AIRWAY MYOCYTE PROLIFERATION & LYSOSOMAL HYDROLASE

平滑肌增生/肥大是一种公认的病理 哮喘的变化，可能在哮喘的发生发展中起重要作用 呼吸道高反应性，哮喘的标志。溶酶体水解酶， 炎症细胞的分泌产物参与了糖尿病的发病机制 过敏原诱导的哮喘和臭氧诱导的毒鼠强反应。这个 建议的研究是基于我们对一种有效的有丝分裂原的初步发现。 纯化的溶酶体水解酶(氨基己糖苷酶)对牛的作用 溶酶体水解酶诱导的气管呼吸道肌细胞。这样做的目的是 建议对呼吸道肌细胞参与的机制进行研究(S 溶酶体水解酶诱导的细胞增殖。具体目标是1)至 确定受体-配体相互作用(特别是甘露糖受体) 是水解酶有丝分裂作用所必需的。2)调查 花生四烯酸代谢产物，特别是前列腺素E2(PGE2) 是溶酶体水解酶诱导有丝分裂的中介物。3)至 研究是否介导了水解酶的促有丝分裂作用 通过激活腺苷环化酶。4)探索可能的 蛋白激酶C(PKC)和二酰甘油(DAG)在心肌梗死中的作用 溶酶体水解酶的促有丝分裂作用。5)确立角色定位 细胞内钙在溶酶体水解酶促有丝分裂中的作用。至 完成特定目标1：a)牛的甘露糖受体的存在 气管肌细胞将通过抗体识别和受体来证明 类型(S)将被刻画。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 捆绑的竞争。B)我们将检查封锁的效果 受体-配体相互作用。受体抑制剂和阻滞剂将是 其作用将与~3H-胸腺嘧啶核苷(TdR)相关 溶酶体水解酶诱导的掺入。C)可能的内部化 作为促有丝分裂作用要求的溶酶体水解酶 学习。甘露糖受体和琼脂糖凝胶结合的抑制剂 将应用水解酶来抑制内化。对于目标2，3，4， PGE_2产生、cAMP水平、DAG水平、PKC活性、 胞浆[Ca++]和三磷酸肌醇(IP3)水平； 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物和激活物 将测试它们对溶酶体诱导的有丝分裂的影响 水解酶。将测量细胞内cAMP水平和PGE2产生 用放射免疫法测定细胞内钙，用Fura 2荧光法测定细胞内钙 指示器。[32P]三磷酸腺苷、磷脂酰丝氨酸和组蛋白III将用于 测定PKC活性。上述测量结果将与~3H-TdR相关 成立为法团。理解受体依赖和受体- 溶酶体水解酶诱导气道肌细胞的独立机制(S) 扩散将使我们能够开发出一种防止 哮喘时可见肌肉增生/肥大。

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.