权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

PROTEIN KINASES IN BRONCHIOLAR EPITHELIAL DEVELOPMENT

细支气管上皮发育中的蛋白激酶

基本信息

批准号：
3353667
负责人：
ALVIN M MALKINSON
金额：
$ 4.02万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-30 至 1990-02-28
项目状态：
已结题

项目摘要

The contribution of Clara cells to the balance of cell types in airway epithelium depends on the stimulus used; for example, NO2 damage of ciliated cells leads mainly to Clara cell differentiation along a pathway which gives rise specifically to ciliated cells. Beta-adrenergic agonists have several effects on the development of airway epithelium, including mitogenic stimulation, release of secretory products, and the conversion of Clara cells into various other cells within the bronchiolar epithelium. The Beta2-subclass of adrenergic agonists stimulates cAMP production, leading to the activation of cAMP-dependent protein kinase (cAK). To understand the mechanism by which Clara cells generate such a diversity of differentiative responses, important intracellular mediators of the response to environmental changes--protein kinase enzymes--will be studied during normal and hormonally accelerated fetal lung development, and during the regenerative repair of NO2 damage in adult lung. Immunohistochemical analysis of the amount, cellular location, and subcellular localization of the regulatory subunits, RI and RII, of cAK will be done in the proximal epithelium of fetal lung in untreated mice, and in fetuses where the mother was injected with dexamethasone (DEX) or the Beta-agonist albuterol (ALB). A fluorescent probe of a protein kinase inhibitor protein (F:PKI) which binds to activated cAK catalytic subunit will be used to estimate the degree of cAK dissociation in different parts of this epithelium. Antibody to cGMP-dependent protein kinase (cGK) will be used to assess the amount and location of this enzyme after DEX and acetylcholine treatment; cGK is in much higher concentration in fetal than in adult lung. Similar studies will be done during stages of repair of adult bronchiolar epithelium in response to NO2 treatment. Clara cells will be isolated from these adult mice, and the phosphorylation of proteins in intact cells determined. The functional status of RI, RII ATP-binding proteins, and GTP-binding proteins, will also be assessed in these Clara cell extracts using nucleotide photoaffinity labeling techniques. Finally, the presence of receptors for glucocorticoids, Beta-agonists, and cholinergic/muscarinic agonists will be determined in isolated Clara cells and in cell lines cloned from Clara cell-derived tumors.

Clara细胞在气道细胞类型平衡中的作用上皮取决于所使用的刺激;例如，纤毛细胞主要沿着沿着一条途径导致Clara细胞分化这就产生了纤毛细胞。 β-肾上腺素能激动剂对气道上皮的发育有几种影响，包括促有丝分裂刺激，分泌产物的释放，以及 Clara细胞分化为细支气管上皮内的各种其他细胞。肾上腺素能激动剂的β 2亚类刺激cAMP产生，导致cAMP依赖性蛋白激酶（cAK）的活化。到了解克拉拉细胞产生这种多样性的机制，分化反应，重要的细胞内介质，将研究蛋白激酶对环境变化的反应在正常和胎肺加速发育期间，成人肺NO2损伤的再生修复。免疫组分析的数量，细胞位置，和亚细胞定位 cAK的调节亚基RI和RII将在近端完成，未处理小鼠的胎肺上皮，以及母体注射地塞米松（DEX）或β-激动剂沙丁胺醇（ALB）。一种蛋白激酶抑制蛋白（F：PKI）的荧光探针，结合活化的cAK催化亚基将用于估计上皮不同部位的cAK解离程度。抗体 cGMP依赖性蛋白激酶（cGK）将用于评估以及DEX和乙酰胆碱处理后这种酶的位置; cGK是在胎儿肺中的浓度远高于成人肺。类似研究将在成人细支气管上皮修复阶段进行， NO2处理的反应。克拉拉细胞将从这些成年人中分离出来，小鼠，并测定完整细胞中蛋白质的磷酸化。的 RI、RII ATP结合蛋白和GTP结合蛋白的功能状态蛋白质，也将在这些克拉拉细胞提取物中进行评估，核苷酸光亲和标记技术。最后，糖皮质激素、β-激动剂和胆碱能/毒蕈碱受体激动剂将在分离的Clara细胞和细胞系中测定克隆自Clara细胞来源的肿瘤。