Mechanisms Regulating Gastrointestinal Hormone Secretion

描述(申请人提供)：胃肠激素是由分散在肠道各处的神经内分泌细胞产生的。大多数含有GI激素的细胞存在于肠粘膜内，通常定位为顶端区域向肠腔开放。胆囊收缩素(CCK)是一种典型的胃肠激素，能调节胆囊收缩、胰酶分泌、延缓胃排空和诱导饱腹感。与大多数胃肠道激素一样，CCK在进食后被分泌到血液中。一般认为营养物质刺激CCK的释放，但调控CCK细胞功能的细胞机制很大程度上不清楚。最近，PI开发了一种分离和鉴定单个、活的、天然的肠道CCK细胞的方法，通过高度浓缩这些细胞，可以在体外研究CCK的分泌，识别这些细胞上的受体，并研究参与调节激素分泌的第二信使信号通路。这些方法结合在一起，能够为营养物质刺激CCK分泌的机制提供独特的见解。重要的是，PI有CCK细胞表达钙敏感受体(CaSR)的初步数据，并且CaSR介导氨基酸诱导的CCK分泌。PI将使用补充技术来研究激素分泌的调节。这些包括：(1)天然CCK细胞的分离和鉴定，(2)体内和体外CCK分泌的测量，(3)细胞内钙荧光的定量，(4)全细胞膜片钳记录测量的电生理特性。这一应用的中心假设是胃肠激素分泌细胞是可电兴奋的细胞，其分泌受受体和离子通道激活的调节。这项建议的总体目的是了解GI内分泌细胞的生理调节，最初的重点是氨基酸如何控制CCK的分泌。CaSR的特性及其与CCK细胞上离子通道激活的关系将通过以下特定的目的来解决：1.研究CaSR在体外和体内对CCK细胞分泌CCK的调节作用。2.研究CaSR激活对CCK细胞钙信号转导的影响。3.研究CCK细胞的电生理特性，探讨CaSR对膜电位、钾通道和钙通道活动的调节作用。这些目标中的每一个都将侧重于调节CaSR，作为调节氨基酸刺激的CCK分泌的关键步骤。在全球范围内，这些目标应该为胃肠道内分泌细胞被已知在激素分泌控制中起重要作用的营养物质调节的机制提供相当大的洞察力。

DESCRIPTION (provided by applicant): Gastrointestinal hormones are produced by discrete neuroendocrine cells which are scattered throughout the intestine. Most GI hormone-containing cells reside within the intestinal mucosa and are often oriented with their apical region open to the lumen of the intestine. Cholecystokinin (CCK) is a prototypical gastrointestinal hormone that regulates gallbladder contraction, pancreatic enzyme secretion, delays gastric emptying, and induces satiety. As is typical of most GI hormones, CCK is secreted into the blood stream after ingestion of a meal. It is generally believed that nutrients stimulate CCK release but the cellular mechanisms regulating CCK cell function are largely unknown. Recently the PI has developed a method for isolating and characterizing individual, viable, native intestinal CCK cells and by highly enriching these cells it has been possible to study CCK secretion in vitro, identify receptors on these cells and investigate second messenger signaling pathways involved in regulated hormone secretion. Together these approaches have the ability to provide unique insights into the mechanisms by which nutrients may stimulate CCK secretion. Importantly, the PI has preliminary data that CCK cells express the calcium-sensing receptor (CaSR) and that CaSR mediates amino acid-induced CCK secretion. The PI will use complementary techniques to study the regulation of hormone secretion. These include: (1) isolation and identification of native CCK cells, (2) measurements of CCK secretion in vivo and in vitro, (3) quantification of intracellular calcium fluorescence, and (4) characterization of electrophysiological properties measured by whole-cell patch clamp recordings. The central hypothesis of this application is that gastrointestinal hormone secreting cels are electrically excitable cells whose secretion is regulated by receptor and ion channel activation. The overall purpose of this proposal is to understand the physiological regulators of GI endocrine cells with the initial focus on how amino acids control CCK secretion. Characterization of CaSR and its relationship to ion channel activation on CCK cells will be addressed by the following Specific Aims: 1. To characterize the role of CaSR in the regulation of CCK secretion in isolated CCK cells in vitro and in mice in vivo. 2. To determine effects of CaSR activation on calcium signaling in CCK cells. 3. To characterize the electrophysiological properties of CCK cells and evaluate CaSR regulation of membrane potential, and potassium channel and calcium channel activities. Each of these aims will focus on regulation of CaSR as a critical step in the regulation of amino acid-stimulated CCK secretion. More globally, these aims should provide considerable insight into the mechanisms by which GI endocrine cells are regulated by nutrients known to be important in the control of hormone secretion.