Ion Transport and Communication in Cultured Mammalian Cells

;R o o t E n t y F C o m p o b j b W o R d d o C u m E n 4 o b j E C t p o o l 1 2 3 4 5 6 7 8 9:;F Microsoft Word 6.0文档MSWordDoc。6;[406646]莱德贝特博士以前的工作是用简单的方法来测量培养的成纤维细胞或上皮细胞通过间隙连接共享小细胞质分子的能力。这种合作可以通过用钠泵抑制剂瓦巴因处理细胞来抑制。速尿是Na+，K+， 2C1-共转运的调制剂，刺激通讯。当肾源性上皮细胞用类固醇激素醛固酮处理时，它们的瓦巴因抑制作用降低。最近的研究表明，钙的变化介导了瓦巴因的作用，醛固酮的作用可能涉及一种不寻常的机制，独立于经典的I型矿物皮质激素受体。莱德贝特博士计划将这些发现作为影响离子转运的调节机制和影响连接介导的通讯的调节机制之间相互作用的例子。她将在自己的实验室开展这些研究，并与斯克里普斯研究所的Norton B. Gilula博士（在她的休假期间）和马萨诸塞大学医学院的Fredic S. Fay博士合作。目标是建立这些和其他离子支持调节剂如何影响细胞通讯。她将利用Gilula博士收集的连接蛋白特异性抗体和核酸探针进行研究。A.免疫荧光染色完整细胞连接蛋白定位；b.细胞提取物中连接蛋白特异性RNA和蛋白的表达以及蛋白的电泳迁移率；c.功能性通讯的快速变化，通过细胞的染料偶联来测量。PI还将使用数字成像显微镜和携带离子敏感指示染料的细胞的荧光光谱法，以及放射性示踪剂实验来关联钙和钾的内流，外排和稳态含量以及细胞质ph对通讯的影响。结果将有助于完善离子运输调节机制和缝隙连接介导的通讯调节之间相互作用的新模式。* * *;如果你不知道你的名字，你的名字，你的名字，你的名字，你的名字，你的名字，你的名字，你的名字，你的名字，你的名字，你的名字。DOT 9406646雪莉·帕克雪莉·帕克@ @ @ @ f#微软Word 6.0 2；lebett博士以前的工作使用简单的方法来测量培养的成纤维细胞或上皮细胞通过间隙连接共享小细胞质分子的能力。这种合作可以通过用钠泵抑制剂瓦巴因处理细胞来抑制。速尿是Na+，K+， 2C1-共转运的调制剂，刺激通讯。当肾源性上皮细胞用类固醇激素醛固酮处理时，它们的瓦巴因抑制作用降低。最近的研究表明，钙的变化介导了这种效应

; R o o t E n t r y F C o m p O b j b W o r d D o c u m e n t 4 O b j e c t P o o l 1 2 3 4 5 6 7 8 9 : ; F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; 9406646 Ledbetter Dr. Ledbetter s previous work has used simple assays to measure the ability of cultured fibroblastic or epithelial cells to share small cytoplasmic molecules through gap junctions. This cooperation can be inhibited by treatment of the cells with the sodium-pump inhibitor ouabain. Furosemide, a modulator of Na+,K+, 2C1- cotransport, stimulates communication. When kidney-derived epithelial cells are treated with the steriod hormone aldosterone, their ouabain inhibition is reduced. Recent work has suggested that calcium changes mediate the effects of ouabain and that aldosterone s action may involve an unusual mechanism independent of the classical Type I mineralocorticoid receptor. Dr. Ledbetter plans to pursue these findings as examples of interactions between regulatory mechanisms that effect ion transport and those that influence junction-mediated communication. She will carry out these studies in her own laboratories as well as in collaboration with Dr. Norton B. Gilula at the Scripps Research Institute (during her sabbatical leave) and with Dr. Fredic S. Fay at the Univ ersity of Massachusetts School of Medicine. The goal is to establish how these and other modulators of ion support influence cell communication. She will use Dr. Gilula s collection of connexin-specific antibodies and nucleic acid probes, to study. a. connexin protein localization by immunofluorescent staining intact cells; b. expression of connexin-specific RNA and protein and protein electrophoretic mobility in cell extracts; and c. rapid changes in functional communication, measured by dye coupling of cells. The PI will also use digital imaging microscopy and spectrofluorimetry of cells loaded with ion-sensitive indicator dyes, along with radioactive tracer experiments to correlate effects on communication with influx, efflux and steady-state content of calcium and potassium and cytoplasmic ph. %%% The results should help to refine new modes for the interaction between ion-transport regulatory mechanisms and regulation of gap-junction-mediated communication. *** ; S u m m a r y I n f o r m a t i o n ( 0 Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT 9406646 Shirley Parker Shirley Parker @ @ @ @ F # Microsoft Word 6.0 2 ; e = e F 4 F j j j j j j j x 1 : Z T 7 x j x j j j j ~ j j j j r 9406646 Ledbetter Dr. Ledbetter s previous work has used simple assays to measure the ability of cultured fibroblastic or epithelial cells to share small cytoplasmic molecules through gap junctions. This cooperation can be inhibited by treatment of the cells with the sodium-pump inhibitor ouabain. Furosemide, a modulator of Na+,K+, 2C1- cotransport, stimulates communication. When kidney-derived epithelial cells are treated with the steriod hormone aldosterone, their ouabain inhibition is reduced. Recent work has suggested that calcium changes mediate the effec