BILE SECRETION AND INTRACELLULAR CA2+ SIGNALS

胆汁分泌和细胞内 CA2 信号

• 批准号: 2144914
• 负责人: MICHAEL H NATHANSON
• 金额: $ 9.15万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144914
• 关键词: apical membrane; basolateral membrane; bile; calcium flux; cell cell interaction; cellular polarity; clone cells; confocal scanning microscopy; gap junctions; hormone regulation /control mechanism; inositol phosphates; intracellular transport; laboratory rat; liver cells; liver metabolism; membrane channels; microinjections; tissue /cell culture; transfection

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.

激素诱导的钙信号调节许多类型细胞的分泌， 包括肝细胞。几乎所有的钙信号都以钙波的形式开始 细胞类型，在肝细胞中，这样的波可能起到调节作用 分泌物。这个项目的目标是定义 Ca~(2+)波在肝细胞内的传播，并确定是否存在钙波 在小管收缩中发挥调节作用，这是一种钙离子介导的事件 对胆汁分泌很重要。我们将检验以下假设： Ca~(2+)波是由钙离子的顺序释放引发的，首先是从 三磷酸肌醇(IP3)敏感的钙离子储存在根尖区域 肝细胞，然后从钙敏感的钙储存在基底外侧 区域。然后，这些钙电波通过缝隙从一个细胞传播到另一个细胞 连接点，取决于缝隙连接点的类型(S) 表达。每个细胞顶端钙离子的增加刺激小管 收缩，因此钙离子波在细胞间的传播导致 肝小管的连续收缩，进而 允许胆汁的蠕动推进。为了检验这一假设， 该项目的具体目标是： 1.确定不同的IP3敏感和IP3不敏感的钙离子 池可以在顶端和基底外侧区被动员 肝细胞亚细胞内钙信号将分别被激发 隔离大鼠微量注射特异性激动剂和拮抗剂 肝细胞内的联体和三联体，以及钙信号将被 用共聚焦显微镜在这些细胞中检测到。 2.确定缝隙连接是否由连接蛋白26、32或 43，每一个都在肝脏内的上皮细胞中表达， 以不同的方式调节细胞间钙波的传播。 人肝癌细胞株SKHep1细胞内钙离子浓度的变化 特定第二信使的显微注射和细胞间偶联 将通过细胞间的钙波传播来测量。这些细胞 通常不表达缝隙连接，但已被每个 这三种类型的缝隙连接蛋白，以及 这三种SKHep1细胞系中的细胞内钙波都将 被量化和比较。 3.确定肝细胞联体中的小管收缩 需要尖端钙离子增加，需要尖端钙离子增加， 以及这种顶端钙离子的增加是否必须在细胞间传播 允许肝细胞内相邻小管的顺序收缩 三胞胎。心尖部和基底侧部的钙离子分别增加 选择性地通过微量注射特定的激动剂和拮抗剂到 肝细胞联体和三联体以及亚细胞内钙离子和小管 将使用共聚焦显微镜同时测量宫缩 和光学平面测量法。 通过以这种方式定义钙离子波的传播机制，这 该项目可能会阐明细胞内和细胞间钙波的重要性 用于调节胆汁分泌。这项工作具有潜在的意义 了解由极化上皮组成的其他器官的分泌 也包括肾脏、外分泌胰腺和胃肠道 由于在细胞中出现类似的极化钙波，包括 那些纸巾。这项研究还可能揭示信号是如何在 沟通的上皮细胞整合，使一个有组织的器官- 对荷尔蒙刺激的水平反应，一个根本的问题 在细胞生理学中的重要性。