Basal-Lateral/Endomembrane Traffic in Lacrimal Acini

泪腺腺泡的基底外侧/内膜交通

• 批准号: 7122431
• 负责人: AUSTIN K MIRCHEFF
• 金额: $ 39.79万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7122431
• 关键词: SDS polyacrylamide gel electrophoresis; Sjogren' s syndrome; acinar cell; autoimmune disorder; basolateral membrane; biological signal transduction; carbachol; confocal scanning microscopy; cysteine endopeptidases; dynein ATPase; enzyme activity; histamine; immunoprecipitation; intracellular transport; laboratory rabbit; lacrimal apparatus; lacrimal disorder; leukocyte adhesion molecules; lysosomes; muscarinic receptor; oxytocin; prostaglandin E; receptor expression; serotonin

DESCRIPTION (provided by applicant): Sjogren's syndrome (SjS) and other immune-related diseases cause lacrimal dysfunction, impaired vision, and ocular surface inflammation in 2 to 4 million Americans. Primary lacrimal deficiency (PLD), which also may have an immune-related component, affects at least 6 million more. These concepts are unified by the hypothesis that: the milieu in the lacrimal glands reflects an immunohomeostasis involving regulatory lymphocytes, autoimmune effector lymphocytes, and secretory epithelial cells; lymphocyte and inflammatory cell mediators modulate epithelial cell functions; and the immunohomeostasis evolves in response to altered epithelial cell functions. However, little is known about the triggers that alter epithelial function to initiate the diseases, the reasons they occur more frequently in women than in men, and the molecular mechanisms that cause lacrimal dysfunction. The investigators have found that chronic stimulation of lacrimal acinar cells with the muscarinic receptor (MAChR) agonist, carbachol, causes epithelial secretory quiescence and also activates an aberrant endomembrane traffic program that blocks movement to lysosomes, potentially increasing exposure of constitutive autoantigens and initiating exposure of previously cryptic epitopes. The G proteins that classically couple to MAChR, Gq and G11, are shared by receptors for a wide variety of mediators, and their signaling is influenced by the sex hormones. Therefore, the investigators propose that: (a) physiological perturbations can initiate inappropriate Gq/G11 signaling that activates the aberrant traffic program, and (b) the local environments in SjS and PLD stimulate inappropriate Gq/G11 signaling that causes functional quiescence underlying lacrimal dysfunction. Specific Aim 1. What are the signals that activate the aberrant membrane traffic program? The central hypothesis is that MAChR remain activated and continue to activate Gq/G11. Specific Aim 2. Can chronic stimulation of other receptors also cause functional quiescence and activate the aberrant traffic program? The central hypothesis is that these changes can be elicited by agonists for receptors that utilize Gq/G11, including histamine, 5-hydroxytryptamine, PGE2, and estradiol. Specific Aim 3. What traffic effectors are responsible for the aberrant program? The central hypothesis is that chronic stimulation decreases dynein motor function but increases p150(Glued) association with kinesin II, thereby increasing kinesin-mediated traffic. Specific Aim 4. How extensively does the aberrant program alter traffic of lysosomal proteins? The investigators will use a GFP-cathepsin S fusion protein and confocal microscopy to test the hypothesis that lysosomal proteins accumulate in the endosomes.

描述（由申请人提供）：干燥综合征 (SjS) 和其他免疫相关疾病导致 2 至 400 万美国人出现泪腺功能障碍、视力受损和眼表炎症。原发性泪腺缺乏症 (PLD) 也可能与免疫相关，影响至少 600 万人。这些概念由以下假设统一起来：泪腺中的环境反映了涉及调节性淋巴细胞、自身免疫效应淋巴细胞和分泌上皮细胞的免疫稳态；淋巴细胞和炎症细胞介质调节上皮细胞功能；免疫稳态随着上皮细胞功能的改变而发生变化。然而，人们对改变上皮功能引发疾病的触发因素、女性比男性更频繁发生这些疾病的原因以及导致泪腺功能障碍的分子机制知之甚少。研究人员发现，用毒蕈碱受体（MAChR）激动剂卡巴胆碱长期刺激泪腺腺泡细胞会导致上皮分泌静止，并激活异常的内膜运输程序，阻止向溶酶体的运动，可能会增加组成型自身抗原的暴露并引发以前神秘的表位。 G 蛋白通常与 MAChR、Gq 和 G11 偶联，由多种介质的受体共享，它们的信号传导受到性激素的影响。因此，研究人员提出：(a) 生理扰动可以启动不适当的 Gq/G11 信号传导，从而激活异常的交通程序，(b) SjS 和 PLD 中的局部环境刺激不适当的 Gq/G11 信号传导，导致泪腺功能障碍的功能静止。具体目标 1. 激活异常膜交通程序的信号是什么？这 中心假设是 MAChR 保持激活状态并继续激活 Gq/G11。具体目标 2. 慢性刺激其他受体是否也会导致功能静止并激活异常的交通程序？核心假设是，这些变化可以由利用 Gq/G11 的受体激动剂引起，包括组胺、5-羟色胺、PGE2 和雌二醇。具体目标 3. 哪些流量效应器对异常程序负责？核心假设是，慢性刺激会降低动力蛋白的运动功能，但会增加 p150(Glued) 与驱动蛋白 II 的关联，从而增加驱动蛋白介导的流量。具体目标 4. 异常程序在多大程度上改变了溶酶体蛋白的运输？研究人员将使用 GFP-组织蛋白酶 S 融合蛋白 和共聚焦显微镜来检验溶酶体蛋白在内体中积累的假设。