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BILE SECRETION AND INTRACELLULAR CA2+ SIGNALS

胆汁分泌和细胞内 CA2 信号

基本信息

批准号：
2856758
负责人：
MICHAEL H NATHANSON
金额：
$ 14.91万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-01 至 1999-09-29
项目状态：
已结题

项目摘要

Hormone-induced Ca2+ signals regulate secretion in many types of cells, including hepatocytes. Ca2+ signals begin as Ca2+ waves in nearly all cell types, and in hepatocytes such waves may play a role in regulation of secretion. The goal of this project is to define the mechanism of Ca2+ wave propagation in hepatocytes, and to determine whether Ca2+ waves play a regulatory role in canalicular contraction, a Ca2+-mediated event important for bile secretion. The following hypothesis will be tested: Ca2+ waves are initiated by sequential release of Ca2+, first from inositol trisphosphate (IP3)-sensitive Ca2+ stores in the apical region of hepatocytes, then from Ca2+-sensitive Ca2+ stores in the basolateral region. These Ca2+ waves then spread from cell to cell across gap junctions, in a fashion that depends on the type(s) of gap junctions expressed. The apical Ca2+ increase in each cell stimulates canalicular contraction, so that intercellular spread of a Ca2+ wave leads to sequential contraction of the hepatocyte canaliculi, which in turn permits peristaltic propulsion of bile. To test this hypothesis, the specific aims of this project are: 1. To determine whether distinct IP3-sensitive and IP3-insensitive Ca2+ pools can be mobilized in the apical and basolateral region of hepatocytes, respectively, Subcellular Ca2+ signals will be elicited by microinjection of specific agonists and antagonists into isolated rat hepatocytes within couplets and triplets, and Ca2+ signals will be detected in these cells using confocal microscopy. 2. To determine whether gap junctions consisting of connexin26, 32, or 43, each of which are expressed in epithelial cells within liver, modulate the spread of intercellular Ca2+ waves in distinct fashions. Ca2+ increases in individual SKHep1 hepatoma cells will be induced by microinjection of specific second messengers, and intercellular coupling will be measured by cell-to-cell spread of the Ca2+ waves. These cells normally do not express gap junctions but have been transfected with each of these three types of gap junction proteins, and features of the intercellular Ca2+ waves in each of these three SKHep1 cell lines will be quantified and compared. 3. To determine whether canalicular contractions in hepatocyte couplets require an apical increase in Ca2+, require an apical increase in Ca2+, and whether such apical Ca2+ increases must propagate from cell to cell to permit sequential contraction of neighboring canaliculi in hepatocyte triplets. Apical and basolateral Ca2+ increases each will be elicited selectively by microinjection of specific agonists and antagonists into hepatocyte couplets and triplets, and subcellular Ca2+ and canalicular contractions will be measured simultaneously using confocal microscopy and optical planimetry. By defining the mechanisms of Ca2+ wave propagation in this fashion, this project may clarify the importance of intra- and intercellular Ca2+ waves for regulating bile secretion. This work is of potential significance for understanding secretion in other organs consisting of polarized epithelia as well, including the kidney, exocrine pancreas and gastrointestinal tract, since similar polarized Ca2+ waves occur in the cells comprising those tissues. This research also may reveal how signals within communicating epithelial cells integrate to permit an organized, organ- level response to hormonal stimulation, a question of fundamental importance in cell physiology.

激素诱导的Ca 2+信号调节许多类型细胞的分泌，包括肝细胞。 Ca 2+信号开始为几乎所有的Ca 2+波细胞类型，在肝细胞中，这种波可能在调节中起作用分泌物。该项目的目标是定义 Ca 2+波在肝细胞中的传播，并确定Ca 2+波是否在小管收缩中起调节作用，小管收缩是一种Ca 2+介导的事件对胆汁分泌很重要。将检验以下假设： Ca 2+波由Ca 2+的顺序释放启动，首先从三磷酸肌醇（IP 3）敏感的Ca 2+储存在顶端区域肝细胞，然后从Ca 2 +-敏感的Ca 2+储存在基底外侧地区然后这些Ca 2+波在细胞间传播以取决于间隙连接类型的方式，表达。每个细胞中顶端Ca 2+的增加刺激小管收缩，因此Ca 2+波的细胞间传播导致肝细胞小管的连续收缩，允许胆汁蠕动推进。为了验证这一假设，该项目的具体目标是： 1. 为了确定是否有不同的IP 3敏感和IP 3不敏感的Ca 2 + 池可以在顶侧和基底侧区域动员，肝细胞，分别，亚细胞Ca 2+信号将引起向离体大鼠中微量注射特异性激动剂和拮抗剂肝细胞内的对联和三联体，和Ca 2+信号将是用共聚焦显微镜检测这些细胞。 2. 为了确定由连接蛋白26、32或 43，每一种都在肝内的上皮细胞中表达，以不同的方式调节细胞间Ca 2+波的传播。在单个SKHep 1肝癌细胞中的Ca 2+增加将由显微注射特异性第二信使和细胞间偶联将通过Ca 2+波的细胞间扩散来测量。这些细胞通常不表达缝隙连接，但已转染了每个这三种类型的间隙连接蛋白，和功能的这三种SKHep 1细胞系中的每一种细胞间Ca 2+波将量化和比较。 3. 为了确定肝细胞偶联中的小管收缩是否需要Ca 2+顶端增加，需要Ca 2+顶端增加，以及这种顶端Ca 2+的增加是否必须从细胞传播到细胞允许肝细胞中相邻小管的顺序收缩三胞胎将分别引起顶侧和基底侧Ca 2+增加选择性地通过显微注射特定的激动剂和拮抗剂进入肝细胞联体和三联体，以及亚细胞Ca 2+和小管使用共聚焦显微镜同时测量收缩和光学平面测量。通过以这种方式定义Ca 2+波传播的机制，该项目可能阐明细胞内和细胞间Ca 2+波的重要性用于调节胆汁分泌。这项工作具有潜在的意义，了解由极化上皮细胞组成的其他器官的分泌以及包括肾脏、外分泌胰腺和胃肠道道，因为类似的极化Ca 2+波发生在细胞，包括这些组织。这项研究也可能揭示出沟通上皮细胞整合，使一个有组织的，器官- 水平反应激素刺激，一个基本的问题，细胞生理学的重要性。