The long-term goal of this project is to understand the precise regulatory mechanisms governing the biosynthesis of mucins in intestinal goblet cells and to define how the mucins are altered, packaged, transported and secreted. These studies will help characterize how mucins are altered in disease states such as cystic fibrosis, CF and eventually contribute to the better clinical management of the disorder. Within the confines of the current proposal, several approaches will be taken. First, studies on the membrane dynamics of goblet cell secretion will be expanded, including a morphometric analysis of goblet cells during and following the rapid release of mucus. Membrane flow will also be characterized by tracer studies at the electron microscopic level using electron dense markers. Membrane alterations after secretion will be characterized by freeze fracture. Second, studies to define the three dimensional makeup of the cytoskeleton in the Golgi region of the goblet cell will be continued using thin section serial reconstructions of Triton models as well as rapid-freeze, deep-etch, rotary-shadow replicas. Third, the intracellular steps involved in stimulus/secretion coupling will be characterized in a mucin-secreting human carcinoma cell line, T-84, using an immunoassay employing a polyclonal or specific monoclonal antibodies against human mucin. Fourth, chloride channels in T-84 cells containing mucin granules and isolated rat goblet cells will be characterized by patch- clamping techniques. Finally, a morphometric model will be used to detect alterations in the mucin species being synthesis in response to pharmacologic manipulation. These changes will be monitored by immunocytochemistry using specific monoclonal antibodies against rat mucin. These studies will be correlated with carbohydrate cytochemistry and staining with fluorescently- labeled lectins and will serve as a basis for studies using cDNA probes. Understanding how goblet cells respond to pharmacologic agents and to varied secretory states will aid in our understanding of the role of mucus in the biology of the intestinal tract. It will also provide necessary background information that may prove useful in the clinical management of disorders which involve this glycoprotein.
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