氧化应激对支气管上皮细胞CFTR门控功能及调控机制研究
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摘要
目的:
在气道炎症或气道高反应(airway hyperresponsiveness,AHR)中,气道粘液分泌量异常增多,粘液组成成分也发生改变,容易形成气道粘液栓,是气道阻力增高的重要病理机制。气道上皮细胞粘液分泌的数量及组成成分取决于某些离子通道的功能活性及表达密度,其中囊性纤维化跨膜转导调节因子(Cystic fibrosis transmembraneconductance regulator,CFTR)尤为人们所关注。CFTR是一种磷酸化调控的上皮细胞Cl~-通道,主要位于气道上皮细胞顶侧膜,借助其对Cl~-的跨膜转运,在跨上皮盐类物质转运、水分流动和离子浓度调节中发挥重要作用。CFTR功能缺陷的患者分泌的粘液成分也与正常人很不一样,粘液包含有细菌感染产物、粘液脂质、肌动蛋白、蛋白酶等,使得其含有更多的非蒸发性的固体成分,增加粘液的稠度,降低粘液清除率和粘液运输效率,容易诱发气道炎症和形成气道粘液栓。基于以上的部分线索,本课题提出“气道上皮细胞CFTR门控功能及表达调节参与气道功能稳态,其缺陷可能与AHR形成机制有关”。
方法与结果:
1)氧化应激抑制支气管上皮细胞CFTR表达及信号机制
我们用臭氧连续应激大鼠3天,每天1小时建立气道高反应动物模型,免疫荧光结果显示,正常大鼠支气管上皮顶膜及胞浆中可见较多的红色染色的CFTR,而臭氧应激的大鼠支气管上皮顶膜及胞浆中CFTR明显减少。我们应用臭氧攻击BECs 30min,建立氧化应激模型。Real-time PCR结果显示,臭氧应激4h后,CFTR mRNA表达明显下降。免疫荧光显示正常BECs胞膜有较强的CFTR染色,臭氧处理后,CFTR。在膜及胞浆均明显下降。Western blot结果显示臭氧应激4h后,CFTR蛋白明显下降。全细胞膜片钳结果发现,在没有激动剂的情况下,记录的CFTR cl~-电流很小,在加入5μM forskolin,记录出明显的CFTR cl~-电流,在forskolin的基础上,可观察到臭氧应激4h后的BECs CFTR cl~-电流下降。在随后的试验中,发现这种cl~-电流能被CFTR cl~-电流抑制剂glibenclamide阻滞,而不能被非CFTR cl~-电流抑制剂DIDS所阻断,说明记录出的cl~-电流是CFTR cl~-电流。
为了研究氧化应激抑制BECs CFTR表达的信号机制,在臭氧应激前,BECs被JAK2激酶抑制剂AG490(10μmol/L)预处理4h,Real-timePCR结果发现臭氧应激后,AG490能增加CFTR mRNA的表达,AG490单独处理对CFTR mRNA的表达没有影响。免疫荧光和Western blot结果均显示AG490预处理能增加CFTR蛋白的表达。随后Western blot结果显示在未受臭氧处理的BECs,STAT1蛋白的磷酸化水平很小,在臭氧处理后,STAT1的磷酸化水平随时间依赖性增加。在前期实验中我们已发现臭氧应激后,小分子NO、CO、ROS增加。为了了解这些小分子是否参与臭氧诱导的STAT1的磷酸化。应用这些小分子抑制剂预处理BECs,免疫荧光和Western blot结果显示AG、ZnPP-9、NAC均降低了臭氧诱导的STAT1的磷酸化。
我们还对信号分子抑制剂是否调节臭氧应激后CFTR的表达进行了研究。Real-time PCR和Western blot结果显示AG,NAC预处理能增加臭氧应激后CFTR的表达,而ZnPP-9则影响不明显。
2) VIP激活支气管上皮细胞CFTR转运及通道活性
CFTR转运涉及到四个过程,最终调节细胞和膜上CFTR的水平。1)CFTR合成及构想成熟后,从ER转运至胞膜表面;2)在细胞膜表面,CFTR通过小泡以及酪氨酸为基础的基序发生内吞作用,CFTR从胞膜内陷至内涵体;3)胞吞的CFTR CFTR循环回胞膜上;4)胞吐的CFTR发生降解。CFTR只有在胞膜上才能对Cl~-的跨膜转运,在跨上皮盐类物质转运、水分流动和离子浓度调节中发挥重要作用。为了解CFTR的转运过程,我们通过以下实验来观察。免疫荧光结果发现VIP作用以后,循环回胞膜的CFTR增多,VIP促进了CFTR向胞膜的循环。免疫共沉淀结果显示正常BECs既表达成熟型CFTR(BandC),又表达非成熟型CFTR(Band B)。臭氧降低了成熟型CFTR的表达,而VIP则增加了成熟型CFTR的表达。Western blot结果显示臭氧降低了胞膜和总CFTR的蛋白水平,VIP增加了胞膜和总CFTR的蛋白水平,VIP促进了CFTR向膜的转运。
随后我们研究VIP对BECs上CFTR依赖的cl~-转运的调控机制进行了研究。采用氯离子敏感荧光染料MQAE检测胞内cl~-浓度和氯化物的分泌,结果发现VIP具有与forskolin一样增加胞内cl~-浓度和氯化物的溢出的特性,并随浓度升高而增大。全细胞记录结果显示在VIP的作用下,CFTR Cl~-电流密度增加,并具有浓度依赖性,在10~(-8)mmol/L为最大半数有效剂量。CFTR Cl~-阻断剂glibenctamide能阻断该电流,而非CFTR Cl~-阻断剂不能阻断该电流。我们应用PKA阻断剂H89,结果发现H89部分阻断了VIP激活的CFTR Cl~-电流;应用PKC阻断剂H7(200μM),同样结果发现H7部分阻断了VIP激活的CFTR Cl~-电流。
在BECs上存在着多种VIP受体(VPAC),我们应用(10~(-8)-10~(-10)M)VIP受体拮抗剂,结果发现VIP受体拮抗剂能剂量依赖性抑制VIP激活的CFTR Cl~-电流。
3) CFTR基因表达调控研究
为了进一步探讨臭氧应激后CFTR的基因表达调控机制,我们对臭氧应激条件下CFTR的转录因子调节谱进行了研究。实验首先用TESS软件对CFTR启动子区转录因子结合位点进行搜索,然后根据搜索结果,设计了6条探针,覆盖所有的转录因子结合位点,应用EMSA和ChIP的方法筛选了调控CFTR表达的转录因子。结果显示探针5、6有探针与蛋白结合形成的滞后带,能被100倍未标记探针所竞争,为特异性结合。通过突变探针结合实验和抗体超迁移实验,证实这2条探针结合的转录因子分别为Spl、ERa。臭氧应激4h后,降低了这两种转录因子的DNA结合活性。ChIP实验显示,Spl和ERa可特异性与CFTR启动子结合。
紧接着,我们用基因定点突变技术观察了两种转录因子对CFTR启动子活性的影响,结果显示Spl与ERa的结合位点突变后,CFTR启动子活性下降,Spl与ERa结合位点同时突变,可几乎到达到臭氧应激对CFTR的抑制。随后,我们用反义寡核苷酸技术观察了两种转录因子对CFTR表达的影响,Western证实了两种ASOs的有效性,他们可分别阻断两种转录因子的蛋白表达。应用两种ASOs后,我们用real-time PCR和Western观察到,Spl和ERa ASO处理可降低CFTR的表达。
通过进一步的研究,我们用EMSA观察到ERa与Spl的结合活性在0-1h之间激活,于应激后2h开始下降。real-time PCR检测CFTR的表达与臭氧应激后ERa和Spl的转录活性一致。我们还用免疫荧光观察到臭氧应激4h后,Spl核转位降低。
臭氧应激4h后,Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录。
综上所述:本研究观察到臭氧应激抑制了BECs上CFTR的表达及功能,这为本课题提出的非CF疾病的气道炎性疾病中CFTR的功能下降提供了依据。研究还观察到臭氧应激后,NO、ROS产生增多,参与并激活了STAT1的磷酸化,抑制了CFTR的表达,臭氧应激还使Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录。另外本研究研究了VIP激活了CFTR的转运及门控特性,PKA和PKC途径通过调节CFTR调节区上的磷酸化位点调节了CFTR的通道功能,为治疗包括CF疾病在内的气道炎性疾病因CFTR功能下调引起的黏液粘稠问题提供了选择。
结论:
臭氧应激抑制了BECs上CFTR的表达及功能。其机制如下:
(1)臭氧应激抑制了CFTR的表达。其途径有早期信号分子NO和ROS的产生,进而激活JAK/STAT途径参与臭氧应激抑制CFTR的表达。臭氧应激还使转录因子Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录;
(2)臭氧应激抑制CFTR cl~-电流,VIP激活了CFTR cl~-电流,PKA和PKC途径参与了其调控过程;
(3)VIP增加了CFTR向胞膜的转运。主要是通过增加CFTR循环回胞膜,增加175kD成熟型CFTR的表达及增加胞膜CFTR蛋白的表达这三个过程来实现的。
Objectives:
The change of mucosal fluid composition result in the decrease of mucosa cilia clearance rate,the increase abnormally of airway mucosal fluid secretory volume,a key pathophysiological process in airway hyperresponsive diseases such as asthma and bronchitis.Cystic fibrosis transmembrane conductance regulator(CFTR),functions as a cAMP-regulated Cl~- channel which controls transepithelial electrolyte transport,fluid flow,ion concentrations in the airway.Loss of CFTR Cl~-function caused a lack of fluid secretion with excessive fluid absorption, which led to watery component reduction in airway surface liquid,mucous thickening,blockage of submucosal gland ducts,and impairment of mucociliary clearance that were followed with infection,inflammation,and ultimately,the tissue destruction characteristic of bronchiectasis.on account of above part cue,we propose that the gating and the expression regulation of CFTR in BECs participate in the homeostasis of airway function,which the defect of CFTR function could be related with AHR.
Contents:
1.Ozone stress down-regulates the expression of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial cells
Wistar rat inhaled 1.5ppm ozone 1h per day for three days to establish a airway hyperresponsiveness animal model.BECs were cultured on slides in DMEM/F12 medium with or without 1.5ppm ozone for 30 minutes to establish oxitative stress model.To investigate abnormalities of cystic fibrosis transmembrane conductance regulator(CFTR) expression in chronic inflammatory airway diseases and its regulation mechanisms,the present study was designed to observe the expression of CFTR,CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR,immunofluorescence, western blot and whole cell patch-clamp.The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells (HBEC).STAT1 signal pathway was checkedto investigate the signal mechanism.It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC,which could be influenced by some signaling molecules related to the early transduction of cellular stress.Furthermore,reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR.Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC.The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules,and subsequently transcription modulator STAT1.
2.Activation of CFTR trafficking and gating by vasoactive intestinal peptide in Human bronchial epithelial cells
The present study was designed to observe the trafficking of CFTR, and channel gating in Human bronchial epithelium cells(HBEC) by using confocal microscopy,western blot,immunoprecipitation and Whole-cell patch clamp.Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. During stimulation with VIP,apical extension of CFTR immunofluorescence into the cell was reduced significantly and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP raised cell surface and total CFTR.Compare with the augmented level of total CFTR,the surface CFTR increased well than that the total CFTR.Immunoprecipitation founded that CFTR band C had an increase markedly in HBEC treated with VIP,compared with the control group.We also observed an increase in the CFTR band B,the immature form of CFTR,which the extent was lower than that band C. VIP led to 4-fold increases in Cl-effiux in HBEC.Glibenclamide-sensitive and DIDS-insensitive CFTR Cl~- currents was consistently observed after stimulation with VIP(10~(-8)mol/L).The augmention of CFTR Cl~- currents enhanced by VIP(10~(-8)mol/L) was reversed,at least in part,by the protein kinase A(PKA) inhibitor,H-89 and the protein kinase C(PKC) inhibitor, H-7,suggesting PKA and PKC participated in the VIP-promoted CFTR Cl~-currents.
3.Study gene expression modulation of CFTR
To probe the mechanisms of the induced expression of CFTR under ozone stress,six oligonucleotide probes corresponding to various regions of the CFTR promoter were used in EMSA studies.Two were found to have a decrease mobility shift with extracts from ozone-stressed cells. Based on the assay of antibody supershift,they were verified as Sp1 and ERα.By ChIP assay,Sp1 and ERαdecreaed the ozone-inducible DNA binding on the CFTR promoter.
Next,site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe the inhibitory effects of the two nuclear factors on CFTR promoter activation and expression.The results also showed that Sp1 and ERαdecreased the ozone-inducible DNA binding on the CFTR promoter and CFTR expression.
The translocation of Sp1 was observed by immunofluorescence assay, which showed that Sp1 nuclear translocation was decreased after ozone exposure.The time courses of Sp1 and ERαactivation and inactivation, followed by CFTR expression were also examined.It was shown that ozone-depression CFTR expression and Sp1 and ERαbinding activity correlated during a-24 hour time course.
In summary:In present study,we observed that ozone stress repressed the eapression and function of CFTR,which provided support for studies investigating CFTR function in inflammatory lung diseases other than cystic fibrosis.We also investigate that the production of NO,ROS were increased and participated in activation of STAT1 after ozone stress,which contributed to the inhibition of CFTR expression,the binding activity and the translocation were inhibited of Sp1,ERαafter ozone stress,which resulted in the repression of CFTR transcription.In addition,we revealed VIP activated the trafficking and gating,the CFTR channel was regulated by PKA and PKC pathway through the phosphorylation of CFTR regulatory domain,which offered a choose to cure inflammatory lung diseases.
Conclusion:
Ozone stress repressed the expression and function of CFTR in BECs. As follow were its mechanisms:
(1) Ozone stress down-regulated the expression of CFTR.Its pathways had NO,ROS,JAK/STAT.Ozone stress depressed the binding activity and the translocation of Sp1,ERα,which resulted in the inhibition of CFTR transcription;
(2) Ozone stress inhibitted CFTR cl~- current,VIP activated CFTR cl~-current.Its signal pathway had PKA and PKC;
(3) VIP raised CFTR trafficking,which included enhance recycling of CFTR,augment the expression of mature CFTR and the protein expression of plasmalemma CFTR.
在气道炎症或气道高反应(airway hyperresponsiveness,AHR)中,气道粘液分泌量异常增多,粘液组成成分也发生改变,容易形成气道粘液栓,是气道阻力增高的重要病理机制。气道上皮细胞粘液分泌的数量及组成成分取决于某些离子通道的功能活性及表达密度,其中囊性纤维化跨膜转导调节因子(Cystic fibrosis transmembraneconductance regulator,CFTR)尤为人们所关注。CFTR是一种磷酸化调控的上皮细胞Cl~-通道,主要位于气道上皮细胞顶侧膜,借助其对Cl~-的跨膜转运,在跨上皮盐类物质转运、水分流动和离子浓度调节中发挥重要作用。CFTR功能缺陷的患者分泌的粘液成分也与正常人很不一样,粘液包含有细菌感染产物、粘液脂质、肌动蛋白、蛋白酶等,使得其含有更多的非蒸发性的固体成分,增加粘液的稠度,降低粘液清除率和粘液运输效率,容易诱发气道炎症和形成气道粘液栓。基于以上的部分线索,本课题提出“气道上皮细胞CFTR门控功能及表达调节参与气道功能稳态,其缺陷可能与AHR形成机制有关”。
方法与结果:
1)氧化应激抑制支气管上皮细胞CFTR表达及信号机制
我们用臭氧连续应激大鼠3天,每天1小时建立气道高反应动物模型,免疫荧光结果显示,正常大鼠支气管上皮顶膜及胞浆中可见较多的红色染色的CFTR,而臭氧应激的大鼠支气管上皮顶膜及胞浆中CFTR明显减少。我们应用臭氧攻击BECs 30min,建立氧化应激模型。Real-time PCR结果显示,臭氧应激4h后,CFTR mRNA表达明显下降。免疫荧光显示正常BECs胞膜有较强的CFTR染色,臭氧处理后,CFTR。在膜及胞浆均明显下降。Western blot结果显示臭氧应激4h后,CFTR蛋白明显下降。全细胞膜片钳结果发现,在没有激动剂的情况下,记录的CFTR cl~-电流很小,在加入5μM forskolin,记录出明显的CFTR cl~-电流,在forskolin的基础上,可观察到臭氧应激4h后的BECs CFTR cl~-电流下降。在随后的试验中,发现这种cl~-电流能被CFTR cl~-电流抑制剂glibenclamide阻滞,而不能被非CFTR cl~-电流抑制剂DIDS所阻断,说明记录出的cl~-电流是CFTR cl~-电流。
为了研究氧化应激抑制BECs CFTR表达的信号机制,在臭氧应激前,BECs被JAK2激酶抑制剂AG490(10μmol/L)预处理4h,Real-timePCR结果发现臭氧应激后,AG490能增加CFTR mRNA的表达,AG490单独处理对CFTR mRNA的表达没有影响。免疫荧光和Western blot结果均显示AG490预处理能增加CFTR蛋白的表达。随后Western blot结果显示在未受臭氧处理的BECs,STAT1蛋白的磷酸化水平很小,在臭氧处理后,STAT1的磷酸化水平随时间依赖性增加。在前期实验中我们已发现臭氧应激后,小分子NO、CO、ROS增加。为了了解这些小分子是否参与臭氧诱导的STAT1的磷酸化。应用这些小分子抑制剂预处理BECs,免疫荧光和Western blot结果显示AG、ZnPP-9、NAC均降低了臭氧诱导的STAT1的磷酸化。
我们还对信号分子抑制剂是否调节臭氧应激后CFTR的表达进行了研究。Real-time PCR和Western blot结果显示AG,NAC预处理能增加臭氧应激后CFTR的表达,而ZnPP-9则影响不明显。
2) VIP激活支气管上皮细胞CFTR转运及通道活性
CFTR转运涉及到四个过程,最终调节细胞和膜上CFTR的水平。1)CFTR合成及构想成熟后,从ER转运至胞膜表面;2)在细胞膜表面,CFTR通过小泡以及酪氨酸为基础的基序发生内吞作用,CFTR从胞膜内陷至内涵体;3)胞吞的CFTR CFTR循环回胞膜上;4)胞吐的CFTR发生降解。CFTR只有在胞膜上才能对Cl~-的跨膜转运,在跨上皮盐类物质转运、水分流动和离子浓度调节中发挥重要作用。为了解CFTR的转运过程,我们通过以下实验来观察。免疫荧光结果发现VIP作用以后,循环回胞膜的CFTR增多,VIP促进了CFTR向胞膜的循环。免疫共沉淀结果显示正常BECs既表达成熟型CFTR(BandC),又表达非成熟型CFTR(Band B)。臭氧降低了成熟型CFTR的表达,而VIP则增加了成熟型CFTR的表达。Western blot结果显示臭氧降低了胞膜和总CFTR的蛋白水平,VIP增加了胞膜和总CFTR的蛋白水平,VIP促进了CFTR向膜的转运。
随后我们研究VIP对BECs上CFTR依赖的cl~-转运的调控机制进行了研究。采用氯离子敏感荧光染料MQAE检测胞内cl~-浓度和氯化物的分泌,结果发现VIP具有与forskolin一样增加胞内cl~-浓度和氯化物的溢出的特性,并随浓度升高而增大。全细胞记录结果显示在VIP的作用下,CFTR Cl~-电流密度增加,并具有浓度依赖性,在10~(-8)mmol/L为最大半数有效剂量。CFTR Cl~-阻断剂glibenctamide能阻断该电流,而非CFTR Cl~-阻断剂不能阻断该电流。我们应用PKA阻断剂H89,结果发现H89部分阻断了VIP激活的CFTR Cl~-电流;应用PKC阻断剂H7(200μM),同样结果发现H7部分阻断了VIP激活的CFTR Cl~-电流。
在BECs上存在着多种VIP受体(VPAC),我们应用(10~(-8)-10~(-10)M)VIP受体拮抗剂,结果发现VIP受体拮抗剂能剂量依赖性抑制VIP激活的CFTR Cl~-电流。
3) CFTR基因表达调控研究
为了进一步探讨臭氧应激后CFTR的基因表达调控机制,我们对臭氧应激条件下CFTR的转录因子调节谱进行了研究。实验首先用TESS软件对CFTR启动子区转录因子结合位点进行搜索,然后根据搜索结果,设计了6条探针,覆盖所有的转录因子结合位点,应用EMSA和ChIP的方法筛选了调控CFTR表达的转录因子。结果显示探针5、6有探针与蛋白结合形成的滞后带,能被100倍未标记探针所竞争,为特异性结合。通过突变探针结合实验和抗体超迁移实验,证实这2条探针结合的转录因子分别为Spl、ERa。臭氧应激4h后,降低了这两种转录因子的DNA结合活性。ChIP实验显示,Spl和ERa可特异性与CFTR启动子结合。
紧接着,我们用基因定点突变技术观察了两种转录因子对CFTR启动子活性的影响,结果显示Spl与ERa的结合位点突变后,CFTR启动子活性下降,Spl与ERa结合位点同时突变,可几乎到达到臭氧应激对CFTR的抑制。随后,我们用反义寡核苷酸技术观察了两种转录因子对CFTR表达的影响,Western证实了两种ASOs的有效性,他们可分别阻断两种转录因子的蛋白表达。应用两种ASOs后,我们用real-time PCR和Western观察到,Spl和ERa ASO处理可降低CFTR的表达。
通过进一步的研究,我们用EMSA观察到ERa与Spl的结合活性在0-1h之间激活,于应激后2h开始下降。real-time PCR检测CFTR的表达与臭氧应激后ERa和Spl的转录活性一致。我们还用免疫荧光观察到臭氧应激4h后,Spl核转位降低。
臭氧应激4h后,Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录。
综上所述:本研究观察到臭氧应激抑制了BECs上CFTR的表达及功能,这为本课题提出的非CF疾病的气道炎性疾病中CFTR的功能下降提供了依据。研究还观察到臭氧应激后,NO、ROS产生增多,参与并激活了STAT1的磷酸化,抑制了CFTR的表达,臭氧应激还使Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录。另外本研究研究了VIP激活了CFTR的转运及门控特性,PKA和PKC途径通过调节CFTR调节区上的磷酸化位点调节了CFTR的通道功能,为治疗包括CF疾病在内的气道炎性疾病因CFTR功能下调引起的黏液粘稠问题提供了选择。
结论:
臭氧应激抑制了BECs上CFTR的表达及功能。其机制如下:
(1)臭氧应激抑制了CFTR的表达。其途径有早期信号分子NO和ROS的产生,进而激活JAK/STAT途径参与臭氧应激抑制CFTR的表达。臭氧应激还使转录因子Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录;
(2)臭氧应激抑制CFTR cl~-电流,VIP激活了CFTR cl~-电流,PKA和PKC途径参与了其调控过程;
(3)VIP增加了CFTR向胞膜的转运。主要是通过增加CFTR循环回胞膜,增加175kD成熟型CFTR的表达及增加胞膜CFTR蛋白的表达这三个过程来实现的。
Objectives:
The change of mucosal fluid composition result in the decrease of mucosa cilia clearance rate,the increase abnormally of airway mucosal fluid secretory volume,a key pathophysiological process in airway hyperresponsive diseases such as asthma and bronchitis.Cystic fibrosis transmembrane conductance regulator(CFTR),functions as a cAMP-regulated Cl~- channel which controls transepithelial electrolyte transport,fluid flow,ion concentrations in the airway.Loss of CFTR Cl~-function caused a lack of fluid secretion with excessive fluid absorption, which led to watery component reduction in airway surface liquid,mucous thickening,blockage of submucosal gland ducts,and impairment of mucociliary clearance that were followed with infection,inflammation,and ultimately,the tissue destruction characteristic of bronchiectasis.on account of above part cue,we propose that the gating and the expression regulation of CFTR in BECs participate in the homeostasis of airway function,which the defect of CFTR function could be related with AHR.
Contents:
1.Ozone stress down-regulates the expression of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial cells
Wistar rat inhaled 1.5ppm ozone 1h per day for three days to establish a airway hyperresponsiveness animal model.BECs were cultured on slides in DMEM/F12 medium with or without 1.5ppm ozone for 30 minutes to establish oxitative stress model.To investigate abnormalities of cystic fibrosis transmembrane conductance regulator(CFTR) expression in chronic inflammatory airway diseases and its regulation mechanisms,the present study was designed to observe the expression of CFTR,CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR,immunofluorescence, western blot and whole cell patch-clamp.The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells (HBEC).STAT1 signal pathway was checkedto investigate the signal mechanism.It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC,which could be influenced by some signaling molecules related to the early transduction of cellular stress.Furthermore,reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR.Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC.The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules,and subsequently transcription modulator STAT1.
2.Activation of CFTR trafficking and gating by vasoactive intestinal peptide in Human bronchial epithelial cells
The present study was designed to observe the trafficking of CFTR, and channel gating in Human bronchial epithelium cells(HBEC) by using confocal microscopy,western blot,immunoprecipitation and Whole-cell patch clamp.Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. During stimulation with VIP,apical extension of CFTR immunofluorescence into the cell was reduced significantly and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP raised cell surface and total CFTR.Compare with the augmented level of total CFTR,the surface CFTR increased well than that the total CFTR.Immunoprecipitation founded that CFTR band C had an increase markedly in HBEC treated with VIP,compared with the control group.We also observed an increase in the CFTR band B,the immature form of CFTR,which the extent was lower than that band C. VIP led to 4-fold increases in Cl-effiux in HBEC.Glibenclamide-sensitive and DIDS-insensitive CFTR Cl~- currents was consistently observed after stimulation with VIP(10~(-8)mol/L).The augmention of CFTR Cl~- currents enhanced by VIP(10~(-8)mol/L) was reversed,at least in part,by the protein kinase A(PKA) inhibitor,H-89 and the protein kinase C(PKC) inhibitor, H-7,suggesting PKA and PKC participated in the VIP-promoted CFTR Cl~-currents.
3.Study gene expression modulation of CFTR
To probe the mechanisms of the induced expression of CFTR under ozone stress,six oligonucleotide probes corresponding to various regions of the CFTR promoter were used in EMSA studies.Two were found to have a decrease mobility shift with extracts from ozone-stressed cells. Based on the assay of antibody supershift,they were verified as Sp1 and ERα.By ChIP assay,Sp1 and ERαdecreaed the ozone-inducible DNA binding on the CFTR promoter.
Next,site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe the inhibitory effects of the two nuclear factors on CFTR promoter activation and expression.The results also showed that Sp1 and ERαdecreased the ozone-inducible DNA binding on the CFTR promoter and CFTR expression.
The translocation of Sp1 was observed by immunofluorescence assay, which showed that Sp1 nuclear translocation was decreased after ozone exposure.The time courses of Sp1 and ERαactivation and inactivation, followed by CFTR expression were also examined.It was shown that ozone-depression CFTR expression and Sp1 and ERαbinding activity correlated during a-24 hour time course.
In summary:In present study,we observed that ozone stress repressed the eapression and function of CFTR,which provided support for studies investigating CFTR function in inflammatory lung diseases other than cystic fibrosis.We also investigate that the production of NO,ROS were increased and participated in activation of STAT1 after ozone stress,which contributed to the inhibition of CFTR expression,the binding activity and the translocation were inhibited of Sp1,ERαafter ozone stress,which resulted in the repression of CFTR transcription.In addition,we revealed VIP activated the trafficking and gating,the CFTR channel was regulated by PKA and PKC pathway through the phosphorylation of CFTR regulatory domain,which offered a choose to cure inflammatory lung diseases.
Conclusion:
Ozone stress repressed the expression and function of CFTR in BECs. As follow were its mechanisms:
(1) Ozone stress down-regulated the expression of CFTR.Its pathways had NO,ROS,JAK/STAT.Ozone stress depressed the binding activity and the translocation of Sp1,ERα,which resulted in the inhibition of CFTR transcription;
(2) Ozone stress inhibitted CFTR cl~- current,VIP activated CFTR cl~-current.Its signal pathway had PKA and PKC;
(3) VIP raised CFTR trafficking,which included enhance recycling of CFTR,augment the expression of mature CFTR and the protein expression of plasmalemma CFTR.
引文
[1] Qin XQ, Xiang Y, Liu C, Tan YR, Qu F, Peng LH, Zhu XL, Qin L. The role of bronchial epithelial cells in airway hyperresponsiveness. Sheng Li Xue Bao, 2007, 59(4): 454-64.
[2] Anderson M, Sheppard DN, Berger HA, Welsh MJ. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am J Physiol, 1992, 263: L1-14.
[3] Gibson R, Burns JL, Ramsey BW. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med, 2003,168: 918-51.
[4] Hyde SC, Emsley P, Hartshorn MJ, Mimmack MM, Gileadi U, Pearce SR, Gallagher MP, Gill DR, Hubbard RE, Higgins CF. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature, 1990, 346: 362-5.
[5] Sheppard D, Welsh MJ. Structure and function of the cystic fibrosis transmembrance conductance regulator chloride channel. Physiol Rev, 1999, 79: S23-S45.
[6] Randak C, Welsh MJ. An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR. Cell, 2003, 115: 837-850.
[7] Mall M, Bleich M, Schurlein M, Kuhr J, Seydewitz HH, Brandis M, Greger R, Kunzelmann K. Cholinergic ion secretion in human colon requires coactivation by cAMP. Am J Physiol Gastrointestinal Liver Physiology, 1998, 38: G1274-G1281.
[8] Ballard S, Trout L, Bebok Z, Sorscher EJ, and Crews A. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol, 1999, 277: L694-L699.
[9] Hug, M, Tamada T, Bridges RJ. CFTR and bicarbonate secretion by epithelial cells. News Physiol Sci, 2003, 18: 38-42.
[10] Pilewski J, Frizzell RA. Role of CFTR in Airway Disease Physiol. Rev., 1999, 79: 215-255.
[11] Anderson M, Berger HA, Rich DP, Gregory RJ, Smith AE, Welsh MJ. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell, 1991,67:775-784.
[12] Drumm M, Wilkinson DJ, Smit LS, Worrell RT, Strong TV, Frizzell RA, Dawson DC, Collins FS. Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes. Science, 1991, 254: 1797-1799.
[13] Tabcharani JA, Chang XB, Riordan JR, Hanrahan JW. Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. Nature, 1991, 352: 628-631.
[14] Bear C, Li CH, Kartner N, Bridges RJ, Jensen TJ, Ramjeesingh M, Riordan JR. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell, 1992, 68: 809-818.
[15] Dahl M, Tybjaerg-Hansen A, Lange P, Nordestgaard BG. F508 heterozygosity in cystic fibrosis and susceptibility to asthma. Lancet, 1998, 351: 1911-1913.
[16] Tzetis M, Efthymiadou A, Strofalis S, Psychou P, Dimakou A, Pouliou E. CFTR gene mutations including three novel nucleotide substitutions and haplotype background in patients with asthma, disseminated brochiectasis and chronic obstructive pulmonary disease. Hum Genet, 2001. 108: p. 216-221.
[17] Taouil K, Hinnrasky J, Hologne C, Corlieu P, Klossek JM, Puchelle E. Stimulation of beta 2-Adrenergic Receptor Increases Cystic Fibrosis Transmembrane Conductance Regulator Expression in Human Airway Epithelial Cells through a cAMP/Protein Kinase A-independent Pathway. J Biol Chem,2003, 278: 17320-27.
[18] Smith J, Travis SM, Gereenberg EP, Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnornmal airway surface fluid. Cell, 1996, 95: 1005-1015.
[19] Di A, Brown ME, Deriy LV, Li C, Szeto FL, Chen Y, Huang P, Tong J, Naren AP, Bindokas V, Palfrey HC, Nelson DJ, CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol, 2006, 8(9): 933-44.
[20] Maggi C, Giachetti A, Dey RD, Said SI. Neuropeptides as regulators of airway function: vasoactive intestinal peptide and the tachykinins. Physiol Rev, 1995, 75(2): 277-322.
[21] Ren YH, Qin XQ, Guan CX, Luo ZQ, Zhang CQ, Sun XH. Temporal and spatial ditribution of VIP, CGRP and their receptors in the development of airway hyperresponsiveness in the lungs. Acta Physiologica Sinica, 2004, 56: 137-146.
[22] Qin XQ, Sun XH, Luo ZQ. Vasoactive intestinal peptide and epidermal growth factor upregulates bcl-2 gene expression in bronchial epithelial cells. Acta Physiologica Sinica, 1999, 51: 419-424.
[23] Lazarus SC, Basbaum CB, Barnes PJ, Gold WM. cAMP immunocytochemistry provides evidence for functional VIP receptors in trachea. Am J Physiol, 1986, 251: C115-C119.
[24] Rogers D. Motor control of airway goblet cells and glands. Respir Physiol, 2000, 125: 129-144.
[25] Groneberg DA, Hartmarm P, Dinh QT, Fischer A. Expression and distribution of vasoactive intestinal polypeptide receptor VPAC(2) mRNA in human airways. Lab Invest, 2001, 81: 749-755.
[26] Qin X, Sun XH, Luo ZQ, Guan CX, Zhang CQ. Affection of epidermal growth factor on VIP secretion and VIPR expression in airway epithel ial cells. Bull of Hunan Med Univ, 1999, 24(2): 99-102.
[27] Joo NS, Saenz Y, Krouse ME, Wine JJ. Mucus secretion from single submucosal glands of pig. Stimulation by carbachol and vasoactive intestinal peptide. J Biol Chem, 2002, 277: 28167-28175.
[28] Tsumura T, Hazama A, Miyoshi T, Ueda S, Okada Y. Activation of cAMP-dependent Cl- currents in guinea-pig Paneth cells without relevant evidence for CFTR expression. The Journal of Physiology, 1998, 512: 765-777.
[29] Ameen N, Marino C, Salas PJI. cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo. Am J Physiol Cell Physiol, 2003, 284: C429-C438.
[30] Li C, Ramjeesingh M, Wang W, Garami E, Hewryk M, Lee D, Rommens JM, Galley K, Bear CE. ATPase activity of the cystic fibrosis transmembrane conductance regulator. J Biol Chem, 1996, 271: 28463-28468.
[31] Gadsby D, Dousmanis AG, Nairn AC. ATP hydrolysis cycles and the gating of CFTR Cl- channels. Acta Physiol Scand, 1998, 643: 247-256.
[32] Jia Y, Mathews CJ, Hanrahan JW. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J Biol Chem, 1997, 272: 4978-4984.
[33] Liedtke C, Cole TS. Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells. Am J Physiol, 1998, 275: C1357-C1364.
[34] Middleton L, Harvey RD. PKC regulation of cardiac CFTR cl- channel function in guinea pig ventricular myocytes. Am J Physiol, 1998, 275: C293-C302.
[35] Bebok Z, Varga K, Hicks JK, Venglarik CJ, Kovacs T, Chen L, Hardiman KM, Collawn JF, Sorscher EJ, Matalon S. Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl- secretion in airway epithelia. J Biol Chem, 2002,277:43041-43049.
[36] Asfaha S, Bell CJ, Wallace JL, MacNaughton WK. Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase. Am J Physiol Gastrointest Liver Physiol, 1999, 276: G703-G710.
[37] MacNaughton W, Lowe SS, Cushing K. Role of nitric oxide in inflammation-induced suppression of secretion in a mouse model of acute colitis. Am J Physiol Gastrointest Liver Physiol, 1998, 275: G1353-G1360.
[38] Skinn A, MacNaughton WK. Nitric oxide inhibits cAMP-dependent CFTR trafficking in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol, 2005, 289: G739-G744.
[39] Kulka M, Dery R, Nahirney D, Duszyk M, Befus AD. Differential Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Interferon gamma in Mast Cells and Epithelial Cells. J Pharmacol Exp Ther, 2005,315:563-570.
[40] Tan Y, Qi MM, Qin XQ, Xiang Y, Li X, Wang Y, Qu F, Liu HJ, Zhang JS. Wound repair and proliferation of bronchial epithelial cells enhanced by bombesin receptor subtype 3 activation. Peptides, 2006, 27: 1852-1858.
[41] Wu H, Guan CX, Qin XQ, Xiang Y, Qi MM, Luo ZQ, Zhang CQ. Upregulation of substance P receptor expression by calcitonin gene-related peptide, a possible cooperative action of two neuropeptides involved in airway hyperresponsiveness. Pulm Pharmacol Ther, 2006, 20: 513-524.
[42] Ren Y, Qin XQ, Guan CX, Luo ZQ, Zhang CQ, Sun XH. The temporal and spatial distribution of vasoactive intestinal peptide and its receptor in the development of airway hyperresponsiveness. Chin J Tuberc Respir Dis, 2004, 27: 224-228.
[43] Qin XQ. Protection from Oxidant Injury on Airway Environment Modulation. Progress in Physiological Sciences, 1999, 30: 129-132.
[44] Qin X, Xiang Y, Luo ZQ, Zhang CQ, Sun XH. Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbit bronchial epithelial cells. Acta Physiologica Sinica, 2000, 52(6): 519-521.
[45] Tamada T, Sasaki T, Saitoh H, Ohkawara Y, Irokawa T, Sasamori K, Oshiro T, Tamura G, Shimura S, Shirato K. A novel function of thyrotropin as a potentiator of electrolyte secretion from the tracheal gland. Am J Respir Cell Mol Biol, 2000, 22: 566-573.
[46] Danahay H, Atherton H, Jones G, Bridges RJ, Poll CT. Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol, 2002, 282: L226-236.
[47] Sasamori K, Sasaki T, Takasawa S, Tamada T, Nara M, Irokawa T, Shimura S, Shirato K, Hattori T. Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operate submucosal gland acinar cells. Am J Physiol Lung Cell Mol Physiol, 2004, 287: L69-78.
[48] Widdicombe J. Regulation of the depth and composition of airway surface liquid. JAnat, 2002, 201: 313-318.
[49] Chmiel J, Davis PB. State of the art: why do the lungs of patients with cystic fibrosis become infected and why can't they clear the infection? Respir Res, 2003, 4: 8.
[50] Regnis J, Robinson M, Bailey DL, Cook P, Hooper P, Chan HK, Gonda I, Bautovich G, Bye PT. Mucociliary clearance in patients with cystic fibrosis and in normal subjects. Am J Respir Crit Care Med, 1994, 150: 66-71.
[51] Chace K, Naziruddin B, Desai VC, Flux M, Schdev GP. Physical properties of purfied human respirstory mucus glycoproteins: effects of sodium chloride concentration on the aggregation properties and shape. Exp Lung Res, 1989, 15: 721-737.
[52] Chace K, Flux M, Sachdev GP. Comparison of physicochemical propreties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosos and asthmatic patients. Biochemistry, 1985, 24: 7334-7341.
[53] Former C, Lorenz JN, Paul RJ, Chloride channel function is linked to epithelium-dependent airway relaxation. Am J Physiol Lung Cell Mol Physiol, 2001, 280:L334-41.
[54] Stutts M, Canessa CM, Olsen JC, Hamrick M, Conn JA, Rossier BC, Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science, 1995, 269: 847-850.
[55] Schwiebert E, Egan ME, Hwang TH, Fulmer SB, Allen SS, Cutting GR, Guggino WB. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell, 1995, 81: 1063-73.
[56] Knowles M, Stutts MJ, Spock A, Fischer N, Gatzy JT, Boucher RC. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science, 1983, 221: 1067-1070.
[57] Cowley E, Linsdell P. Oxidant stress stimulates anion secretion from the human airway epithelial cell line Calu-3: implications for cystic fibrosis lung disease. J Physiol, 2002, 543: 201-209.
[58] Jung J, Lee JY, Oh SO, Jang PG, Bae HR, Kim YK, Lee SH. Effect of t-butylhydroperoxide on chloride secretion in rat tracheal epithelia. Pharmacol Toxicol, 1998,82:236-242.
[59] Nguyen T, Canada AT. Modulation of human colonic T84 cell secretion by hydrogen peroxide. Biochem Pharmacol, 1994,47: 403-410.
[60] Tamai H, Kachur JF, Baron DA, Grisham MB, Gaginella TS. Monochloramine, a neutrophil-derived oxidant, stimulates rat colonic secretion. J Pharmacol Exp Ther, 1991,257:887-894.
[61] Cantin A, Bilodeau G, Ouellet C, Liao J, Hanrahan JW. Oxidant stress suppresses CFTR expression. Am J Physiol Cell Physiol, 2006, 290: C262-C270.
[62] Besancon F, Przewlocki G, Baro I, Hongre AS, Escande D, Edelman A. Interferon-y downregulates CFTR gene expression in epithelial cells. Am J Physiol Cell Physiol, 1994, 267: C1398-C1404.
[63] Nakamura H, Yoshimura K, Bajocchi G, Trapnell BC, Pavirani A, Crystal RG. Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene. FEBS Lett, 1992, 314: 366-370.
[64] Koh J, Sferra TJ, Collins FS. Characterization of the cystic fibrosis transmembrane conductance regulator promoter region. Chromatin context and tissue-specificity. J Biol Chem, 1993, 268: 15912-21.
[65] Yoshimura K, Nakamura H, Trapnell BC, Chu CS, Dalemans W, Pavirani A, Lecocq JP, Crystal RG. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin. Nucleic Acids Res, 1991,19:5417-23.
[66] Chou J, Rozmahel R, Tsui LC. Characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene. J Biol Chem, 1991, 266: 24471-6.
[67] Qin XQ, Xiang Y, Luo ZQ, Zhang CQ, Sun XH. Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbit bronchial epithelial cells. Acta Physiologica Sinica, 2000, 52(6): 519-521.
[68] Chen L, Patel RP, Teng X, Bosworth CA, Lancaster JR, Matalon S. Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione. J Biol Chem, 2006, 981: 9190-9.
[69] Rahman I, MacNee W. Regulation of redox glutathione levels and gene transcription in lung inflammation: therapeutic approaches. Free Radic. Biol. Med, 2000, 28: 1405-1420.
[70] Andreadis A, Hazen SL, Comhair SA, Erzurum SC. Oxidative and nitrosative events in asthma. Free Radic Biol Med, 2003, 35: p. 213-25.
[71] Chen P, Illsley NP, Verkman AS. Renal brush-border chloride transport mechanisms characterized using a fluorescent indicator. Am J Physiol, 1988, 254:F114-F120.
[72] Soroceanu L, Manning TJ , Sontheimer H. Modulation of glioma cell migration and invasion using Cl - and K+ ion channel blockers. J Neurosci, 1999, 19: 5942-5954.
[73] Groneberg D, Springer J, Fischer A. Vasoactive intestinal polypeptide as mediator of asthma. Pulm Pharmacol Ther, 2001, 14: 391-401.
[74] Haws C, Finkbeiner WE, Widdicombe JH, Wine JJ. CFTR in Calu-3 human airway cells, channel properties and role in cAMP- activated Cl- conductance. Am J Physiol, 1994, 266: L502-L512.
[75] Shen B, Finkbeiner WE, Wine JJ, Mrsny RJ, Widdicombe JH. Calu-3, a human airway epithelial cell line that shows cAMP-dependent Cl- secretion. Am J Physiol, 1994, 266: L493-L501.
[76] Joo N, Irokawa T, Wu JV, Robbins RC, Whyte RI, Wine JJ. Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands. J Biol Chem, 2002, 277:50710-50715.
[77] Lucchini R, Facchini F, Turato G, Saetta M, Caramori G, Ciaccia A, Maestrelli P, Springall DR, Polak JM, Fabbri L, Mapp CE. Increased VIP-positive nerve fibers in the mucous glands of subjects with chronic bronchitis. Am J Respir Crit Care Med, 1997, 156: 1963-1968.
[78] Chanez P, Springall D, Vignola AM, Moradoghi-hattvani A, Polak JM, Godard P, Bousquet J. Bronchial mucosal immunoreactivity of sensory neuropeptides in severe airway diseases. Am J Respir Crit Care Med, 1998, 158: 985-990.
[79] Lazarus S, Basbaum CB, Barnes PJ, Gold WM. cAMP immunocytochemistry provides evidence for functional VIP receptors in trachea. Am J Physiol, 1986, 251: C115-C119.
[80] Sreedharan SP, Robichon A, Peterson KE, Goetzl EJ. Cloning and expression of the human vasoactive intestinal peptide receptor. Proc Natl Acad Sci USA, 1991, 88: 4986-4990.
[81] Harmar A, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase- activating polypeptide. Pharmacol Rev, 1998, 50: 265-270.
[82] Laburthe M, Couvineau A. Molecular pharmacology and structure of VPAC receptors for VIP and PACAP. Regul Pept, 2002, 108: 165-173.
[83] Busto R, Carrero I, Guijarro LG, Solano R.M, Zapatero J, Noguerales F, Prieto JC, Expression, pharmacological, and functional evidence for PACAP/VIP receptors in human lung. Am J Physiol, 1999, 277: L42-L48.
[84] Fischer A, Kummer W, Couraud JY, Adler D, Branscheid D, Heym C. Immunohistochemical localization of receptors for vasoactive intestinal peptide and substance P in human trachea. Lab Invest, 1992, 67: 387-393.
[85] Ameen N, Martensson B, Bourguinon L, Marino C, Isenberg J, Mclaughlin GE. CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo. J Cell Sci, 1999,112: 887-894.
[86] Gadsby D, Nairn A. Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol Rev 1999, 79: S77-S107.
[87] Kopito R. Biosynthesis and Degradation of CFTR. Physiol Rev, 1999, 79: S167-S173.
[88] Yoo J, Moyer BD, Bannykh S, Yoo HM, Riordan JR, Balch WE. Non-conventional Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator through the Early Secretory Pathway. J Biol Chem, 2002,277:11401-11409.
[89] Weixel K, Bradbury NA. The Carboxyl Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Binds to AP-2 Clathrin Adaptors. J Biol Chem, 2000, 275: 3655-3660.
[90] Prince L, Peter K, Hatton SR, Zaliauskiene L, Cotlin LF, Clancy JP, Marchase RB, Collawn JF. Efficient Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator Requires a Tyrosine-based Signal. J Biol Chem, 1999, 274: 3602-3609
[91] Lukacs G, Segal G, Kartner N, Grinstein S, Zhang, F. Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem J, 1997, 328: 353-361.
[92] Gentzsch M, Chang XB, Cui L, Wu Y, Ozols W, Choudhury A, Pagano RE, Riordan JR. Endocytic Trafficking Routes of Wild Type and F508 Cystic Fibrosis Transmembrane Conductance Regulator. Mol Biol Cell, 2004, 15: 2684-2696.
[93] Benharouga M, Haardt M, Kartner N, Lukacs GL. COOH-terminal Truncations Promote Proteasome-dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments. J Cell Biol, 2001, 153: 957-970.
[94] Bradbury N, Clark JA, Watkins SC, Widnell CC, Smith HST, Bridges RJ. Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator. Am J Physiol, 1999, 276: L659-L668.
[95] Hu W, Howard M, Lukacs GL. Multiple endocytic signals in the C-terminal tail of the cystic fibrosis transmembrane conductance regulator. Biochem J, 2001, 354: 561-572.
[96] Weixel K, Bradbury NA. Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway. J Biol Chem, 2001, 276: 46251-46259.
[97] Picciano J, Ameen N, Grant B, Bradbury NA. Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator. Am J Physiol, 2003, 285: C1009-C1018.
[98] Hanrahan J, Tabcharani JA, Becq F, Mathews CJ, Augustinas O, Jensen TJ, Chang X-B, Riordan JR. Function and dysfunction of the CFTR chloride channel. In: Ion Channels and Genetic Diseases, edited by Dawson DC, and Frizzell RA, Vol 50, 1995, New York: Rockefeller University Press.
[99] Kunzelman K, Mall M. Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev, 2001, 82: 245-289.
[100] Howard M, Jiang X, Stolz D, Hill W, Johnson J, Watkins S, Frizzell R, Bruton C, Robbins P, Weisz O. Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells. Am J Physiol Cell Physiol, 2000, 279: C375-C382.
[101] Weber W, Segal A, Simaels J, Vankeerberghen A, Cassiman JJ, Driessche WV. Functional integrity of the vesicle transporting machinery is required for complete activation of CFTR expressed in Xenopus laevis oocytes. Pflugers Arch, 2001, 441: 850-859.
[102]Romey M, Pallares-Ruiz N, Mange A, Mettling C, Peytavi R, Demaille J, Claustres M. A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression. J Biol Chem, 2000, 275:3561-3567.
[103] Rowntree, R, Harris A. DNA polymorphisms in potential regulatory elements of the CFTR gene alter transcription factor binding. Hum Genet, 2002, 111: 66-74.
[104] Wittwer J, Marti-Jaun J, Hersberger M. Functional polymorphism in ALOX15 results in increased allele-specific transcription in macrophages through binding of the transcription factor SPI1. Hum Mutat, 2006, 27: 78-87.
[105] Rene C, Taulan M , Iral F, Doudement J, Honore AL, Gerbon C, Demaille J, Claustres M, Romey MC. Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway. Nucleic Acids Res, 2005, 33: 5271-5290.
[106] Taulana M, Lopeza E, Guittarda C, Renea C, Bauxa D, Altieria JP, DesGeorgesa M, Claustresa M, Romeya MC. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding Biochemical and Biophysical Research Communications, 2007, 361: 775-781.
[107] Koh J, Sferra TJ, Collins FS. Characterization of the CFTR promoter region: chromatin context and tissue-specificity. J Biol Chem, 1993, 268: 15912-15921.
[108] Brugmann S, Tapadia MD, Helms JA. The molecular origins of species-specific facial pattern. Curr Top Dev Biol, 2006, 73: 1-42.
[109]Kadonaga J, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell, 1987,51:1079-1090.
[110] Yoshimura K, Nakamura H, Trapnell BC, Dalemans W, Pavirani A, Lecocq JP, Crystal RG. The cystic fibrosis gene has a "housekeeping"-type promoter and is expressed at low levels in cells of epithelial origin. J Biol Chem, 1991, 266: 9140-9144.
[111]Kwon H, Kim MS, Edenberg HJ, Hur MW. Sp3 and Sp4 can repress transcription by competing with Spl for core cis-elements on the human ADH5/FDH minimal promoter. J Biol Chem, 1999, 274: 20-28.
[112]Shafer G, Cramer T, Suske G, Kemmner W, Wiedenmann B, H"ocker M. Oxidative stress regulates vascular endothelial growth factor A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements. J Biol Chem, 2003, 278: 8190-8198.
[113] Kelley K, Wang H, Ratnam M. Dual regulation of ets-activated gene expression by SP1. Gene, 2003, 307: 87-97.
[114] Couse J, Lindzey J, Grandien K, Gustafsson JA, Korach KS. Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology, 1997, 138: 4613-4621.
[115] Carey M, Cared JW, Bradbury J. Spontaneous airway hyperresponsiveness in estrogen receptor alpha deficient mice. Am J Respir Crit Care Med, 2007, 175: 126-135.
[116]Gavett S, Madison SL, Chulada PC, Scarborough PE, Qu W, Boyle JE, Tiano HF, Lee CA, Langenbach R, Roggli VL. Allergic lung responses are increased in prostaglandin H synthase-deficient mice. J Clin Invest, 1999, 104: 721-732.
[117]Wilder J, Collie DD, Wilson BS, Bice DE, Lyons CR, Lipscomb MF. Dissociation of airway hyperresponsiveness from immunoglobulin E and airway eosinophilia in a murine model of allergic asthma. Am J Respir Cell Mol Biol, 1999,20: 1326-1334.
[118]Takeda K, Haczku A, Lee JJ, Irvin CG, Gelfand EW. Strain dependence of airway hyperresponsiveness reflects differences in eosinophil localization in the lung. Am J Physiol Lung Cell Mol Physiol, 2001, 281: L394-L401.
[119]Dijkstra A, Howard TD, Vonk JM, Ampleford EJ, Lange LA, Bleecker ER, Meyers DA, Postma DS. Estrogen receptor 1 polymorphisms are associated with airway hyperresponsiveness and lung function decline, particularly in female subjects with asthma. Allergy Clin Immunol, 2006,117: 604-611.
[120]Cuzzocrea S, Mazzon E, Sautebin L. The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat. Mol Med, 2001, 7: 478-487.
[121]Coakley R, Sun HR, Chines LA, Rasmussen JE, Stackhouse JR, Okada SF, Fricks I, Young SL, Tarran R. 17β-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia. J Clin Invest, 2008,118: 4025-4035.
[122] Mollerup S, Jorgensen K, Berge G, Haugen A. Expression of estrogen receptors alpha and beta in human lung tissue and cell lines. Lung Cancer, 2002, 37: 153-159.
[123]Thomas L, Doyle, LA, Edelman MJ. Lung cancer in women: emerging differences in epidemiology, biology, and therapy. Chest, 2005, 128: 370-381.
[124]Riordan J, Rommens JM, Kerem BS, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FS, Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 1989,245: 1066-1073.
[125]Anderson M, Gregory RJ, Thompson S, Souza DW, Paul S, Mulligan RC, Smith AE, Welsh MJ. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science, 1991, 253: 202-205.
[126]Quinton P. Chloride impermeability in cystic fibrosis. Nature, 1983. 301: 421-422.
[127] Cai Z, Scott-Ward TS, Sheppard DN. Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Clchanne. J Gen Physiol, 2003, 122: 605-620.
[128] Dorwart M, Thibodeau P, Thomas P. Cystic fibrosis: recent structural insights. J Cyst Fibres, 2004, 3: 91-94.
[129] Gong X, Burbridge SM, Cowley EA, Linsdell P. Molecular determinants of Au(CN)2 - binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl- channel pore. J Physiol, 2002, 540: 39-47.
[130] Ge N, Muise CN, Gong X, Linsdell P. Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore. J Biol Chem, 2004, 279: 55283-55289.
[131] St Aubin C, Linsdell P. Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel. J Gen Physiol, 2006, 128: 535-545.
[132] Wright A, Gong X, Verdon B. Novel regulation of cystic fibrosis transmembrane conductance regulator (CFTR) channel gating by external chloride. J B iol Chem, 2004, 279: 41658-41663.
[133]Riordan J. Assembly of functional chloride channels. Arm Rev Physiol, 2005, 67:701-718.
[134] McCarty N. Permeation through the CFTR chloride channel. J Exp Biol, 2000, 203: 1947-1962.
[135] Linsdell P. Mechanism of chloride permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. Exp Physiol, 2006, 91: 123-129.
[136]Randak C, Welsh MJ. ADP inhibits function of the ABC transponter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity. Proc Natl Aead Sci USA, 2005,102: 2216-2220.
[137]Kidd J, Ramjeesingh M, Straiford F. A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator(CFTR) mediates ATP ase activity. J Biol Chem, 2004, 279: 41664-41669.
[138]Vergani P, Lockless SW, Nairn AC. Channel opening by ATP-driven tight dimerization of its nueleotide-binding domains. Nature, 2005, 433: 876-880.
[139] Lewis H, Buehanan SG, Burley SK. Structure of nueleotide-binding domain 1 of the cystic fibrosis trans-membrane conductance regulator. EMBO J, 2004, 23: 282-293.
[140] Vergani P, Nairn AC, Gadsby DC. On the mechanism of MgATP-dependent gating of CFTR cl- channels. J Gen Physiol, 2003,121: 17-36.
[141] Berger A, Lkuma M, Welsh MJ. Nomal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nueleotide- binding domain. Proc Natl Acad Sci USA, 2005, 102: 455-460.
[142] Cheng S, Rich DP, Marshall J, Gregory RJ, Welsh MJ, Smith AE. Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell, 1991, 66:1027-36
[143] Quinton P, Reddy MM. Regulation of absorption by phosphorylation of CFTR. Jpn J Physiol, 1994, 44 : S207-S213.
[144] Schwencke C, Yamamoto M, Okumura S, Toya Y, Kim SJ, Ishikawa Y. Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae. Mol Endocrinol, 1999,13: 1061-1070.
[145] Ostrom R, Violin JD, Coleman S, Insel PA. Selective enhancement of beta-adrenergic receptor signaling by overexpression of adenylyl cyclase type 6: colocalization of receptor and adenylyl cyclase in caveolae of cardiac myocytes. Mol Pharmacol Toxicol, 2000, 57: 1075-1079.
[146] Rich D, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain. J Biol Chem,1993, 268:20259-20267.
[147] Gadsby D, Nairn AC. Regulation of CFTR channel gating. Trends Biochem Sci, 1994,19:513-518.
[148] Dahan D, Evagelidis A, Hanrahan JW, Hinkson DA, Jia Y, Luo J, Zhu T. Regulation of the CFTR channel by phosphorylation. Pflugers Arch, 2001, 443 (Suppl ): S92-S96.
[149] Chang X, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, Hanrahan JW, Riordan JR. Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. J Biol Chem, 1993, 268: 11304-11311.
[150] Seibert F, Chang XB, Aleksandrov AA, Clarke DM, Hanrahan JW, Riordan JR. Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum. Biochim Biophys Acta, 1999, 1461: 275-283.
[151] Bajnath R, Groot JA, De Jonge HR, Kansen M, Bijman J. Synergistic activation of non-rectifying small-conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl.19A. Pflugers Arch, 1993, 425: 100-108.
[152] Vaandrager A, Ehlert EM, Jarchau T, Lohmann SM, de Jonge HR. N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II. J Biol Chem, 1996, 271: 7025-7029.
[153]Reddy M, Quinton PM. Functional interaction of CFTR and ENaC in sweat glands. Pflugers Arch, 2003,445: 499-503.
[154] Vaandrager A, Smolenski A, Tilly BC, Houtsmuller AB, Ehlert EM, Bot AG, Edixhoven M, Boomaars WE, Lohmann SM, de Jonge HR. Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation. Proc Natl Acad Sci USA, 1998, 95: 1466-1471.
[155] Csanady L, Chan KW, Angel BB, Nairn AC, Gadsby DC. Negative regulation of CFTR chloride channel gating through R-domain serine 768. Ped Pulmon, 1998,17:205.
[156] Travis S, Berger HA, Welsh MJ. Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA, 1997,94:11055-11060.
[157] Nairn A, Qin J, Chait BT, Gadsby DC. Indentification of sites in the R domain of CFTR phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatase 2A and 2C. Ped Pulmon, 1996, Suppl 13:21.
[158]Reddy M, Quinton PM. Deactivation of CFTR-C1 conductance by endogenous phosphatases in the native sweat duct. Am J Physiol, 1996, 270: C474-C480.
[159]Reddy M, Quinton PM. Cytosolic potassium controls CFTR deactivation in human sweat duct. Am J Physiol Cell Physiol, 2006, 291: C122-C129.
[160] Zhu T, Dahan D, Evagelidis A, Zheng SX, Luo J, Hanrahan JW. Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C. J Biol Chem, 1999, 274: 29102-29107.
[161] Fischer H, Machen TE. The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel. Biophys J, 1996, 71: 3073-3082.
[162] Nguyen T, Canada AT, Heintz GG, Gettys TW, Conn JA. Stimulation of secretion by the T84 colonic epithelial cell line with dietary flavonols. Biochem Pharmacol, 1991,41: 1879-1886.
[163] Sears C, Firoozmand F, Mellander A, Chambers FG, Eromar IG, Bot AG, Scholte B, De Jonge HR, Donowitz M. Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers. Am J Physiol, 1995, 269: G874-G882.
[164] French P, Bijman J, Bot AG, Boomaars WE, Scholte BJ, de Jonge HR. Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism. Am J Physiol, 1997, 273: C747-C753.
[165] Wang F, Zeltwanger S, Yang IC, Nairn AC, Hwang TC. Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating. Evidence for two binding sites with opposite effects. J Gen Physiol, 1998,111: 477-490.
[166] Bulteau-Pignoux, L, Derand R, Metaye T, Joffre M, Becq F. Genistein modifies the activation kinetics and magnitude of phosphorylated wild-type and G551D-CFTR chloride currents. J Membr Biol, 2002,188: 175-182.
[167]Berger A, Randak CO, Ostedgaard LS, Karp PH, Vermeer DW, Welsh MJ. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. J Biol Chem, 2005, 280: 5221-5226.
[168] Lansdell K, Cai Z, Kidd JF, Sheppard DN. Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in murine cell line. J Physiol, 2000, 524: 317-330.
[169] Moran O, Zegarra-Moran O, A quantitative description of the activation and inhibition of CFTR by potentiators: Genistein. FEBS Lett, 2005, 579: 3979-3983.
[170] Linsdell P, Hanrahan JW. Flickery block of single CFTR chloride channels by intracellular anions and osmolytes. Am J Physiol, 1996, 271: C628-C634.
[171] Quinton P, Reddy MM. Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding. Nature, 1992, 360: 79-81.
[172] Bell C, Quinton PM. Regulation of CFTR Cl- conductance in secretion by cellular energy levels. Am J Physiol, 1993, 264: C925-C931.
[173] Egan M, Pearson M, Weiner SA, Rajendran V, Rubin D, Glockner-Pagel J, Canny S, Du K, Lukacs GL, Caplan MJ. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science, 2004, 304: 600-602.
[174] Mall M, Kunzelmann K. Correction of the CF defect by curcumin: hypes and disappointments. Bioessays, 2005, 27: 9-13.
[175] Scott O. Investigation of CFTR intermolecular structure and protein protein interaction. Diss Abstr Int, 2002, 62: 3593.
[176] Chappe V, Irvine T, Liao J. Phosphorylation of CFTR by PKA promotes binding of the regulatory domain. EMBO J, 2005, 24: 2730-2740.
[177] Amaral M. Processing of CFTR: traversing the cellular maze-how much CFTR needs to go through to avoid cystic fibrosis? Pediatr Pulmonol, 2005, 39: 479-491.
[178] Winter M, Welsh MJ. Stimulation of CFTR activity by its phosphorylated R domain. Nature, 1997, 389: 294-296.
[179] Grimard V, Li C, Ramjeesingh M, Bear CE, Goormaghtigh E, Ruysschaert JM. Phosphorylation-induced conformational changes of cystic fibrosis transmembrane conductance regulator monitored by attenuated total reflection- fourier transform IR spectroscopy and fluorescence spectroscopy. J Biol Chem, 2004, 279: 5528-5536.
[180]Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC. In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer. EMBO J, 2006, 25: 4728-4739.
[181] Mehta A, CFTR: more than just a chloride channel. Pediatr Pulmonol, 2005. 39: 292-298.
[182] Guggino W, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol, 2006, 7: 426-436.
[183] Short D, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem, 1998, 273:19797-19801.
[184] Naren A, Cobb B, Li C, Roy K, Nelson D, Heda GD, Liao J, Kirk KL, Sorscher EJ, Hanrahan J, Clancy JP. A macromolecular complex of β2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA. Proc Natl Acad Sci USA, 2003, 100: 342-346.
[185] Vastiau, A., Cao L, Jaspers M, Owsianik G, Janssens V, Cuppens H, Goris J, Nilius B, Cassiman JJ. Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel. FEBS Lett, 2005, 579: 3392-3396.
[186] Pouyssegur J. Molecular biology and hormonal regulation of vertebrate Na+/H+ exchanger isoforms. isoforms. Renal Physiol Biochem, 1994,17: 190-193.
[187] Guggino W, Banks-Schlegel SP. Macromolecular interactions and ion transport in cystic fibrosis. Am J Respir Crit Care Med, 2004,170: 815-820.
[188]Bachhuber T, Konig J, Voelcker T. Cl~- interference with the epithelial Na~+ Channel ENaC. J Biol Chem, 2005, 280: 31587-31594.
[189] Cuppoletti J, Tewari KP, SherryAM. C1C-2C1- channels in human lung epithelia: activation by arachidonic acid, amidation, and acid activated omep razole. Am J Physiol Cell Physiol, 2001, 281: C46-C54.
[190] Burghardt B, ElkaermL, Kwon TH. Distribution of aquaporin water channelsAQP1 and AQP5 in the ductal system of the human pancreas. Gut, 2003,52:1008-1016.
[191] Welsh M, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell, 1993, 73: 1251-1254.
[192]Rowe S, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med, 2005, 352:1992-2001.
[193]Zielenski J. Genotype and phenotype in cystic fibrosis. Respiration, 2000, 67: 127-133.
[194] Ahmed N, Corey M, Forstner G, Zielenski J, Tsui LC, Ellis L, Tullis E, Durie P. Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas. Gut, 2003, 52: 1159-1164.
[195] Rowntree R, Harris A. The phenotypic consequences of CFTR mutations. Ann Hum Genet, 2003, 67: 471-485.
[196]Kopelman H, Ferretti E, Gauthier C, Goodyer PR. Rabbit pancreatic acini express CFTR as a cAMP-activated chloride efflux pathway. Am J Physiol, 1995, 269:C626-C631.
[197] Zeng W, Lee MG, Yan M, Diaz J, Benjamin I, Marino CR, Kopito R, Freedman S, Cotton C, Muallem S, Thomas P. Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells. Am J Physiol, 1997, 273:C442-C455.
[198] Novak I, Greger R. Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane. Pflugers Arch, 1988,411:58-68.
[199] Novak I, Greger R. Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport. Pflugers Arch, 1988, 411: 546-553.
[200] Zabner J, Smith JJ, Karp PH, Widdicombe JH, Welsh MJ. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol Cell, 1998, 2: 397-403.
[201]Ishiguro H, Steward MC, Wilson RW, Case RM. Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. J Physiol, 1996. 495: 179-191.
[202] Ishiguro H, Steward MC, Lindsay AR, Case RM. Accumulation of intracellular HCO3- by Na+-HCO3- cotransport in interlobular ducts from guinea-pig pancreas. J Physiol, 1996, 495: 169-178.
[203] Shumaker H, Amlal H, Frizzell R, Ulrich CD. CFTR drives Na+-nHCO3- cotransport in pancreatic duct cells: a basis for defective HCO3- secretion in CF. Am J Physiol, 1999, 276: C16-C25.
[204] Marino C, Matovcik LM, Gorelick FS, Cohn JA. Localization of the cystic fibrosis transmembrane conductance regulator in pancreas. J Clin Invest, 1991, 88:712-716.
[205] Illek B, Tarn AW, Fischer H, Machen TE. Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium. Pflugers Arch, 1999, 437: 812-822.
[206] Ko S, Shcheynikov N, Choi JY, Luo X, Ishibashi K, Thomas PJ, Kim JY, Kim KH, Lee MG, Naruse S, Muallem S. A molecular mechanism for aberrant CFTR-dependent HCO3- transport in cystic fibrosis. EMBO J, 2002, 21: 5662-5672.
[207] Ishiguro H, Namkung W, Yamamoto A, Wang Z, Worrell RT, Xu J, Lee MG, Soleimani M. Effect of Slc26a6 deletion on apical C1-/HCO3 - exchanger activity and cAMP-stimulated bicarbonate secretion in pancreatic duct. Am J Physiol Gastrointest Liver Physiol, 2007, 292: G447-G455.
[208] Sohma Y, Gray MA, Imai Y, Argent BE. HCO3- transport in a mathematical model of the pancreatic ductal epithelium. J Membr Biol, 2000, 176: 77-100.
[209] Lee M, Choi JY, Luo X, Strickland E, Thomas PJ, Muallem S. Cystic fibrosis transmembrane conductance regulator regulates luminal C1-/HCO3- exchange in mouse submandibular and pancreatic ducts. J Biol Chem, 1999, 274:14670-14677.
[210] Whitcomb D. Pancreatic bicarbonate secretion: role of CFTR and the sodium-bicarbonate cotransporter. Gastroenterology, 1999,117: 275-277.
[211]Devor D, Singh AK, Lambert LC, DeLuca A, Frizzell RA, Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol, 1999,113: 743-760.
[212] Abuladze N, Lee I, Newman D, Hwang J, Boorer K, Pushkin A, Kurtz I. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J Biol Chem, 1998, 273: 17689-17695.
[213]Malinowska D, Kupert EY, Bahiniski A, Sherry AM. Cuppoletti J Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel. Am J Physiol, 1995, 268: C191-C200.
[214] Choi J, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis. Nature, 2001,410: 94-97.
[215] Choi J, Lee MG, Ko S, Muallem S. Cl~--dependent HCO3- transport by cystic fibrosis transmembrane conductance regulator. JOP, 2001, 2: 243-246.
[216] Chan H, Goldstein J, Nelson DJ. Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells. Am J Physiol, 1992, 262:C1273-83.
[217] Namkung W, Lee JA, Ahn W, Han W, Kwon SW, Ahn DS, Kim KH, Lee MG. Ca2+ activates cystic fibrosis transmembrane conductance regulator- and Cl-dependent HCO3- transport in pancreatic duct cells. J Biol Chem, 2003, 278: 200-207.
[218]Kasai H, Angustine GJ. Cystosolic Ca~(2+) gradients triggering uniderectional fluid secretion from exocrine pancreas. Nature, 1990, 348: 735-738.
[219] Quinton P. Viscosity versus composition in airway pathology. Am J Respir Crit Care Med, 1994, 149:6-7.
[220] Joris L, Quinton PM. Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid. Am J Physiol, 1992, 263: L243-L248.
[221] Noah T, Black HR, Cheng PW, Wood RE, Leigh MW. Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis. J Infect Dis, 1997, 175: 638-647.
[222] Bhaskar K, Gong DH, Bansil R, Pajevic S, Hamilton JA, Turner BS, LaMont JT. Profound increase in viscosity and aggregation of pig gastric mucin at low pH. Am J Physiol, 1991, 261: G827-G832.
[223] Cao X, Bansil R, Bhaskar KR, Turner BS, LaMont JT, Niu N, Afdhal NH. pH-dependent conformational change of gastric mucin leads to sol-gel transition. BiophysJ, 1999, 76:1250-1258.
[224] Muhlebach M, Stewart PW, Leigh MW, Noah TL. Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients. Am J Respir Crit Care Med, 1999,160: 186-191.
[225] Armstrong, D, Grimwood K, Carlin JB, Carzino R, Gutierrez JP, Hull J, Olinsky A, Phelan EM, Robertson CF, Phelan PD. Lower airway inflammation in infants and young children with cystic fibrosis. Am J Respir Crit Care Med, 1997, 156: 1197-1204.
[226] Luk C, Dulfano MJ. Effect of pH, viscosity and ionic-strength changes on ciliary beating frequency of human bronchial explants. Clin Sci, 1983, 64: 449-451.
[227] Sanderson M, Dirksen ER. Mechanosensitive and beta-adrenergic control of the ciliary beat frequency of mammalian respiratory tract cells in culture. Am Rev Respir Dis, 1989, 139: 432-440.
[228] Khan T, Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DW. Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med, 1995,151:1075-1082.
[229] Armstrong D, Hook SM, Jamsen KM, Nixon GM, Carzino R, Carlin JB, Robertson CF, Grimwood K., Lower airway inflammation in infants with cystic fibrosis detected by newborn screening. Pediatr Pulmonol, 2005, 40: 500-510.
[230] Joseph T, Look D, Ferkol T. NF-kappaB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol, 2005,288: L471-L479.
[231] Rubin B. CFTR is a modulator of airway inflammation. Am J Physiol Lung Cell Mol Physiol, 2007, 292: L381-L382.
[232] Machen T. Innate immune response in CF airway epithelia: hyperinfiammatory? Am J Physiol Cell Physiol, 2006, 291: C218-C230.
[233] Aldallal N, McNaughton EE, Manzel LJ, Richards AM, Zabner J, Ferkol TW, Look DC. Inflammatory response in airway epithelial cells isolated from patients with cystic fibrosis. Am J Respir Crit Care Med, 2002, 166: 1248-1256.
[234] Becker M, Sauer MS, Muhlebach MS, Hirsh AJ, Wu Q, Verghese MW, Randell SH. Cytokine secretion by cystic fibrosis airway epithelial cells. Am J Respir Crit Care Med, 2004, 169: 645-653.
[235] Perez A, Issler AC, Cotton CU, Kelley TJ, Verkman AS, Davis PB. CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol, 2007, 292: L383-L395.
[236] Chan M, Chmura K, Chan ED. Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the deltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene. Cytokine, 2006, 33: 309-316.
[237] de Jonge H. Cystic fibrosis mice rehabilitated for studies of airway gland dysfunction. J Physiol, 2007, 580: 301-314.
[238] Quinton P. Viscosity versus composition in airway pathology. Am J Respir Crit Care Med, 1994, 149:6-7.
[239] Hull J, Skinner W, Robertson C, Phelan P. Elemental content of airway surface liquid from infants with cystic fibrosis. Am J Respir Crit Care Med, 1998, 157: 10-14.
[240] Song Y, Thiagarajah J, Verkman AS. Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways. J Gen Physiol, 2003, 122: 511-519.
[241]Cowley E, Govindaraju K, Lloyd DK, Eidelman DH. Airway surface fluid composition in the rat determined by capillary electrophoresis. Am J Physiol, 1997,273: L895-L899.
[242] Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis W, Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airway disease. Cell, 1998. 95: 1005-1015.
[243] Knowles M, Robinson JM, Wood RE, Pue CA, Mentz WM, Wager GC, Gatzy JT, Boucher RC. Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J Clin Invest, 1997, 100:2588-2595.
[244] Singh P, Tack BF, McCray PB Jr, Welsh MJ. Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L799-L805.
[245] Trout L, King M, Feng W, Inglis SK, Ballard ST. Inhibition of airway liquid secretion and its effect on the physical properties of airway mucus. Am J Physiol, 1998, 274: L258-L263.
[246] Joo N, Krouse ME, Wu JV, Saenz Y, Jayaraman S, Verkman AS, Wine JJ. HCO3- transport in relation to mucus secretion from submucosal glands. JOP, 2001,2:280-284.
[247] Cowley E, Linsdell P. Oxidant stress stimulates anion secretion from the human airway epithelial cell line Calu-3: implications for cystic fibrosis lung disease. Journal of Physiology, 2002, 543: 201-209.
[2] Anderson M, Sheppard DN, Berger HA, Welsh MJ. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am J Physiol, 1992, 263: L1-14.
[3] Gibson R, Burns JL, Ramsey BW. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med, 2003,168: 918-51.
[4] Hyde SC, Emsley P, Hartshorn MJ, Mimmack MM, Gileadi U, Pearce SR, Gallagher MP, Gill DR, Hubbard RE, Higgins CF. Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature, 1990, 346: 362-5.
[5] Sheppard D, Welsh MJ. Structure and function of the cystic fibrosis transmembrance conductance regulator chloride channel. Physiol Rev, 1999, 79: S23-S45.
[6] Randak C, Welsh MJ. An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR. Cell, 2003, 115: 837-850.
[7] Mall M, Bleich M, Schurlein M, Kuhr J, Seydewitz HH, Brandis M, Greger R, Kunzelmann K. Cholinergic ion secretion in human colon requires coactivation by cAMP. Am J Physiol Gastrointestinal Liver Physiology, 1998, 38: G1274-G1281.
[8] Ballard S, Trout L, Bebok Z, Sorscher EJ, and Crews A. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol, 1999, 277: L694-L699.
[9] Hug, M, Tamada T, Bridges RJ. CFTR and bicarbonate secretion by epithelial cells. News Physiol Sci, 2003, 18: 38-42.
[10] Pilewski J, Frizzell RA. Role of CFTR in Airway Disease Physiol. Rev., 1999, 79: 215-255.
[11] Anderson M, Berger HA, Rich DP, Gregory RJ, Smith AE, Welsh MJ. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell, 1991,67:775-784.
[12] Drumm M, Wilkinson DJ, Smit LS, Worrell RT, Strong TV, Frizzell RA, Dawson DC, Collins FS. Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes. Science, 1991, 254: 1797-1799.
[13] Tabcharani JA, Chang XB, Riordan JR, Hanrahan JW. Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. Nature, 1991, 352: 628-631.
[14] Bear C, Li CH, Kartner N, Bridges RJ, Jensen TJ, Ramjeesingh M, Riordan JR. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell, 1992, 68: 809-818.
[15] Dahl M, Tybjaerg-Hansen A, Lange P, Nordestgaard BG. F508 heterozygosity in cystic fibrosis and susceptibility to asthma. Lancet, 1998, 351: 1911-1913.
[16] Tzetis M, Efthymiadou A, Strofalis S, Psychou P, Dimakou A, Pouliou E. CFTR gene mutations including three novel nucleotide substitutions and haplotype background in patients with asthma, disseminated brochiectasis and chronic obstructive pulmonary disease. Hum Genet, 2001. 108: p. 216-221.
[17] Taouil K, Hinnrasky J, Hologne C, Corlieu P, Klossek JM, Puchelle E. Stimulation of beta 2-Adrenergic Receptor Increases Cystic Fibrosis Transmembrane Conductance Regulator Expression in Human Airway Epithelial Cells through a cAMP/Protein Kinase A-independent Pathway. J Biol Chem,2003, 278: 17320-27.
[18] Smith J, Travis SM, Gereenberg EP, Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnornmal airway surface fluid. Cell, 1996, 95: 1005-1015.
[19] Di A, Brown ME, Deriy LV, Li C, Szeto FL, Chen Y, Huang P, Tong J, Naren AP, Bindokas V, Palfrey HC, Nelson DJ, CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol, 2006, 8(9): 933-44.
[20] Maggi C, Giachetti A, Dey RD, Said SI. Neuropeptides as regulators of airway function: vasoactive intestinal peptide and the tachykinins. Physiol Rev, 1995, 75(2): 277-322.
[21] Ren YH, Qin XQ, Guan CX, Luo ZQ, Zhang CQ, Sun XH. Temporal and spatial ditribution of VIP, CGRP and their receptors in the development of airway hyperresponsiveness in the lungs. Acta Physiologica Sinica, 2004, 56: 137-146.
[22] Qin XQ, Sun XH, Luo ZQ. Vasoactive intestinal peptide and epidermal growth factor upregulates bcl-2 gene expression in bronchial epithelial cells. Acta Physiologica Sinica, 1999, 51: 419-424.
[23] Lazarus SC, Basbaum CB, Barnes PJ, Gold WM. cAMP immunocytochemistry provides evidence for functional VIP receptors in trachea. Am J Physiol, 1986, 251: C115-C119.
[24] Rogers D. Motor control of airway goblet cells and glands. Respir Physiol, 2000, 125: 129-144.
[25] Groneberg DA, Hartmarm P, Dinh QT, Fischer A. Expression and distribution of vasoactive intestinal polypeptide receptor VPAC(2) mRNA in human airways. Lab Invest, 2001, 81: 749-755.
[26] Qin X, Sun XH, Luo ZQ, Guan CX, Zhang CQ. Affection of epidermal growth factor on VIP secretion and VIPR expression in airway epithel ial cells. Bull of Hunan Med Univ, 1999, 24(2): 99-102.
[27] Joo NS, Saenz Y, Krouse ME, Wine JJ. Mucus secretion from single submucosal glands of pig. Stimulation by carbachol and vasoactive intestinal peptide. J Biol Chem, 2002, 277: 28167-28175.
[28] Tsumura T, Hazama A, Miyoshi T, Ueda S, Okada Y. Activation of cAMP-dependent Cl- currents in guinea-pig Paneth cells without relevant evidence for CFTR expression. The Journal of Physiology, 1998, 512: 765-777.
[29] Ameen N, Marino C, Salas PJI. cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo. Am J Physiol Cell Physiol, 2003, 284: C429-C438.
[30] Li C, Ramjeesingh M, Wang W, Garami E, Hewryk M, Lee D, Rommens JM, Galley K, Bear CE. ATPase activity of the cystic fibrosis transmembrane conductance regulator. J Biol Chem, 1996, 271: 28463-28468.
[31] Gadsby D, Dousmanis AG, Nairn AC. ATP hydrolysis cycles and the gating of CFTR Cl- channels. Acta Physiol Scand, 1998, 643: 247-256.
[32] Jia Y, Mathews CJ, Hanrahan JW. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J Biol Chem, 1997, 272: 4978-4984.
[33] Liedtke C, Cole TS. Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells. Am J Physiol, 1998, 275: C1357-C1364.
[34] Middleton L, Harvey RD. PKC regulation of cardiac CFTR cl- channel function in guinea pig ventricular myocytes. Am J Physiol, 1998, 275: C293-C302.
[35] Bebok Z, Varga K, Hicks JK, Venglarik CJ, Kovacs T, Chen L, Hardiman KM, Collawn JF, Sorscher EJ, Matalon S. Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl- secretion in airway epithelia. J Biol Chem, 2002,277:43041-43049.
[36] Asfaha S, Bell CJ, Wallace JL, MacNaughton WK. Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase. Am J Physiol Gastrointest Liver Physiol, 1999, 276: G703-G710.
[37] MacNaughton W, Lowe SS, Cushing K. Role of nitric oxide in inflammation-induced suppression of secretion in a mouse model of acute colitis. Am J Physiol Gastrointest Liver Physiol, 1998, 275: G1353-G1360.
[38] Skinn A, MacNaughton WK. Nitric oxide inhibits cAMP-dependent CFTR trafficking in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol, 2005, 289: G739-G744.
[39] Kulka M, Dery R, Nahirney D, Duszyk M, Befus AD. Differential Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Interferon gamma in Mast Cells and Epithelial Cells. J Pharmacol Exp Ther, 2005,315:563-570.
[40] Tan Y, Qi MM, Qin XQ, Xiang Y, Li X, Wang Y, Qu F, Liu HJ, Zhang JS. Wound repair and proliferation of bronchial epithelial cells enhanced by bombesin receptor subtype 3 activation. Peptides, 2006, 27: 1852-1858.
[41] Wu H, Guan CX, Qin XQ, Xiang Y, Qi MM, Luo ZQ, Zhang CQ. Upregulation of substance P receptor expression by calcitonin gene-related peptide, a possible cooperative action of two neuropeptides involved in airway hyperresponsiveness. Pulm Pharmacol Ther, 2006, 20: 513-524.
[42] Ren Y, Qin XQ, Guan CX, Luo ZQ, Zhang CQ, Sun XH. The temporal and spatial distribution of vasoactive intestinal peptide and its receptor in the development of airway hyperresponsiveness. Chin J Tuberc Respir Dis, 2004, 27: 224-228.
[43] Qin XQ. Protection from Oxidant Injury on Airway Environment Modulation. Progress in Physiological Sciences, 1999, 30: 129-132.
[44] Qin X, Xiang Y, Luo ZQ, Zhang CQ, Sun XH. Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbit bronchial epithelial cells. Acta Physiologica Sinica, 2000, 52(6): 519-521.
[45] Tamada T, Sasaki T, Saitoh H, Ohkawara Y, Irokawa T, Sasamori K, Oshiro T, Tamura G, Shimura S, Shirato K. A novel function of thyrotropin as a potentiator of electrolyte secretion from the tracheal gland. Am J Respir Cell Mol Biol, 2000, 22: 566-573.
[46] Danahay H, Atherton H, Jones G, Bridges RJ, Poll CT. Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol, 2002, 282: L226-236.
[47] Sasamori K, Sasaki T, Takasawa S, Tamada T, Nara M, Irokawa T, Shimura S, Shirato K, Hattori T. Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operate submucosal gland acinar cells. Am J Physiol Lung Cell Mol Physiol, 2004, 287: L69-78.
[48] Widdicombe J. Regulation of the depth and composition of airway surface liquid. JAnat, 2002, 201: 313-318.
[49] Chmiel J, Davis PB. State of the art: why do the lungs of patients with cystic fibrosis become infected and why can't they clear the infection? Respir Res, 2003, 4: 8.
[50] Regnis J, Robinson M, Bailey DL, Cook P, Hooper P, Chan HK, Gonda I, Bautovich G, Bye PT. Mucociliary clearance in patients with cystic fibrosis and in normal subjects. Am J Respir Crit Care Med, 1994, 150: 66-71.
[51] Chace K, Naziruddin B, Desai VC, Flux M, Schdev GP. Physical properties of purfied human respirstory mucus glycoproteins: effects of sodium chloride concentration on the aggregation properties and shape. Exp Lung Res, 1989, 15: 721-737.
[52] Chace K, Flux M, Sachdev GP. Comparison of physicochemical propreties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosos and asthmatic patients. Biochemistry, 1985, 24: 7334-7341.
[53] Former C, Lorenz JN, Paul RJ, Chloride channel function is linked to epithelium-dependent airway relaxation. Am J Physiol Lung Cell Mol Physiol, 2001, 280:L334-41.
[54] Stutts M, Canessa CM, Olsen JC, Hamrick M, Conn JA, Rossier BC, Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science, 1995, 269: 847-850.
[55] Schwiebert E, Egan ME, Hwang TH, Fulmer SB, Allen SS, Cutting GR, Guggino WB. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell, 1995, 81: 1063-73.
[56] Knowles M, Stutts MJ, Spock A, Fischer N, Gatzy JT, Boucher RC. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science, 1983, 221: 1067-1070.
[57] Cowley E, Linsdell P. Oxidant stress stimulates anion secretion from the human airway epithelial cell line Calu-3: implications for cystic fibrosis lung disease. J Physiol, 2002, 543: 201-209.
[58] Jung J, Lee JY, Oh SO, Jang PG, Bae HR, Kim YK, Lee SH. Effect of t-butylhydroperoxide on chloride secretion in rat tracheal epithelia. Pharmacol Toxicol, 1998,82:236-242.
[59] Nguyen T, Canada AT. Modulation of human colonic T84 cell secretion by hydrogen peroxide. Biochem Pharmacol, 1994,47: 403-410.
[60] Tamai H, Kachur JF, Baron DA, Grisham MB, Gaginella TS. Monochloramine, a neutrophil-derived oxidant, stimulates rat colonic secretion. J Pharmacol Exp Ther, 1991,257:887-894.
[61] Cantin A, Bilodeau G, Ouellet C, Liao J, Hanrahan JW. Oxidant stress suppresses CFTR expression. Am J Physiol Cell Physiol, 2006, 290: C262-C270.
[62] Besancon F, Przewlocki G, Baro I, Hongre AS, Escande D, Edelman A. Interferon-y downregulates CFTR gene expression in epithelial cells. Am J Physiol Cell Physiol, 1994, 267: C1398-C1404.
[63] Nakamura H, Yoshimura K, Bajocchi G, Trapnell BC, Pavirani A, Crystal RG. Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene. FEBS Lett, 1992, 314: 366-370.
[64] Koh J, Sferra TJ, Collins FS. Characterization of the cystic fibrosis transmembrane conductance regulator promoter region. Chromatin context and tissue-specificity. J Biol Chem, 1993, 268: 15912-21.
[65] Yoshimura K, Nakamura H, Trapnell BC, Chu CS, Dalemans W, Pavirani A, Lecocq JP, Crystal RG. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin. Nucleic Acids Res, 1991,19:5417-23.
[66] Chou J, Rozmahel R, Tsui LC. Characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene. J Biol Chem, 1991, 266: 24471-6.
[67] Qin XQ, Xiang Y, Luo ZQ, Zhang CQ, Sun XH. Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbit bronchial epithelial cells. Acta Physiologica Sinica, 2000, 52(6): 519-521.
[68] Chen L, Patel RP, Teng X, Bosworth CA, Lancaster JR, Matalon S. Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione. J Biol Chem, 2006, 981: 9190-9.
[69] Rahman I, MacNee W. Regulation of redox glutathione levels and gene transcription in lung inflammation: therapeutic approaches. Free Radic. Biol. Med, 2000, 28: 1405-1420.
[70] Andreadis A, Hazen SL, Comhair SA, Erzurum SC. Oxidative and nitrosative events in asthma. Free Radic Biol Med, 2003, 35: p. 213-25.
[71] Chen P, Illsley NP, Verkman AS. Renal brush-border chloride transport mechanisms characterized using a fluorescent indicator. Am J Physiol, 1988, 254:F114-F120.
[72] Soroceanu L, Manning TJ , Sontheimer H. Modulation of glioma cell migration and invasion using Cl - and K+ ion channel blockers. J Neurosci, 1999, 19: 5942-5954.
[73] Groneberg D, Springer J, Fischer A. Vasoactive intestinal polypeptide as mediator of asthma. Pulm Pharmacol Ther, 2001, 14: 391-401.
[74] Haws C, Finkbeiner WE, Widdicombe JH, Wine JJ. CFTR in Calu-3 human airway cells, channel properties and role in cAMP- activated Cl- conductance. Am J Physiol, 1994, 266: L502-L512.
[75] Shen B, Finkbeiner WE, Wine JJ, Mrsny RJ, Widdicombe JH. Calu-3, a human airway epithelial cell line that shows cAMP-dependent Cl- secretion. Am J Physiol, 1994, 266: L493-L501.
[76] Joo N, Irokawa T, Wu JV, Robbins RC, Whyte RI, Wine JJ. Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands. J Biol Chem, 2002, 277:50710-50715.
[77] Lucchini R, Facchini F, Turato G, Saetta M, Caramori G, Ciaccia A, Maestrelli P, Springall DR, Polak JM, Fabbri L, Mapp CE. Increased VIP-positive nerve fibers in the mucous glands of subjects with chronic bronchitis. Am J Respir Crit Care Med, 1997, 156: 1963-1968.
[78] Chanez P, Springall D, Vignola AM, Moradoghi-hattvani A, Polak JM, Godard P, Bousquet J. Bronchial mucosal immunoreactivity of sensory neuropeptides in severe airway diseases. Am J Respir Crit Care Med, 1998, 158: 985-990.
[79] Lazarus S, Basbaum CB, Barnes PJ, Gold WM. cAMP immunocytochemistry provides evidence for functional VIP receptors in trachea. Am J Physiol, 1986, 251: C115-C119.
[80] Sreedharan SP, Robichon A, Peterson KE, Goetzl EJ. Cloning and expression of the human vasoactive intestinal peptide receptor. Proc Natl Acad Sci USA, 1991, 88: 4986-4990.
[81] Harmar A, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase- activating polypeptide. Pharmacol Rev, 1998, 50: 265-270.
[82] Laburthe M, Couvineau A. Molecular pharmacology and structure of VPAC receptors for VIP and PACAP. Regul Pept, 2002, 108: 165-173.
[83] Busto R, Carrero I, Guijarro LG, Solano R.M, Zapatero J, Noguerales F, Prieto JC, Expression, pharmacological, and functional evidence for PACAP/VIP receptors in human lung. Am J Physiol, 1999, 277: L42-L48.
[84] Fischer A, Kummer W, Couraud JY, Adler D, Branscheid D, Heym C. Immunohistochemical localization of receptors for vasoactive intestinal peptide and substance P in human trachea. Lab Invest, 1992, 67: 387-393.
[85] Ameen N, Martensson B, Bourguinon L, Marino C, Isenberg J, Mclaughlin GE. CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo. J Cell Sci, 1999,112: 887-894.
[86] Gadsby D, Nairn A. Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol Rev 1999, 79: S77-S107.
[87] Kopito R. Biosynthesis and Degradation of CFTR. Physiol Rev, 1999, 79: S167-S173.
[88] Yoo J, Moyer BD, Bannykh S, Yoo HM, Riordan JR, Balch WE. Non-conventional Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator through the Early Secretory Pathway. J Biol Chem, 2002,277:11401-11409.
[89] Weixel K, Bradbury NA. The Carboxyl Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Binds to AP-2 Clathrin Adaptors. J Biol Chem, 2000, 275: 3655-3660.
[90] Prince L, Peter K, Hatton SR, Zaliauskiene L, Cotlin LF, Clancy JP, Marchase RB, Collawn JF. Efficient Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator Requires a Tyrosine-based Signal. J Biol Chem, 1999, 274: 3602-3609
[91] Lukacs G, Segal G, Kartner N, Grinstein S, Zhang, F. Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem J, 1997, 328: 353-361.
[92] Gentzsch M, Chang XB, Cui L, Wu Y, Ozols W, Choudhury A, Pagano RE, Riordan JR. Endocytic Trafficking Routes of Wild Type and F508 Cystic Fibrosis Transmembrane Conductance Regulator. Mol Biol Cell, 2004, 15: 2684-2696.
[93] Benharouga M, Haardt M, Kartner N, Lukacs GL. COOH-terminal Truncations Promote Proteasome-dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments. J Cell Biol, 2001, 153: 957-970.
[94] Bradbury N, Clark JA, Watkins SC, Widnell CC, Smith HST, Bridges RJ. Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator. Am J Physiol, 1999, 276: L659-L668.
[95] Hu W, Howard M, Lukacs GL. Multiple endocytic signals in the C-terminal tail of the cystic fibrosis transmembrane conductance regulator. Biochem J, 2001, 354: 561-572.
[96] Weixel K, Bradbury NA. Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway. J Biol Chem, 2001, 276: 46251-46259.
[97] Picciano J, Ameen N, Grant B, Bradbury NA. Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator. Am J Physiol, 2003, 285: C1009-C1018.
[98] Hanrahan J, Tabcharani JA, Becq F, Mathews CJ, Augustinas O, Jensen TJ, Chang X-B, Riordan JR. Function and dysfunction of the CFTR chloride channel. In: Ion Channels and Genetic Diseases, edited by Dawson DC, and Frizzell RA, Vol 50, 1995, New York: Rockefeller University Press.
[99] Kunzelman K, Mall M. Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev, 2001, 82: 245-289.
[100] Howard M, Jiang X, Stolz D, Hill W, Johnson J, Watkins S, Frizzell R, Bruton C, Robbins P, Weisz O. Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells. Am J Physiol Cell Physiol, 2000, 279: C375-C382.
[101] Weber W, Segal A, Simaels J, Vankeerberghen A, Cassiman JJ, Driessche WV. Functional integrity of the vesicle transporting machinery is required for complete activation of CFTR expressed in Xenopus laevis oocytes. Pflugers Arch, 2001, 441: 850-859.
[102]Romey M, Pallares-Ruiz N, Mange A, Mettling C, Peytavi R, Demaille J, Claustres M. A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression. J Biol Chem, 2000, 275:3561-3567.
[103] Rowntree, R, Harris A. DNA polymorphisms in potential regulatory elements of the CFTR gene alter transcription factor binding. Hum Genet, 2002, 111: 66-74.
[104] Wittwer J, Marti-Jaun J, Hersberger M. Functional polymorphism in ALOX15 results in increased allele-specific transcription in macrophages through binding of the transcription factor SPI1. Hum Mutat, 2006, 27: 78-87.
[105] Rene C, Taulan M , Iral F, Doudement J, Honore AL, Gerbon C, Demaille J, Claustres M, Romey MC. Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway. Nucleic Acids Res, 2005, 33: 5271-5290.
[106] Taulana M, Lopeza E, Guittarda C, Renea C, Bauxa D, Altieria JP, DesGeorgesa M, Claustresa M, Romeya MC. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding Biochemical and Biophysical Research Communications, 2007, 361: 775-781.
[107] Koh J, Sferra TJ, Collins FS. Characterization of the CFTR promoter region: chromatin context and tissue-specificity. J Biol Chem, 1993, 268: 15912-15921.
[108] Brugmann S, Tapadia MD, Helms JA. The molecular origins of species-specific facial pattern. Curr Top Dev Biol, 2006, 73: 1-42.
[109]Kadonaga J, Carner KR, Masiarz FR, Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell, 1987,51:1079-1090.
[110] Yoshimura K, Nakamura H, Trapnell BC, Dalemans W, Pavirani A, Lecocq JP, Crystal RG. The cystic fibrosis gene has a "housekeeping"-type promoter and is expressed at low levels in cells of epithelial origin. J Biol Chem, 1991, 266: 9140-9144.
[111]Kwon H, Kim MS, Edenberg HJ, Hur MW. Sp3 and Sp4 can repress transcription by competing with Spl for core cis-elements on the human ADH5/FDH minimal promoter. J Biol Chem, 1999, 274: 20-28.
[112]Shafer G, Cramer T, Suske G, Kemmner W, Wiedenmann B, H"ocker M. Oxidative stress regulates vascular endothelial growth factor A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements. J Biol Chem, 2003, 278: 8190-8198.
[113] Kelley K, Wang H, Ratnam M. Dual regulation of ets-activated gene expression by SP1. Gene, 2003, 307: 87-97.
[114] Couse J, Lindzey J, Grandien K, Gustafsson JA, Korach KS. Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology, 1997, 138: 4613-4621.
[115] Carey M, Cared JW, Bradbury J. Spontaneous airway hyperresponsiveness in estrogen receptor alpha deficient mice. Am J Respir Crit Care Med, 2007, 175: 126-135.
[116]Gavett S, Madison SL, Chulada PC, Scarborough PE, Qu W, Boyle JE, Tiano HF, Lee CA, Langenbach R, Roggli VL. Allergic lung responses are increased in prostaglandin H synthase-deficient mice. J Clin Invest, 1999, 104: 721-732.
[117]Wilder J, Collie DD, Wilson BS, Bice DE, Lyons CR, Lipscomb MF. Dissociation of airway hyperresponsiveness from immunoglobulin E and airway eosinophilia in a murine model of allergic asthma. Am J Respir Cell Mol Biol, 1999,20: 1326-1334.
[118]Takeda K, Haczku A, Lee JJ, Irvin CG, Gelfand EW. Strain dependence of airway hyperresponsiveness reflects differences in eosinophil localization in the lung. Am J Physiol Lung Cell Mol Physiol, 2001, 281: L394-L401.
[119]Dijkstra A, Howard TD, Vonk JM, Ampleford EJ, Lange LA, Bleecker ER, Meyers DA, Postma DS. Estrogen receptor 1 polymorphisms are associated with airway hyperresponsiveness and lung function decline, particularly in female subjects with asthma. Allergy Clin Immunol, 2006,117: 604-611.
[120]Cuzzocrea S, Mazzon E, Sautebin L. The protective role of endogenous estrogens in carrageenan-induced lung injury in the rat. Mol Med, 2001, 7: 478-487.
[121]Coakley R, Sun HR, Chines LA, Rasmussen JE, Stackhouse JR, Okada SF, Fricks I, Young SL, Tarran R. 17β-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia. J Clin Invest, 2008,118: 4025-4035.
[122] Mollerup S, Jorgensen K, Berge G, Haugen A. Expression of estrogen receptors alpha and beta in human lung tissue and cell lines. Lung Cancer, 2002, 37: 153-159.
[123]Thomas L, Doyle, LA, Edelman MJ. Lung cancer in women: emerging differences in epidemiology, biology, and therapy. Chest, 2005, 128: 370-381.
[124]Riordan J, Rommens JM, Kerem BS, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FS, Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 1989,245: 1066-1073.
[125]Anderson M, Gregory RJ, Thompson S, Souza DW, Paul S, Mulligan RC, Smith AE, Welsh MJ. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science, 1991, 253: 202-205.
[126]Quinton P. Chloride impermeability in cystic fibrosis. Nature, 1983. 301: 421-422.
[127] Cai Z, Scott-Ward TS, Sheppard DN. Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Clchanne. J Gen Physiol, 2003, 122: 605-620.
[128] Dorwart M, Thibodeau P, Thomas P. Cystic fibrosis: recent structural insights. J Cyst Fibres, 2004, 3: 91-94.
[129] Gong X, Burbridge SM, Cowley EA, Linsdell P. Molecular determinants of Au(CN)2 - binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl- channel pore. J Physiol, 2002, 540: 39-47.
[130] Ge N, Muise CN, Gong X, Linsdell P. Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore. J Biol Chem, 2004, 279: 55283-55289.
[131] St Aubin C, Linsdell P. Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel. J Gen Physiol, 2006, 128: 535-545.
[132] Wright A, Gong X, Verdon B. Novel regulation of cystic fibrosis transmembrane conductance regulator (CFTR) channel gating by external chloride. J B iol Chem, 2004, 279: 41658-41663.
[133]Riordan J. Assembly of functional chloride channels. Arm Rev Physiol, 2005, 67:701-718.
[134] McCarty N. Permeation through the CFTR chloride channel. J Exp Biol, 2000, 203: 1947-1962.
[135] Linsdell P. Mechanism of chloride permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. Exp Physiol, 2006, 91: 123-129.
[136]Randak C, Welsh MJ. ADP inhibits function of the ABC transponter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity. Proc Natl Aead Sci USA, 2005,102: 2216-2220.
[137]Kidd J, Ramjeesingh M, Straiford F. A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator(CFTR) mediates ATP ase activity. J Biol Chem, 2004, 279: 41664-41669.
[138]Vergani P, Lockless SW, Nairn AC. Channel opening by ATP-driven tight dimerization of its nueleotide-binding domains. Nature, 2005, 433: 876-880.
[139] Lewis H, Buehanan SG, Burley SK. Structure of nueleotide-binding domain 1 of the cystic fibrosis trans-membrane conductance regulator. EMBO J, 2004, 23: 282-293.
[140] Vergani P, Nairn AC, Gadsby DC. On the mechanism of MgATP-dependent gating of CFTR cl- channels. J Gen Physiol, 2003,121: 17-36.
[141] Berger A, Lkuma M, Welsh MJ. Nomal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nueleotide- binding domain. Proc Natl Acad Sci USA, 2005, 102: 455-460.
[142] Cheng S, Rich DP, Marshall J, Gregory RJ, Welsh MJ, Smith AE. Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell, 1991, 66:1027-36
[143] Quinton P, Reddy MM. Regulation of absorption by phosphorylation of CFTR. Jpn J Physiol, 1994, 44 : S207-S213.
[144] Schwencke C, Yamamoto M, Okumura S, Toya Y, Kim SJ, Ishikawa Y. Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae. Mol Endocrinol, 1999,13: 1061-1070.
[145] Ostrom R, Violin JD, Coleman S, Insel PA. Selective enhancement of beta-adrenergic receptor signaling by overexpression of adenylyl cyclase type 6: colocalization of receptor and adenylyl cyclase in caveolae of cardiac myocytes. Mol Pharmacol Toxicol, 2000, 57: 1075-1079.
[146] Rich D, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain. J Biol Chem,1993, 268:20259-20267.
[147] Gadsby D, Nairn AC. Regulation of CFTR channel gating. Trends Biochem Sci, 1994,19:513-518.
[148] Dahan D, Evagelidis A, Hanrahan JW, Hinkson DA, Jia Y, Luo J, Zhu T. Regulation of the CFTR channel by phosphorylation. Pflugers Arch, 2001, 443 (Suppl ): S92-S96.
[149] Chang X, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, Hanrahan JW, Riordan JR. Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. J Biol Chem, 1993, 268: 11304-11311.
[150] Seibert F, Chang XB, Aleksandrov AA, Clarke DM, Hanrahan JW, Riordan JR. Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum. Biochim Biophys Acta, 1999, 1461: 275-283.
[151] Bajnath R, Groot JA, De Jonge HR, Kansen M, Bijman J. Synergistic activation of non-rectifying small-conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl.19A. Pflugers Arch, 1993, 425: 100-108.
[152] Vaandrager A, Ehlert EM, Jarchau T, Lohmann SM, de Jonge HR. N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II. J Biol Chem, 1996, 271: 7025-7029.
[153]Reddy M, Quinton PM. Functional interaction of CFTR and ENaC in sweat glands. Pflugers Arch, 2003,445: 499-503.
[154] Vaandrager A, Smolenski A, Tilly BC, Houtsmuller AB, Ehlert EM, Bot AG, Edixhoven M, Boomaars WE, Lohmann SM, de Jonge HR. Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation. Proc Natl Acad Sci USA, 1998, 95: 1466-1471.
[155] Csanady L, Chan KW, Angel BB, Nairn AC, Gadsby DC. Negative regulation of CFTR chloride channel gating through R-domain serine 768. Ped Pulmon, 1998,17:205.
[156] Travis S, Berger HA, Welsh MJ. Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA, 1997,94:11055-11060.
[157] Nairn A, Qin J, Chait BT, Gadsby DC. Indentification of sites in the R domain of CFTR phosphorylated by cAMP-dependent protein kinase and dephosphorylated by protein phosphatase 2A and 2C. Ped Pulmon, 1996, Suppl 13:21.
[158]Reddy M, Quinton PM. Deactivation of CFTR-C1 conductance by endogenous phosphatases in the native sweat duct. Am J Physiol, 1996, 270: C474-C480.
[159]Reddy M, Quinton PM. Cytosolic potassium controls CFTR deactivation in human sweat duct. Am J Physiol Cell Physiol, 2006, 291: C122-C129.
[160] Zhu T, Dahan D, Evagelidis A, Zheng SX, Luo J, Hanrahan JW. Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C. J Biol Chem, 1999, 274: 29102-29107.
[161] Fischer H, Machen TE. The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel. Biophys J, 1996, 71: 3073-3082.
[162] Nguyen T, Canada AT, Heintz GG, Gettys TW, Conn JA. Stimulation of secretion by the T84 colonic epithelial cell line with dietary flavonols. Biochem Pharmacol, 1991,41: 1879-1886.
[163] Sears C, Firoozmand F, Mellander A, Chambers FG, Eromar IG, Bot AG, Scholte B, De Jonge HR, Donowitz M. Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers. Am J Physiol, 1995, 269: G874-G882.
[164] French P, Bijman J, Bot AG, Boomaars WE, Scholte BJ, de Jonge HR. Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism. Am J Physiol, 1997, 273: C747-C753.
[165] Wang F, Zeltwanger S, Yang IC, Nairn AC, Hwang TC. Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating. Evidence for two binding sites with opposite effects. J Gen Physiol, 1998,111: 477-490.
[166] Bulteau-Pignoux, L, Derand R, Metaye T, Joffre M, Becq F. Genistein modifies the activation kinetics and magnitude of phosphorylated wild-type and G551D-CFTR chloride currents. J Membr Biol, 2002,188: 175-182.
[167]Berger A, Randak CO, Ostedgaard LS, Karp PH, Vermeer DW, Welsh MJ. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. J Biol Chem, 2005, 280: 5221-5226.
[168] Lansdell K, Cai Z, Kidd JF, Sheppard DN. Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in murine cell line. J Physiol, 2000, 524: 317-330.
[169] Moran O, Zegarra-Moran O, A quantitative description of the activation and inhibition of CFTR by potentiators: Genistein. FEBS Lett, 2005, 579: 3979-3983.
[170] Linsdell P, Hanrahan JW. Flickery block of single CFTR chloride channels by intracellular anions and osmolytes. Am J Physiol, 1996, 271: C628-C634.
[171] Quinton P, Reddy MM. Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding. Nature, 1992, 360: 79-81.
[172] Bell C, Quinton PM. Regulation of CFTR Cl- conductance in secretion by cellular energy levels. Am J Physiol, 1993, 264: C925-C931.
[173] Egan M, Pearson M, Weiner SA, Rajendran V, Rubin D, Glockner-Pagel J, Canny S, Du K, Lukacs GL, Caplan MJ. Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science, 2004, 304: 600-602.
[174] Mall M, Kunzelmann K. Correction of the CF defect by curcumin: hypes and disappointments. Bioessays, 2005, 27: 9-13.
[175] Scott O. Investigation of CFTR intermolecular structure and protein protein interaction. Diss Abstr Int, 2002, 62: 3593.
[176] Chappe V, Irvine T, Liao J. Phosphorylation of CFTR by PKA promotes binding of the regulatory domain. EMBO J, 2005, 24: 2730-2740.
[177] Amaral M. Processing of CFTR: traversing the cellular maze-how much CFTR needs to go through to avoid cystic fibrosis? Pediatr Pulmonol, 2005, 39: 479-491.
[178] Winter M, Welsh MJ. Stimulation of CFTR activity by its phosphorylated R domain. Nature, 1997, 389: 294-296.
[179] Grimard V, Li C, Ramjeesingh M, Bear CE, Goormaghtigh E, Ruysschaert JM. Phosphorylation-induced conformational changes of cystic fibrosis transmembrane conductance regulator monitored by attenuated total reflection- fourier transform IR spectroscopy and fluorescence spectroscopy. J Biol Chem, 2004, 279: 5528-5536.
[180]Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC. In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer. EMBO J, 2006, 25: 4728-4739.
[181] Mehta A, CFTR: more than just a chloride channel. Pediatr Pulmonol, 2005. 39: 292-298.
[182] Guggino W, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol, 2006, 7: 426-436.
[183] Short D, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem, 1998, 273:19797-19801.
[184] Naren A, Cobb B, Li C, Roy K, Nelson D, Heda GD, Liao J, Kirk KL, Sorscher EJ, Hanrahan J, Clancy JP. A macromolecular complex of β2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA. Proc Natl Acad Sci USA, 2003, 100: 342-346.
[185] Vastiau, A., Cao L, Jaspers M, Owsianik G, Janssens V, Cuppens H, Goris J, Nilius B, Cassiman JJ. Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel. FEBS Lett, 2005, 579: 3392-3396.
[186] Pouyssegur J. Molecular biology and hormonal regulation of vertebrate Na+/H+ exchanger isoforms. isoforms. Renal Physiol Biochem, 1994,17: 190-193.
[187] Guggino W, Banks-Schlegel SP. Macromolecular interactions and ion transport in cystic fibrosis. Am J Respir Crit Care Med, 2004,170: 815-820.
[188]Bachhuber T, Konig J, Voelcker T. Cl~- interference with the epithelial Na~+ Channel ENaC. J Biol Chem, 2005, 280: 31587-31594.
[189] Cuppoletti J, Tewari KP, SherryAM. C1C-2C1- channels in human lung epithelia: activation by arachidonic acid, amidation, and acid activated omep razole. Am J Physiol Cell Physiol, 2001, 281: C46-C54.
[190] Burghardt B, ElkaermL, Kwon TH. Distribution of aquaporin water channelsAQP1 and AQP5 in the ductal system of the human pancreas. Gut, 2003,52:1008-1016.
[191] Welsh M, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell, 1993, 73: 1251-1254.
[192]Rowe S, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med, 2005, 352:1992-2001.
[193]Zielenski J. Genotype and phenotype in cystic fibrosis. Respiration, 2000, 67: 127-133.
[194] Ahmed N, Corey M, Forstner G, Zielenski J, Tsui LC, Ellis L, Tullis E, Durie P. Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas. Gut, 2003, 52: 1159-1164.
[195] Rowntree R, Harris A. The phenotypic consequences of CFTR mutations. Ann Hum Genet, 2003, 67: 471-485.
[196]Kopelman H, Ferretti E, Gauthier C, Goodyer PR. Rabbit pancreatic acini express CFTR as a cAMP-activated chloride efflux pathway. Am J Physiol, 1995, 269:C626-C631.
[197] Zeng W, Lee MG, Yan M, Diaz J, Benjamin I, Marino CR, Kopito R, Freedman S, Cotton C, Muallem S, Thomas P. Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells. Am J Physiol, 1997, 273:C442-C455.
[198] Novak I, Greger R. Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane. Pflugers Arch, 1988,411:58-68.
[199] Novak I, Greger R. Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport. Pflugers Arch, 1988, 411: 546-553.
[200] Zabner J, Smith JJ, Karp PH, Widdicombe JH, Welsh MJ. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol Cell, 1998, 2: 397-403.
[201]Ishiguro H, Steward MC, Wilson RW, Case RM. Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. J Physiol, 1996. 495: 179-191.
[202] Ishiguro H, Steward MC, Lindsay AR, Case RM. Accumulation of intracellular HCO3- by Na+-HCO3- cotransport in interlobular ducts from guinea-pig pancreas. J Physiol, 1996, 495: 169-178.
[203] Shumaker H, Amlal H, Frizzell R, Ulrich CD. CFTR drives Na+-nHCO3- cotransport in pancreatic duct cells: a basis for defective HCO3- secretion in CF. Am J Physiol, 1999, 276: C16-C25.
[204] Marino C, Matovcik LM, Gorelick FS, Cohn JA. Localization of the cystic fibrosis transmembrane conductance regulator in pancreas. J Clin Invest, 1991, 88:712-716.
[205] Illek B, Tarn AW, Fischer H, Machen TE. Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium. Pflugers Arch, 1999, 437: 812-822.
[206] Ko S, Shcheynikov N, Choi JY, Luo X, Ishibashi K, Thomas PJ, Kim JY, Kim KH, Lee MG, Naruse S, Muallem S. A molecular mechanism for aberrant CFTR-dependent HCO3- transport in cystic fibrosis. EMBO J, 2002, 21: 5662-5672.
[207] Ishiguro H, Namkung W, Yamamoto A, Wang Z, Worrell RT, Xu J, Lee MG, Soleimani M. Effect of Slc26a6 deletion on apical C1-/HCO3 - exchanger activity and cAMP-stimulated bicarbonate secretion in pancreatic duct. Am J Physiol Gastrointest Liver Physiol, 2007, 292: G447-G455.
[208] Sohma Y, Gray MA, Imai Y, Argent BE. HCO3- transport in a mathematical model of the pancreatic ductal epithelium. J Membr Biol, 2000, 176: 77-100.
[209] Lee M, Choi JY, Luo X, Strickland E, Thomas PJ, Muallem S. Cystic fibrosis transmembrane conductance regulator regulates luminal C1-/HCO3- exchange in mouse submandibular and pancreatic ducts. J Biol Chem, 1999, 274:14670-14677.
[210] Whitcomb D. Pancreatic bicarbonate secretion: role of CFTR and the sodium-bicarbonate cotransporter. Gastroenterology, 1999,117: 275-277.
[211]Devor D, Singh AK, Lambert LC, DeLuca A, Frizzell RA, Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol, 1999,113: 743-760.
[212] Abuladze N, Lee I, Newman D, Hwang J, Boorer K, Pushkin A, Kurtz I. Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. J Biol Chem, 1998, 273: 17689-17695.
[213]Malinowska D, Kupert EY, Bahiniski A, Sherry AM. Cuppoletti J Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel. Am J Physiol, 1995, 268: C191-C200.
[214] Choi J, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis. Nature, 2001,410: 94-97.
[215] Choi J, Lee MG, Ko S, Muallem S. Cl~--dependent HCO3- transport by cystic fibrosis transmembrane conductance regulator. JOP, 2001, 2: 243-246.
[216] Chan H, Goldstein J, Nelson DJ. Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells. Am J Physiol, 1992, 262:C1273-83.
[217] Namkung W, Lee JA, Ahn W, Han W, Kwon SW, Ahn DS, Kim KH, Lee MG. Ca2+ activates cystic fibrosis transmembrane conductance regulator- and Cl-dependent HCO3- transport in pancreatic duct cells. J Biol Chem, 2003, 278: 200-207.
[218]Kasai H, Angustine GJ. Cystosolic Ca~(2+) gradients triggering uniderectional fluid secretion from exocrine pancreas. Nature, 1990, 348: 735-738.
[219] Quinton P. Viscosity versus composition in airway pathology. Am J Respir Crit Care Med, 1994, 149:6-7.
[220] Joris L, Quinton PM. Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid. Am J Physiol, 1992, 263: L243-L248.
[221] Noah T, Black HR, Cheng PW, Wood RE, Leigh MW. Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis. J Infect Dis, 1997, 175: 638-647.
[222] Bhaskar K, Gong DH, Bansil R, Pajevic S, Hamilton JA, Turner BS, LaMont JT. Profound increase in viscosity and aggregation of pig gastric mucin at low pH. Am J Physiol, 1991, 261: G827-G832.
[223] Cao X, Bansil R, Bhaskar KR, Turner BS, LaMont JT, Niu N, Afdhal NH. pH-dependent conformational change of gastric mucin leads to sol-gel transition. BiophysJ, 1999, 76:1250-1258.
[224] Muhlebach M, Stewart PW, Leigh MW, Noah TL. Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients. Am J Respir Crit Care Med, 1999,160: 186-191.
[225] Armstrong, D, Grimwood K, Carlin JB, Carzino R, Gutierrez JP, Hull J, Olinsky A, Phelan EM, Robertson CF, Phelan PD. Lower airway inflammation in infants and young children with cystic fibrosis. Am J Respir Crit Care Med, 1997, 156: 1197-1204.
[226] Luk C, Dulfano MJ. Effect of pH, viscosity and ionic-strength changes on ciliary beating frequency of human bronchial explants. Clin Sci, 1983, 64: 449-451.
[227] Sanderson M, Dirksen ER. Mechanosensitive and beta-adrenergic control of the ciliary beat frequency of mammalian respiratory tract cells in culture. Am Rev Respir Dis, 1989, 139: 432-440.
[228] Khan T, Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DW. Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med, 1995,151:1075-1082.
[229] Armstrong D, Hook SM, Jamsen KM, Nixon GM, Carzino R, Carlin JB, Robertson CF, Grimwood K., Lower airway inflammation in infants with cystic fibrosis detected by newborn screening. Pediatr Pulmonol, 2005, 40: 500-510.
[230] Joseph T, Look D, Ferkol T. NF-kappaB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol, 2005,288: L471-L479.
[231] Rubin B. CFTR is a modulator of airway inflammation. Am J Physiol Lung Cell Mol Physiol, 2007, 292: L381-L382.
[232] Machen T. Innate immune response in CF airway epithelia: hyperinfiammatory? Am J Physiol Cell Physiol, 2006, 291: C218-C230.
[233] Aldallal N, McNaughton EE, Manzel LJ, Richards AM, Zabner J, Ferkol TW, Look DC. Inflammatory response in airway epithelial cells isolated from patients with cystic fibrosis. Am J Respir Crit Care Med, 2002, 166: 1248-1256.
[234] Becker M, Sauer MS, Muhlebach MS, Hirsh AJ, Wu Q, Verghese MW, Randell SH. Cytokine secretion by cystic fibrosis airway epithelial cells. Am J Respir Crit Care Med, 2004, 169: 645-653.
[235] Perez A, Issler AC, Cotton CU, Kelley TJ, Verkman AS, Davis PB. CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol, 2007, 292: L383-L395.
[236] Chan M, Chmura K, Chan ED. Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the deltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene. Cytokine, 2006, 33: 309-316.
[237] de Jonge H. Cystic fibrosis mice rehabilitated for studies of airway gland dysfunction. J Physiol, 2007, 580: 301-314.
[238] Quinton P. Viscosity versus composition in airway pathology. Am J Respir Crit Care Med, 1994, 149:6-7.
[239] Hull J, Skinner W, Robertson C, Phelan P. Elemental content of airway surface liquid from infants with cystic fibrosis. Am J Respir Crit Care Med, 1998, 157: 10-14.
[240] Song Y, Thiagarajah J, Verkman AS. Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways. J Gen Physiol, 2003, 122: 511-519.
[241]Cowley E, Govindaraju K, Lloyd DK, Eidelman DH. Airway surface fluid composition in the rat determined by capillary electrophoresis. Am J Physiol, 1997,273: L895-L899.
[242] Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis W, Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airway disease. Cell, 1998. 95: 1005-1015.
[243] Knowles M, Robinson JM, Wood RE, Pue CA, Mentz WM, Wager GC, Gatzy JT, Boucher RC. Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J Clin Invest, 1997, 100:2588-2595.
[244] Singh P, Tack BF, McCray PB Jr, Welsh MJ. Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Am J Physiol Lung Cell Mol Physiol, 2000, 279: L799-L805.
[245] Trout L, King M, Feng W, Inglis SK, Ballard ST. Inhibition of airway liquid secretion and its effect on the physical properties of airway mucus. Am J Physiol, 1998, 274: L258-L263.
[246] Joo N, Krouse ME, Wu JV, Saenz Y, Jayaraman S, Verkman AS, Wine JJ. HCO3- transport in relation to mucus secretion from submucosal glands. JOP, 2001,2:280-284.
[247] Cowley E, Linsdell P. Oxidant stress stimulates anion secretion from the human airway epithelial cell line Calu-3: implications for cystic fibrosis lung disease. Journal of Physiology, 2002, 543: 201-209.