Gap junctions are thought to be responsible for transmitting intercellular messages. While abundant in some tissues, their prevalence in mammalian respiratory epithelium is negligible except during early development and re-epithelialization subsequent to injury. The particular cell types responsible for elaborating gap junctional proteins and establishing intercellular traffic during these events have not been identified, nor have the patterns of gap junction synthesis and the initiation of intercellular coupling in these two situations been compared. Furthermore, gap junctions may provide a direct route for the exchange of small hormones, growth factors, short transcripts, and other biologically significant molecules. Studies to confirm this hypothesis and to ascertain the properties of molecules that might transverse airway epithelial gap junctions are few. We propose developmental studies to identify the sites of gap junction biosynthesis and intercellular coupling in: 1) post-natal ferret airways, 2) human fetal airways, and 3) ferret and human airway mucosa subsequent to acute injury. Additionally, we propose studies aimed at characterizing the size and nature of nucleic acid to which gap junctions may be permeable. Gap junction synthesis during development and in response to injury will be localized by light microscopic in situ hybridization experiments using cDNA probes that encode gap junctional proteins. These experiments will be compared to light and electron immunocytochemical studies using specific peptide antibodies to identify cell types containing gap junction translation products. Investigations of the size and characteristics of nucleic acid sequences which traverse gap junctional channels will involve microinjection of nucleic acids ranging from simple linear to more complex structures into developing ferret tracheal epithelial cells maintained in vitro. If these studies suggest that native transcripts might traverse gap junction channels, we will microinject mRNA encoding reporter proteins. This will allow the target cell and communicating cells to be identified by conventional light microscopic histochemistry. We anticipate that these studies will provide fundamental information necessary for elucidation of the role of gap junctions in airways epithelial development and repair.
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