INTRACELLULAR PATHWAYS MEDIATING AIRWAY MUCIN SECRETION

粘蛋白分泌过多是阻塞性气道疾病的常见组成部分，也是患者及其主治医生面临的一个主要问题。该实验室的一个主要长期目标是确定针对粘蛋白高分泌的药物治疗的分子靶点。利用SPOC1细胞作为气道粘蛋白分泌的模型，我们发现定位于顶膜的磷脂酶c偶联P2Y2受体在这些细胞中形成了主要的调节途径。由于所有气道上皮细胞都可能具有细胞外ATP和UTP的P2Y2受体，因此选择性抑制粘蛋白分泌的靶点需要位于PLC、IP3和DAG生成激活的远端。PLC通路的两个下游元件，Ca2+-蛋白激酶C (PKC)，在激活粘蛋白释放方面似乎是独立的，初步数据强烈表明，nPKCdelta，一种Ca2+不敏感的异构体，被激动剂唯一激活。该提案将测试Ca2+和PKC途径是否导致粘蛋白分泌是完全加性和独立的，或者它们是否在接近粘蛋白颗粒/质膜融合和胞吐的限速步骤收敛。这种近端分泌屏障可能是由肌动蛋白微丝组成的皮质细胞骨架。特异性Aim 1将使用野生型和突变型nPKCdelta逆转录病毒表达载体来测试nPKCdelta是否实际上介导了粘蛋白分泌激动剂的作用，并将检查nPKCdelta被激活的方式。特异性Aim II通过探测粘蛋白颗粒穿过皮质微丝屏障和质膜的运输来检测Ca2+-和pkc激活的粘蛋白分泌途径之间的独立性。我们分别关注了scinderin（一种凝胶相关的f -作用切断酶）和Rab3异构体的作用。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外分泌调节，以及它们是否位于由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+在胞外作用的最后步骤中的独立程度。

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.