Mechanisms Regulating Gastrointestinal Hormone Secretion

描述（由申请人提供）：胃肠道激素是由分散在整个肠道的离散神经内分泌细胞产生的。大多数含GI激素的细胞位于肠粘膜内，它们的根尖区域通常面向肠管腔。胆囊收缩素（CCK）是一种典型的胃肠激素，可调节胆囊收缩、胰酶分泌、延迟胃排空和诱导饱腹感。与大多数胃肠道激素一样，CCK在进食后分泌到血液中。一般认为，营养物质刺激CCK释放，但调节CCK细胞功能的细胞机制在很大程度上是未知的。最近，PI开发了一种方法来分离和表征个体，有活力的，天然肠道CCK细胞，并通过高度富集这些细胞，使体外研究CCK分泌，鉴定这些细胞上的受体和研究参与调节激素分泌的第二信使信号通路成为可能。总之，这些方法有能力为营养物质刺激CCK分泌的机制提供独特的见解。重要的是，PI有初步数据表明CCK细胞表达钙敏感受体（CaSR），并且CaSR介导氨基酸诱导的CCK分泌。PI将使用互补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)细胞内钙荧光的量化，以及(4)通过全细胞膜片钳记录测量的电生理特性的表征。本应用的中心假设是胃肠激素分泌细胞是电兴奋细胞，其分泌受受体和离子通道激活调节。本提案的总体目的是了解胃肠道内分泌细胞的生理调节因子，最初的重点是氨基酸如何控制CCK分泌。CaSR的表征及其与CCK细胞离子通道激活的关系将通过以下具体目标来解决：在离体CCK细胞和小鼠体内研究CaSR在CCK分泌调控中的作用。2. 确定CaSR激活对CCK细胞钙信号传导的影响。3. 研究CCK细胞的电生理特性，评价CaSR对膜电位、钾通道和钙通道活性的调控作用。这些目标都将集中在CaSR的调控上，作为氨基酸刺激CCK分泌调控的关键步骤。从更广泛的角度来看，这些目标应该为胃肠道内分泌细胞受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见解。

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.