INTRACELLULAR PATHWAYS MEDIATING AIRWAY MUCIN SECRETION

介导气道粘蛋白分泌的细胞内途径

• 批准号: 6027990
• 负责人: C. William Davis
• 金额: $ 20.02万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6027990
• 关键词: athymic mouse; calcium flux; chickens; chronic obstructive pulmonary disease; exocytosis; gelsolin; microfilaments; molecular pathology; mucins; phospholipase C; protein binding; protein kinase C; second messengers; secretion; tissue /cell culture; trachea; transfection /expression vector

Mucin hyperproduction is a common component of obstructive airways disease and comprises a principle problem faced by patients and their attending physicians. A major long-term goal of this laboratory is the identification of molecular targets for pharmacologic therapies against mucin hypersecretion. Using SPOC1 cells as a model for airway mucin secretion, we have shown that phospholipase C-coupled P2Y2 receptors localized in the apical membrane form the dominate regulatory pathway in these cells. Because all airway epithelial cells likely possess P2Y2 receptors for extracellular ATP and UTP, targets for the selective inhibition of mucin secretion need to be located at points distal to activation of PLC, and IP3 and DAG generation. The two downstream elements of the PLC pathway, Ca2+-protein kinase C (PKC), appear to be independent in their activation of mucin release, and preliminary data suggest strongly that nPKCdelta, a Ca2+-insensitive isoform, is uniquely activated by agonist. This proposal will test whether the Ca2+ and PKC pathways leading to mucin secretion are fully additive and independent, or whether they converge at a rate limiting step proximal to mucin granule/plasma membrane fusion and exocytosis. A likely candidate for this proximal barrier to secretion is the cortical cytoskeleton, comprised of actin microfilaments. Specific Aim 1 will use wild-type and mutant nPKCdelta retroviral expression vectors to test whether nPKCdelta, in fact, mediates the effects of agonist of mucin secretion, and it will examine the means by which nPKCdelta is activated. Specific Aim II tests the independence between Ca2+- and PKC-activated mucin secretory pathways by probing the transit of mucin granules across the cortical microfilament barrier and the plasma membrane. We focus on the roles of scinderin, a gelsolin-related, F-acting severing enzyme, and Rab3 isoforms, respectively. In each case, we will test whether these elements participate in the regulation of agonist-stimulated exocytosis and whether they lie in the pathways modulated by nPKCdelta and/or Ca2+. Lastly, we will exploit the different binding kinetics of BAPTA and EGTRA to test the degree of independence between PKC and Ca2+ in the final steps of exocytosis.

粘蛋白高生产是阻塞气道疾病的常见组成部分，包括患者及其主治医生所面临的主要问题。该实验室的一个主要长期目标是鉴定针对粘蛋白过度分泌的药理疗法的分子靶标。使用SPOC1细胞作为气道粘蛋白分泌的模型，我们表明磷脂酶C偶联的P2Y2受体位于顶部膜中的磷脂酶偶联受体形成了这些细胞中主导的调节途径。由于所有气道上皮细胞都可能具有用于细胞外ATP和UTP的P2Y2受体，因此需要在PLC激活的远端以及IP3和DAG产生的端点位于选择性抑制粘蛋白分泌的靶标。 PLC途径的两个下游元件Ca2+ - 蛋白激酶C（PKC）似乎在粘蛋白释放的激活中独立，并且初步数据强烈表明NPKCDELTA是Ca2+ - 不敏感的同工型，是由aremonist唯一激活的。该提案将测试导致粘蛋白分泌的Ca2+和PKC途径是否完全添加且独立，或者它们是否以限制速率的步骤融合到粘蛋白颗粒/质膜膜融合和胞吐作用。该分泌近端障碍的可能候选者是由肌动蛋白微丝组成的皮质细胞骨架。具体目标1将使用野生型和突变的NPKCDELTA逆转录病毒表达向量来测试Npkcdelta实际上是否介导了粘蛋白分泌的激动剂的作用，并将检查Npkcdelta激活NPKCDELTA的手段。特定的AIM II通过探测粘蛋白颗粒在皮质微丝屏障和质膜之间的转运，测试Ca2+ - 和PKC激活的粘蛋白分泌途径之间的独立性。我们专注于Scinderin的作用，Scinderin，分别与凝胶素相关的，F-Ot的切断酶和Rab3同工型的作用。在每种情况下，我们将测试这些元素是否参与激动剂刺激的胞吐作用的调节，以及它们是否位于NPKCDELTA和/或Ca2+调节的途径中。最后，我们将利用BAPTA和EGTRA的不同结合动力学来测试PKC和Ca2+之间的独立性，在胞吞作用的最后步骤中。