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1、Human cystic fibrosis airway epithelia have reduced Cl conductance but not increased Na+ conductance,Omar A.Itana,1, Jeng-Huar Chena,b, Philip H. Karpa,b, Sarah Ernsta, Shaf Keshavjeec, Kalpaj Parekhd, Julia Klesney-Taita, Joseph Zabnera,2,and Michael J.Welsha,b,e,2 Departments of aInternal Medicine
2、,dCardiothoracic Surgery, and eMolecular Physiology and Biophysics and bThe Howard Hughes Medical Institute, The RoyJ, and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA52242; and cToronto Lung Transplant Program, University of Toronto, Toronto, ON, Canada M5G 2C4 PNAS. 201
3、1 Jun 21;108(25):10260-5.,Michael J. Welsh, M.D. Professor of Medicine, Molecular Physiology and Biophysics, and Neurosurgery Roy J. Carver Biomedical Chair in Internal Medicine Investigator, Howard Hughes Medical Institute Director, Pappajohn Institute Director, Cystic Fibrosis Research Center Dr.
4、Welshs clinical interests include the diagnosis and therapy of lung disease, especially airway diseases. His research focuses in three main areas. First: Biology and pathogenesis of CF and new treatments. Second: Physiology and cell biology of airway epithelia. Third: Acid sensing ion channels in th
5、e central and peripheral nervous systems with an emphasis on their role in fear and neurological diseases.,Contents,Background Materials and Methods Hypothesis Results Discussion,Background,Cl- channel c-AMP-PKA CFTRinh-172,CF disease not only a CFTR problem, but also with hyper-absorb Na+. The stud
6、y of Welsh, denied this view. Welsh used short-circuit current method to do this article.,短路电流装置,短路电流 (ISC) 定义为穿过上皮并使得跨上皮电压降为零的电流。 钳制模式 1. 电流钳模式跨上皮电流被钳制在0A，测量细胞两侧的电势差。 2. 电压钳模式组织的跨上皮电压被设定在一个特定的值，然后接通电压钳，跨上皮电流将此电压维持在预设值上。这种模式用于测定由上皮主动转运的离子。,短路电流的实验方法,研究上皮正常或病理情况下的离子转运,研究药物样品 液对上皮细 胞Isc的影响,阻断剂法,离子替代法,
7、打孔法,阻断剂法,在加药反应之前加入某种通道的阻断剂（如Cl-通道阻断剂DPC等）对组织进行一段时间的孵育，然后再加药，或在加药反应到高峰的时候再加阻断剂，看此时药物的反应是否会被抑制。,离子替代法,把正常K-H溶液中的某种离子替换掉（如Cl-用葡萄糖酸根替代），看此时加药后的反应比起在正常K-H中的反应是否有变化。,打孔法,在细胞两侧先加入某种离子浓度不相同的溶液（如做K+导就在顶膜侧加入高K+，基膜面加入低K+溶液），然后有目的性地在细胞的某侧加入打孔剂（如制霉毒素，两性霉素等），将该侧打穿，使该侧外界与细胞内相通（即细胞内液与该侧外溶液相同），待其稳定后，再加入药物，看此时是否能引起进一
8、步的反应。,上皮细胞,生物体内依靠不同组织系统的作用有选择性地将必须的非电解质和电解质从外环境吸收或者从内环境分泌出去，所有这些组织系统由大量形态较规则，排列紧密的细胞组成，这些多面体细胞组成的几乎没有细胞间质的薄层称之为上皮。 具有极性：顶膜面和基底膜面 特点 具有紧密连接，在形态上彼此紧密的相连续 不具有毛细血管，依赖附近的结缔组织供给营养和氧气 主要功能：屏障功能和有方向性物质转运功能。,Cl- 分泌,NKCC cotransporter,CFTR,Na+ channel,基底膜面,顶膜面,New born CFTR/ and CFTRF508/F508 pigs increased n
9、asal Vt and Vtamil like persons who have CF. But have no more Na+ absorption than controls. In CF, elevations in basal Vt, basal Isc, Vtamil, and Iscamil are caused by the lack of apical Cl conductance, not by increased Na+ conductance. Measured Gt, Isc, and Vt.,Hypothesis,How lack of CFTR changes E
10、lectrolyte transport in primary cultures of differentiated human Airway epithelia? Decreased Cl conductance, increased Na+ conductance, or a combination of the two?,Materials and Methods,Primary Cultures of Human Airway Epithelia. In vitro Human Airway Epithelia. Electrophysiological Measurements.,R
11、esults,The effects of these compounds on CF and Non-CF Epithelia: 1、 Observations about Gt:,Fig.2.,Na+ channel activity increased in CF epithelia ?,Amiloride sensitive current in epithelia,Fig.3.A,Na+ absorption in CF not exceed that in non-CF.,Open-circuit Na+ unidirectional and net Na+ ux rates,Fi
12、g.3.,2、 Observations about Isc,Fig.1.,Fig.2.,3、 Observations about Vt,Fig.2.,Conclusion: Amiloride causes greater Vt and Isc changes in CF Epithelia,Excised Non-CF Epithelia Show Properties Similar to Those in Cultured Epithelia.,Fig.2.,Paradoxical Finding: Vtamil and Iscamil Not Na+ Conductance Inc
13、reased in CF. Conclusion: CF/non-CF differences in Basal Vt, Vtamil, and Iscamil ?,Fig.2.,Fig.4.,Values of basal Vt, Vtamil,and Iscamil did not exceed or were only slightly greater than those in non-CF,Fig.5.,In Cl-free solutions,basal Vt, Vtamil,and Iscamil did not differ between CF and non-CF.Clfr
14、ee solutions reduced basal Isc in both non-CF and CF,Effects of CFTRinh-172,Fig.6.,Gt :Same in non-CF and CF,Fig.7.,Discussion,CF Airway Epithelia Have Reduced Anion Conductance but Not Increased Na+ Conductance. The data allow four main conclusions about electrolyte transport in CF vs. non-CF airwa
15、y epithelia.,First, CF airway epithelia have a markedly reduced trans-epithelial Cl conductance under basal conditions. Second, non-CF epithelia had some constitutive CFTR Cl Conductance under basal conditions. Third, the measurements of Gt, apical Na+ current, and 22Na+ uxes indicate that Na+ conductance in CF epithelia did not exceed that in non-CF epithelia. Fourth, the paper found that basal Vt, Vtamil and Iscamil were greater in CF than in non-CF epithelia.,Thank you!,娄本光 2011-11-3,
