptosis or induction of a cyclin/CDK kinase inhibitor, P21.