Much of the pathology associated with cystic fibrosis is due to the obstruction of ducts and passageways by an abnormally viscous and sticky mucus. The direct characterization of this secretory process along with the study of native condensed mucins have been hampered by the inability to isolate intact and stable mucin granules. Other granules have been successfully isolated, including the fragile pancreatic zymogen granule. This proposal is designed to incorporate methods and information from the zymogen granule system into a scheme to harvest mucin granules in a physiologically stable state. The approach will utilize mechanically gentle Percoll fractionation procedures along with delipidated BSA buffering of endogenous surface active agents (fatty acids) which have been shown to cause granule instability. The transport properties of the stable mucin granule membrane can be studied by monitoring granule lysis in suspended solutions. Lysis rate is correlated to the influx of osmotically active solutes and thus to swelling by osmosis. Useful probes of the transport characteristics include the use of electrogenic and electroneutral cation ionophores and the use of fluorescent agents such as NBD-taurine to study the anion pathways. These procedures are mechanically gentle and therefore suitable for use with the delicate mucin granule. Additionally, the technique will provide a method to isolate native and condensed mucus for chemical and physical studies of structure and conformations.
