REGULATION OF NA-K-CL COTRANSPORT IN AIRWAY EPITHELIA

气道上皮中 NA-K-CL 协同转运的调节

• 批准号: 3245463
• 负责人: MARK HAAS
• 金额: $ 10.14万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245463
• 关键词: adenosine triphosphate; affinity labeling; alpha adrenergic agent; beta adrenergic agent; bradykinin; bumetanide; calcium; cell morphology; chemical binding; chloride channels; chlorine; cystic fibrosis; dogs; fluorescence microscopy; histamine; human tissue; immunoelectron microscopy; ion transport; laboratory mouse; membrane transport proteins; molecular site; organ culture; phosphorylation; posttranslational modifications; potassium; respiratory epithelium; respiratory pharmacology; secretion; sodium; uridine triphosphate

The specific aims of this project are to determine the cellular and molecular mechanisms involved in the regulation of Na-K-C1 cotransport in mammalian (including human) airway epithelial cells, and whether the regulation of this cotransporter is intact in airway cells from patients with cystic fibrosis (CF). Na-K-C1 cotransport is the major pathway for basolateral NaC1 entry into both human and canine airway cells, and the activity of this pathway must be increased simultaneously with apical C1 channel activity during stimulation of net salt and fluid secretion by beta-adrenergic hormones and other secretagogues. Cyclic AMP (cAMP)- mediated stimulation of apical C1 channels does not occur in CF airways, and one potential therapeutic approach to the airway disease in CF is to develop alternative means to activate C1 channels and net salt secretion in these airways. As such, it is important to know if these means are capable of activating basolateral C1 entry via Na-K-C1 cotransport as well as apical C1 channels; it may also be possible to secondarily activate apical C1 channels and net secretion by first stimulating the cotransporter. Cultured canine and human (normal and CF) airway epithelial cells will be employed in this project. [3H]bumetanide binding and tracer ion flux assays will be used to determine Na-K-C1 cotransport and transepithelial ion transport in these cultures, and their responses to potential stimuli including beta- and alpha-adrenergic agonists, changes in cell volume, bradykinin, histamine, ATP, UTP, and A23187 + Ca. It will be determined if stimulation of basolateral cotransport by these treatments represents a primary effect on the cotransporter, or if such stimulation is secondary to activation of apical C1 channels and possibly a resultant decrease in cell volume. Protein kinase and phosphatase inhibitors will be employed to determine the role of phosphorylation reactions in regulation of Na-K-C1 cotransport by both cAMP-dependent and cAMP-independent mechanisms, and whether different kinases and phosphatases are involved in regulation of apical C1 channels and basolateral cotransporters. Using a photoaffinity label for the Na-K-C1 cotransporter and specific antibodies directed against this transporter, it will be determined if the cotransporter itself is phosphorylated in response to stimuli of net C1 secretion in airway epithelia, and if changes in phosphorylation are correlated with changes in ion transport and [3H]bumetanide binding.

该项目的具体目的是确定细胞和 参与Na-K-C1共转运调节的分子机制 哺乳动物（包括人）气道上皮细胞，以及是否是否 该共转运蛋白的调节在患者的气道细胞中是完整的 与囊性纤维化（CF）。 Na-k-C1共晶是主要途径 基底外侧NAC1进入人类和犬类气道细胞，以及 该途径的活性必须与顶端C1同时增加 刺激净盐和液体分泌过程中的通道活动 β-肾上腺素能激素和其他促促血压。 循环放大器（营地） - 在CF气道中不发生介导的顶端C1通道的刺激， CF中气道疾病的一种潜在治疗方法是 开发替代手段，以激活C1通道和净盐分泌物 这些气道。 因此，重要的是要知道这些手段是否有能力 通过NA-K-C1共晶体激活基底外侧C1进入 顶端C1通道；也有可能其次激活顶端 首先刺激共转运蛋白，C1通道和净分泌。 培养的犬和人（正常和CF）气道上皮细胞将是 在这个项目中使用。 [3H] bumetanide结合和示踪离子通量 测定将用于确定NA-K-C1共晶和transepithialial 这些培养物中的离子运输及其对潜在刺激的反应 包括β-和α-肾上腺素能激动剂，细胞体积的变化， Bradykinin，组胺，ATP，UTP和A23187 + CA。 将确定是否 通过这些治疗刺激基底外侧共转移代表 对共转运蛋白的主要作用，或者此类刺激是继发的 顶端C1通道的激活，可能导致细胞的减少 体积。 蛋白激酶和磷酸酶抑制剂将用于 确定磷酸化反应在调节Na-K-C1中的作用 依赖营和独立的机制的共同传输，以及 不同的激酶和磷酸酶是否参与调节 顶端C1通道和基底外侧共转运蛋白。 使用光性 NA-K-C1共转运蛋白的标签和定向的特定抗体 在此转运蛋白的情况下，将确定持续运输蛋白本身是否 响应气道中净C1分泌的刺激而被磷酸化 上皮，如果磷酸化的变化与变化有关 离子转运和[3H] bumetanide结合。