PROTEIN KINASES IN BRONCHIOLAR EPITHELIAL DEVELOPMENT

细支气管上皮发育中的蛋白激酶

• 批准号: 3353665
• 负责人: ALVIN M MALKINSON
• 金额: $ 9.05万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1989-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3353665
• 关键词: adenosine triphosphate; affinity labeling; beta adrenergic agent; bronchioles; cell differentiation; cholinergic agents; cyclic AMP; cyclic GMP; dexamethasone; embryo /fetus pharmacology; guanosine triphosphate; histochemistry /cytochemistry; histogenesis; immunochemistry; mammalian embryology; mature animal; neoplastic cell culture for noncancer research; newborn animals; phosphorylation; protein kinase; regeneration; respiratory epithelium; respiratory toxin; tissue /cell culture

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.

克拉拉细胞对气道平衡平衡的贡献 上皮取决于所用的刺激。例如，No2损坏 纤毛细胞主要导致沿途径的克拉拉细胞分化 这是专门引起的纤毛细胞。 β-肾上腺素能激动剂 对气道上皮的发展有多种影响，包括 有丝分泌物刺激，分泌产物的释放和转换 克拉拉细胞进入支气管上皮中的其他各种细胞。 肾上腺素能激动剂的β2-贝克斯刺激营地的产量， 导致依赖CAMP的蛋白激酶（CAK）激活。 到 了解克拉拉细胞产生如此多样性的机制 区分反应，重要的细胞内介体 对环境变化的反应 - 蛋白酶激酶酶 - 将研究 在正常和荷尔蒙加速的胎儿肺发育期间以及期间 成年肺No2损伤的再生修复。 免疫组织化学 分析数量，细胞位置和亚细胞定位 CAK的监管亚基RI和RII将在近端完成 未经治疗的小鼠和母亲的胎儿肺上皮 注射地塞米松（Dex）或β-激动剂白斑酚（ALB）。 蛋白激酶抑制剂蛋白（F：PKI）的荧光探针，该探针 与激活的CAK催化亚基结合将用于估计 该上皮不同部分的CAK解离程度。 抗体 将CGMP依赖性蛋白激酶（CGK）用于评估量 DEX和乙酰胆碱治疗后这种酶的位置； CGK是 胎儿的浓度比成年肺更高。 类似的研究 将在成年支气管上皮的修复阶段完成 对NO2处理的反应。 克拉拉细胞将与这些成年人分离 小鼠，以及确定完整细胞中蛋白质的磷酸化。 这 RI，RII ATP结合蛋白和GTP结合的功能状态 蛋白质也将在这些克拉拉细胞提取物中评估 核苷酸光附为标记技术。 最后，存在 糖皮质激素，β-激动剂和胆碱能/毒蕈碱的受体 激动剂将在孤立的克拉拉细胞和细胞系中确定 从克拉拉细胞衍生的肿瘤克隆。