PROTEIN KINASES IN BRONCHIOLAR EPITHELIAL DEVELOPMENT

细支气管上皮发育中的蛋白激酶

• 批准号: 3353668
• 负责人: ALVIN M MALKINSON
• 金额: $ 9.88万
• 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/3353668
• 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.

Clara细胞对气道细胞类型平衡的贡献 上皮细胞取决于所使用的刺激；例如，NO2 损害 纤毛细胞主要导致 Clara 细胞沿一条途径分化 它专门产生纤毛细胞。 β-肾上腺素能激动剂 对气道上皮的发育有多种影响，包括 有丝分裂刺激、分泌产物的释放以及 克拉拉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 Beta2 亚类可刺激 cAMP 产生， 导致 cAMP 依赖性蛋白激酶 (cAK) 的激活。 到 了解克拉拉细胞产生如此多样性的机制 分化反应，重要的细胞内介质 将研究对环境变化的反应——蛋白激酶 在正常和激素加速胎儿肺部发育期间，以及在 成人肺中 NO2 损伤的再生修复。 免疫组织化学 的数量、细胞位置和亚细胞定位分析 cAK 的调节亚基 RI 和 RII 将在近端完成 未经治疗的小鼠以及母亲的胎儿的胎儿肺上皮细胞 注射了地塞米松（DEX）或β-激动剂沙丁胺醇（ALB）。 蛋白激酶抑制剂蛋白 (F:PKI) 的荧光探针 与激活的 cAK 催化亚基的结合将用于估计 该上皮不同部分的 cAK 解离程度。 抗体 cGMP依赖性蛋白激酶（cGK）将用于评估量 以及 DEX 和乙酰胆碱处理后该酶的位置； cGK 是 胎儿肺中的浓度远高于成人肺中的浓度。 类似的研究 将在成人细支气管上皮修复阶段进行 对 NO2 治疗的反应。 克拉拉细胞将从这些成人中分离出来 小鼠，并测定了完整细胞中蛋白质的磷酸化。 这 RI、RII ATP 结合蛋白和 GTP 结合蛋白的功能状态 蛋白质，也将使用这些克拉拉细胞提取物进行评估 核苷酸光亲和标记技术。 最后，存在 糖皮质激素、β 激动剂和胆碱能/毒蕈碱受体 将在分离的 Clara 细胞和细胞系中测定激动剂 从 Clara 细胞衍生的肿瘤中克隆。