INTESTINAL MUSCLE EFFECTS OF CLOSTRIDIUM DIFFICILE TOXIN

来自艰难梭菌的毒素是主要的 引起腹泻的原因 尽管众所周知， 这种和其他感染性腹泻的临床表现 是由于肠肌肉的参与，病理生理基础 肌肉细胞功能障碍的原因尚不清楚。 我们认为C. 艰难梭菌对肠平滑肌的直接作用是： 毒素B对平滑肌膜电生理和细胞内 钙代谢，2）毒素A通过炎症反应的间接作用， 激活的白细胞释放的介质，和3）调节 肠神经活动导致神经递质或肽的变化 release. 本提案的总体目标是确定 这些现象中的每一种都是肠道对C. difficile，并确定其潜在的细胞机制。 首先，我们的初步数据表明毒素B 的钙传导在肠平滑肌，我们将评估 膜电生理反应毒素的研究兴奋- 组织条的收缩耦合和膜电生理学 孤立的细胞 毒素B的作用程度 将通过将细胞内钙离子浓度的变化与细胞内钙离子浓度的变化 酶分散肌细胞中的细胞大小和游离胞质钙，以及 测量皂苷透化的细胞中的钙释放。 二是 毒素对肠神经的影响将通过以下方法直接研究： 细胞内膜电生理学测量，突触 对细胞外纤维刺激的反应电位，以及对 肌间和粘膜下神经原位的外源性激动剂。 第三、 因为我们已经表明毒素A在体内导致 类花生酸和体外白三烯导致肌膜 去极化，我们将专门研究白三烯C4的作用， D4介导离体平滑肌细胞膜兴奋性的变化 细胞和肠神经。 这些研究有望增加我们对细胞的理解， 运动反应的基础。艰难，并提高 了解感染因子作用于 胃肠肌

Toxins derived from the bacterium Clostridium difficile are the principal cause of antibiotic-associated diarrhea. Although it is known that many of the clinical manifestations of this and other infectious diarrheal states are due to involvement of intestinal muscle, the pathophysiological basis of muscle cell dysfunction is unknown. We propose that the effects of C. difficile on intestinal smooth muscle result from: 1) The direct effects of toxin B on smooth muscle membrane electrophysiology and intracellular calcium metabolism, 2) The indirect effect of toxin A via inflammatory mediators released by activated leukocytes, and 3) The modulation of enteric nerve activity resulting in changes of neurotransmitter or peptide release. The overall goal of this proposal is to determine the role of each of these phenomenon in the motility response of the intestine to C. difficile, and to ascertain their underlying cellular mechanisms. First, since our preliminary data suggests that toxin B causes inhibition of calcium conductance in intestinal smooth muscle, we will evaluate the membrane electrophysiological response to toxin by studies of excitation- contraction coupling in tissue strips and membrane electrophysiology in isolated cells. The degree to which the effects of toxin B are a function of changes of cystosolic calcium will be studied by correlating changes of cell size and free cytosolic calcium in enzyme-dispersed muscle cells, and measurement of calcium release in saponin-permeabilized cells. Second, the effects of toxins on enteric nerves will be directly studied by measurements of intracellular membrane electrophysiology, synaptic potentials in response to extracellular fiber stimulation, and response to exogenous agonists in myenteric and submucosal nerves in situ. Third, inasmuch as we have shown that toxin A in vivo results in the release of eicosanoids and that in vitro leukotriene results in muscle membrane depolarization, we will specifically study the role of leukotrienes C4 and D4 in mediating changes of membrane excitability in isolated smooth muscle cells and enteric nerves. These studies are expected to increase our understanding of the cellular basis of the motility response to C. difficile, and to enhance understanding of the general mechanisms by which infectious agents act on gastrointestinal muscle.