Accessory cholera enterotoxin, ACE, Mechanism of action

副霍乱肠毒素，ACE，作用机制

基本信息

批准号：
6619038
负责人：
MICHELE TRUCKSIS
金额：
$ 11.93万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
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：
1995-09-15 至 2007-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6619038
关键词：
SDS polyacrylamide gel electrophoresis Vibrio cholerae apical membrane bacterial proteins bacterial toxins bicarbonates biological signal transduction calcium carbachol chloride ion cholera cholera toxin emerging infectious disease enterotoxins gastrointestinal epithelium high performance liquid chromatography laboratory rabbit mass spectrometry molecular genetics second messengers

SDS polyacrylamide gel electrophoresis Vibrio cholerae apical membrane bacterial proteins bacterial toxins bicarbonates biological signal transduction calcium carbachol chloride ion cholera cholera toxin emerging infectious disease enterotoxins gastrointestinal epithelium high performance liquid chromatography laboratory rabbit mass spectrometry molecular genetics second messengers

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项目摘要

DESCRIPTION (provided by applicant): Colonization of the small intestine by Vibrio cholerae causes the potentially lethal disease cholera due to massive salt and water secretion. Although the dehydrating diarrhea of cholera is attributed to secretion stimulated by cholera toxin, two other toxins of V. cholerae that alter short circuit current or resistance in Ussing chambers have been identified. V. cholerae ACE (accessory cholera enterotoxin) was initially described in 1993 as a toxin which increased short circuit current and potential difference in rabbit ileum mounted in Ussing chambers and caused fluid secretion in ligated rabbit ileal loops. We have investigated the mechanism of action of ACE utilizing monolayers of polarized intestinal epithelial cells (T84 cells) mounted in modified Ussing chambers. In these studies we identified novel physiology stimulated by ACE including that ACE is dependent on calcium as a second messenger; that although it is a calcium-dependent agonist it is unique in that it has a prolonged current response and acts synergistically with other calcium-dependent agonists; and finally, that it stimulates secretion that is equally dependent on chloride and bicarbonate ions (a newly emerging mechanism of secretion). We hypothesize that ACE interacts with the polarized intestinal epithelial cell, through a receptor present on the apical surface and that through this interaction it initiates signal transduction with calcium as a second messenger. We also hypothesize that ACE potentiates the Cl- secretory activity of carbachol by blocking the normal inhibitory pathway stimulated by carbachol. The Specific Aims are 1) Identify the ACE receptor; 2) Examine the signal transduction pathways stimulated by ACE; and 3) Examine the interaction of ACE with calcium-mediated inhibitory pathways. We will use molecular genetic, biochemical and cell physiology methods to examine the mechanism of action of ACE. The long term objectives of this proposal are to enhance our understanding of the role of ACE in cholera pathogenesis and to identify novel mechanisms of action of bacterial toxins. The utility of ACE in investigating the epithelial transport pathways in the intestine, and perhaps other tissue types lies in ACE's novel physiology. The characterization of the ion channels activated by ACE may provide valuable information, which can be used to develop pharmacological modulators of chloride and bicarbonate secretion useful in the treatment of diarrheal diseases (excessive secretion) or cystic fibrosis (defective secretion).

描述（由申请人提供）：小肠对小肠的定殖导致可能致命的霍乱霍乱，这是由于大量的盐和水分分泌而导致的。尽管霍乱的脱水腹泻归因于霍乱毒素刺激的分泌，但已经鉴定出改变了短路电流的V.霍乱的另外两种毒素，或者已经鉴定出了USSING腔室中的电阻。 V. Cholerae Ace（辅助霍乱肠毒素）最初在1993年被描述为一种毒素，该毒素增加了短路电流和安装在Ussing Chombers中的兔回肠的电势差，并在结扎的兔回肠环中引起流体分泌。我们已经研究了利用修饰的Ussing腔室中的极化肠上皮细胞（T84细胞）的ACE的作用机理。在这些研究中，我们确定了ACE刺激的新生理学，其中包括ACE取决于钙作为第二信使。尽管它是一种依赖钙的激动剂，但它的独特之处在于它具有延长的电流响应，并与其他依赖性钙依赖性激动剂协同作用。最后，它刺激了分泌，该分泌同样依赖于氯化物和碳酸氢盐离子（一种新出现的分泌机制）。我们假设ACE通过存在的受体与偏振肠上皮细胞相互作用，并且通过这种相互作用，它可以启动与钙作为第二信使的信号转导。我们还假设ACE通过阻止卡尔巴乔刺激的正常抑制途径来增强卡尔巴醇的cl分构活性。具体目的是1）识别ACE受体； 2）检查ACE刺激的信号转导途径； 3）检查ACE与钙介导的抑制途径的相互作用。我们将使用分子遗传，生化和细胞生理方法来检查ACE的作用机理。该提案的长期目标是增强我们对ACE在霍乱发病机理中的作用的理解，并确定细菌毒素作用的新颖机制。 ACE在研究肠道中的上皮运输途径以及其他组织类型的实用性在于Ace的新生理学。 ACE激活的离子通道的表征可能会提供有价值的信息，可用于开发氯化物和碳酸氢盐分泌的药理学调节剂，可用于治疗腹泻疾病（过度分泌）或囊性纤维化（有缺陷的分泌）。