Ion Transport and Communication in Cultured Mammalian Cells

；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 L 1 2 3 4 5 6 7 7 8 9：；F Microsoft Word 6.0文档MSWordDoc Word.Document.6；9406646 LedBetter S博士以前的工作是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缝隙连接共享小分子细胞质的能力。这种合作可以通过用钠泵抑制剂哇巴因处理细胞来抑制。速尿是一种Na+，K+，2Cl-共转运的调节剂，它刺激交流。当肾脏来源的上皮细胞被类固醇激素醛固酮处理时，它们的哇巴因抑制作用减弱。最近的研究表明，钙离子的变化介导哇巴因的作用，而醛固酮S的作用可能涉及一种不依赖于经典的I型盐皮质激素受体的不寻常的机制。莱德贝特博士计划将这些发现作为影响离子转运的调控机制和影响连接介导的通讯的调控机制之间相互作用的例子。她将在自己的实验室以及与斯克里普斯研究所的诺顿·B·吉鲁拉博士(在她休假期间)和马萨诸塞大学医学院的弗雷迪奇·S·费伊博士合作进行这些研究。我们的目标是确定这些和其他离子支持调节器如何影响细胞通讯。她将用吉鲁拉博士收集的S博士收集的连接蛋白特异性抗体和核酸探针，进行研究。A.通过免疫荧光染色对完整细胞进行连接蛋白蛋白定位；b.细胞提取物中连接蛋白特异性RNA的表达以及蛋白质和蛋白质的电泳率；以及c.通过细胞染料偶联来测量功能通讯的快速变化。PI还将使用数字成像显微镜和离子敏感指示剂染料细胞的荧光光谱，以及放射性示踪剂实验，以关联钙、钾的流入、流出和稳态含量以及细胞质ph值对通讯的影响。%这一结果将有助于完善离子转运调控机制和缝隙连接介导的通讯调控之间的相互作用的新模式。*；S u m a r y In f r m a r n o t i on(0 oh+‘0$H L D h R：\WWUSER\TEMPLATE\NORMAL.DOT 9406646雪莉·帕克Shirley Parker@@@F#Microsoft Word 6.0 2；e=e F 4 F j x 1：Z T 7 x j x j~j r 9406646 LedBetter博士S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缝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哇巴因处理细胞来抑制。速尿是一种Na+，K+，2Cl-共转运的调节剂，它刺激交流。当肾脏来源的上皮细胞被类固醇激素醛固酮处理时，它们的哇巴因抑制作用减弱。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