AIRWAY MYOCYTE PROLIFERATION AND LYSOSOMAL HYDROLASE

气道肌细胞增殖和溶酶体水解酶

• 批准号: 2223335
• 负责人: DUKHEE Betty LEW
• 金额: $ 10.21万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2223335
• 关键词: adenylate cyclase; asthma; beta N acetylhexosaminidase; binding proteins; calcium; carbohydrate receptor; cell differentiation; cow; cyclic AMP; diacylglycerols; enzyme inhibitors; enzyme mechanism; fluorescent dye /probe; glycoproteins; hyperplasia; inhibitor /antagonist; mannose; mitogens; muscle cells; prostaglandin E; protein kinase C; radioimmunoassay; radiotracer; respiratory hypersensitivity; smooth muscle; tissue /cell culture; trachea

Smooth muscle hyperplasia/hypertrophy is a well recognized pathological change of asthma and may play an important role in the development of airway hyperreactivity, the hallmark of asthma. Lysosomal hydrolases, secretory products of inflammatory cells, are implicated in pathogenesis of allergen-induced asthma and ozone-induced muscarinic hyperreactivity. The proposed study is based on our preliminary findings of a potent mitogenic action of purified lysosomal hydrolases (hexosaminidases) on bovine tracheal airway myocyte by lysosomal hydrolases. The objective of this proposal is to investigate the mechanism(s) involved in airway myocyte proliferation induced by lysosomal hydrolases. Specific aims are 1) To determine if a receptor-ligand interaction (specifically mannose receptor) is required for the mitogenic action of hydrolases. 2) To investigate whether arachidonic acid metabolites, particularly prostaglandin E2 (PGE2) is a mediator of lysosomal hydrolase induced mitogenesis. 3) To investigate whether or not mitogenic action of hydrolases is mediated through the activation of adenylate cyclase. 4) To explore a possible contribution of protein kinase C (PKC) and diacylglycerol (DAG) in the mitogenic effects of lysosomal hydrolases. 5) To establish the role cytosolic calcium in the mitogenic effects of lysosomal hydrolases. To accomplish specific aim 1: a) The presence of mannose receptor on bovine tracheal myocytes will be documented by antibody recognition and receptor type(s) will be characterized. Iodinated glycoproteins will be used for binding study and mannan and fructose-1-phosphate will be used for competition of binding. b) We will examine the effect of blocking receptor-ligand interaction. Receptor inhibitors and blockers will be utilized and their effect will be correlated to 3H-thymidine (TdR) incorporation induced by lysosomal hydrolases. c) Possible internalization of lysosomal hydrolases as a requirement in mitogenic action will be studied. Inhibitors of mannose receptor and agarose and sepharose bound hydrolases will be applied to inhibit internalization. For aims 2, 3, 4, and 5, changes of PGE2 production, cAMP level, DAG level, PKC activity, cytosolic [Ca++] and inositol-triphosphate (IP3) levels; respectively will be studied in the presence and the absence of lysosomal hydrolases. Inhibitors and activators of second messenger systems and cyclooxygenase will be tested for their effects on mitogenesis induced by lysosomal hydrolases. Intracellular cAMP levels and PGE2 production will be measured by RIA and cytosolic calcium will be measured using Fura 2 fluorescent indicator. [32P] ATP, phosphatidyl serine and Histone IIIS will be used to assay PKC activity. Above measurements will be correlated to 3H-TdR incorporation. Understanding the receptor-dependent and the receptor- independent mechanism(s) of lysosomal hydrolase-induced airway myocyte proliferation would allow us to develop a method of prevention of smooth muscle hyperplasia/hypertrophy seen in asthma.

平滑肌增生/肥大是一种公认​​的病理学 哮喘的变化并可能在哮喘的发展中发挥重要作用 气道高反应性，哮喘的标志。 溶酶体水解酶， 炎症细胞的分泌产物，与炎症的发病机制有关 过敏原诱发的哮喘和臭氧诱发的毒蕈碱高反应性。 这 拟议的研究是基于我们对强有丝分裂剂的初步发现 纯化的溶酶体水解酶（己糖胺酶）对牛的作用 气管气道肌细胞通过溶酶体水解酶作用。 此举的目的 提案是研究气道肌细胞涉及的机制 溶酶体水解酶诱导的增殖。 具体目标是 1) 确定受体-配体是否相互作用（特别是甘露糖受体） 是水解酶促有丝分裂作用所必需的。 2）调查 是否有花生四烯酸代谢物，特别是前列腺素 E2 (PGE2) 是溶酶体水解酶诱导有丝分裂发生的介质。 3) 至 研究水解酶的促有丝分裂作用是否是介导的 通过腺苷酸环化酶的激活。 4）探索一种可能 蛋白激酶 C (PKC) 和二酰甘油 (DAG) 在 溶酶体水解酶的有丝分裂作用。 5）建立角色 溶酶体水解酶的有丝分裂作用中的胞质钙。 到 实现特定目标 1：a) 牛身上存在甘露糖受体 气管肌细胞将被抗体识别和受体记录 类型将被表征。 碘化糖蛋白将用于 结合研究和甘露聚糖和果糖-1-磷酸将用于 绑定的竞争。 b) 我们将检查阻塞的效果 受体-配体相互作用。 受体抑制剂和阻滞剂将 其作用与 3H-胸苷 (TdR) 相关 由溶酶体水解酶诱导的掺入。 c) 可能的内化 溶酶体水解酶作为有丝分裂作用的必要条件将是 研究过。 甘露糖受体抑制剂与琼脂糖和琼脂糖结合 将应用水解酶来抑制内化。 对于目标 2、3、4， 5、PGE2产量、cAMP水平、DAG水平、PKC活性的变化， 胞质 [Ca++] 和肌醇三磷酸 (IP3) 水平；分别会 在存在和不存在溶酶体水解酶的情况下进行研究。 第二信使系统和环氧合酶的抑制剂和激活剂 将测试它们对溶酶体诱导的有丝分裂发生的影响 水解酶。 将测量细胞内 cAMP 水平和 PGE2 产量 通过 RIA 进行测定，并使用 Fura 2 荧光灯测量胞质钙 指标。 [32P] ATP、磷脂酰丝氨酸和组蛋白 IIIS 将用于 测定 PKC 活性。 上述测量结果将与 3H-TdR 相关 合并。 了解受体依赖性和受体依赖性 溶酶体水解酶诱导气道肌细胞的独立机制 扩散将使我们能够开发出一种防止顺利发生的方法 哮喘中出现的肌肉增生/肥大。