RECEPTOR ACTIVATED CHLORIDE SECRETION IN HT 29 CELLS
RECEPTOR ACTIVATED CHLORIDE SECRETION IN HT 29 CELLS
批准号:
3710305
负责人:
GERDA BREITWIESER
金额:
$0.0万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
--
资助国家:
美国
项目状态:
未结题
起止时间:
至
中文摘要
活跃的氯化物分泌是体内正常体液平衡的基础。
肠道,引起病理后果的原因之一是
高分泌,如腹泻,或分泌不足,如囊性纤维化。
明确参与活性氯离子分泌的途径,并理解
囊性纤维化跨膜电导调节因子(CFTR)的作用
这一进程是制定治疗制度的关键一步
治疗各种分泌障碍。我们已经确定了一个表面上的
结肠癌细胞系HT29-18-氯离子电导的整流
由cAMP和钙离子介导的信号转导激活的CL
小路。我们将利用电生理(全上皮、跨上皮)
细胞和单通道膜片钳技术)、细胞生物学
(细胞内钙离子和氯离子的荧光成像)、生化和
分子生物学(反义寡核苷酸)方法
解决两个目标。(1)我们将确定向外整顿是否
HT29-18-Cl细胞的氯离子电导是由CFTR介导的。我们会
表征单通道的外向整流氯离子电导
水平;我们将用CFTR反义寡核苷酸处理细胞和
确定CFTR蛋白水平是否同时下降,以及
向外整流氯离子电导;我们将使用佛波酯来
下调CFTR,并确定这是否也消除了外部
氯离子电导的整流。如果我们观察到两个CFTR的平行下降
和向外整流的氯离子电导,我们假设CFtr是
调解海流,然后我们将对
控制CFTR激活的信号转导通路。如果我们确定
CFTR蛋白水平与外向整流性氯离子电导
不相关,我们将进一步描述
向外整流氯离子电导,因为它代表肠道氯离子
不受慢性阻塞性肺疾病影响的经络,因此其药理作用
调制可为CF患者提供治疗上的好处。(2)我们会
确定钙离子和cAMP介导的信号转导途径
外向整流氯离子电导的激活。在单层,
整个小区和单通道级别,我们将定义第二信使,
参与外向激活的蛋白激酶和磷酸酶
氯离子电导的整流。
英文摘要
Active chloride secretion is fundamental to normal fluid balance in the
intestine, with pathological consequences resulting from either
hypersecretion, as in diarrhea, or hyposecretion, as in cystic fibrosis.
Defining the pathways involved in active Cl- secretion, and understanding
the role of the cystic fibrosis transmembrane conductance regulator (CFTR)
in the process is a crucial step in the development of treatment regimes
for a variety of secretory disorders. We have identified an outwardly
rectifying Cl- conductance in a colon carcinoma-derived cell line, HT29-18-
Cl, which is activated by both cAMP and Ca2+-mediated signal transduction
pathways. We will utilize electrophysiological (transepithelial, whole
cell and single channel patch clamp techniques), cell biological
(fluorescence imaging of intracellular Ca2+ and Cl-), biochemical, and
molecular biological (antisense oligodeoxynucleotides) approaches to
address two aims. (1) We will determine whether the outwardly rectifying
Cl- conductance of HT29-18-Cl cells is mediated by CFTR. We will
characterize the outwardly rectifying Cl- conductance at the single channel
level; we will treat cells with CFTR antisense oligodeoxynucleotides and
determine whether there is a parallel decrease in CFTR protein levels and
outwardly rectifying Cl- conductance; and we will use phorbol esters to
down-regulate CFTR and determine whether this also eliminates the outwardly
rectifying Cl- conductance. If we observe parallel decreases in both CFTR
and the outwardly rectifying Cl- conductance, we will assume that CFTR is
mediating the current, and we will then undertake a characterization of the
signal transduction pathways controlling CFTR activation. If we determine
that the levels of CFTR protein and outwardly rectifying Cl- conductance
are not correlated, we will further characterize the properties of the
outwardly rectifying Cl- conductance, since it represents an intestinal Cl-
channel which is not affected in CF, and hence its pharmacological
modulation may provide therapeutic benefits to CF patients. (2) We will
define the signal transduction pathways for Ca2+ and cAMP-mediated
activation of the outwardly rectifying Cl- conductance. At the monolayer,
whole cell and single channel level, we will define second messengers,
protein kinases and phosphatases involved in activation of the outwardly
rectifying Cl- conductance.
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RECEPTOR ACTIVATED CHLORIDE SECRETION IN HT 29 CELLS
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批准号:3776761
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项目类别:
-
资助金额:$0.0万
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财政年份:--
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负责人:GERDA BREITWIESER
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依托单位:
RECEPTOR ACTIVATED CHLORIDE SECRETION IN HT 29 CELLS
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批准号:3754614
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项目类别:
-
资助金额:$0.0万
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财政年份:--
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负责人:GERDA BREITWIESER
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依托单位:
RECEPTOR ACTIVATED CHLORIDE SECRETION IN HT 29 CELLS
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批准号:3840368
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项目类别:
-
资助金额:$0.0万
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财政年份:--
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负责人:GERDA BREITWIESER
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依托单位:
海外基金