Ion Transport and Communication in Cultured Mammalian Cells

培养哺乳动物细胞中的离子传输和通讯

• 批准号: 9406646
• 负责人: Mary Lee Ledbetter
• 金额: --
• 依托单位: College of the Holy Cross
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9406646&HistoricalAwards=false
• 关键词: Ion; Transport; Communication; Cultured; Mammalian

; 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

; 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 8 9：; F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; 9406646 Ledbetter博士以前的工作使用简单的测定来测量培养的成纤维细胞或上皮细胞共享小细胞质的能力。分子通过缝隙连接。这种合作可以通过用钠泵抑制剂哇巴因处理细胞来抑制。呋塞米是Na+、K+、2C 1-共转运的调节剂，可刺激通讯。当用类固醇激素醛固酮处理肾源性上皮细胞时，它们的哇巴因抑制作用降低。最近的研究表明，钙的变化介导哇巴因的作用，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型盐皮质激素受体。Ledbetter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信的调节机制之间相互作用的例子。她将在自己的实验室以及与Norton B博士合作开展这些研究。吉鲁拉在斯克里普斯研究所（在她的休假期间），并与Fredic S。费伊在马萨诸塞州大学医学院。目标是确定这些和其他离子支持调节剂如何影响细胞通讯。她将使用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.通过免疫荧光染色完整细胞的连接蛋白蛋白定位; B.细胞提取物中连接蛋白特异性RNA和蛋白质的表达以及蛋白质电泳迁移率;以及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荧光分光光度法的细胞加载离子敏感指示染料，沿着放射性示踪剂实验，以关联的流入，流出和稳态含量的钙和钾和细胞质的pH值的通信的影响。%的结果应该有助于完善离子转运调节机制和调节间隙连接介导的通信之间的相互作用的新模式。*** ;例如，您可以使用以下命令：{\wwuser\TEMPLATE\NORMAL.DOT 9406646} Shirley帕克Shirley帕克@ F# Microsoft Word 6.0 2 ; e = e F 4 F j j j j j j j j x 1：Z T 7 x j x j j j ~ Ledbetter博士以前的工作使用简单的分析来测量培养的成纤维细胞或上皮细胞共享小细胞质分子的能力通过缝隙连接。这种合作可以通过用钠泵抑制剂哇巴因处理细胞来抑制。呋塞米是Na+、K+、2C 1-共转运的调节剂，可刺激通讯。当用类固醇激素醛固酮处理肾源性上皮细胞时，它们的哇巴因抑制作用降低。最近的研究表明钙的变化介导了这种效应