Jonathan Widdicombe, Ph.D. - US grants

The purpose of this proposal is to determine the role of cAMP- dependent protein phosphorylation in C1 secretion across tracheal epithelia. Using cultured tracheal epithelial cells, proteins will be phosphorylated by an hour's incubation with 32PO4. Cytosolic and membrane proteins will be separated. The time course of changes in phosphorylation of individual proteins in response to agents which increase intracellular cAMP will be followed, and compared to the time course of change in Cl secretion. The catalytic subunit of cAMP-dependent protein kinase, AT32P and cAMP will be introduced into apical membrane vesicles. Changes in phosphorylation will be correlated with changes in Cl influx measured with the C1-sensitive fluorescent probe, SPQ. Apical membranes will be used because they are relatively enriched in membrane proteins. To this same end, we will try and repeat these studies on vesicles from which cytoskeletal elements have been removed. Phosphorylation changes in normal cultured cells and vesicles will be compared with those of CF cells. Levels of cAMP-dependent protein kinase and its sensitivity to cAMP will be compared in normal and CF cells. Endogenous substrates will be measured in cell extracts. The various form of the RI and RII regulatory subunits of cAMP-dependent protein kinase will be covalently labelled with a photo-affinity derivative of cAMP, and their levels determined following separation on two-D gels. All experiments will be performed initially on dog or cow cells. Once the techniques are established, non-CF and CF human cells will be compared. Because, CF may involve a defective interaction between Ca- and cAMP-dependent systems, similar experiments to those on cAMP-dependent protein kinase and phosphorylation will be performed on the calmodulin-dependent protein kinase and protein kinase C. We hope to obtain information on how normal C1 secretion is regulated. Membrane proteins which change their phosphorylation as rapidly as the change in C1 secretion are candidates for the apical membrane C1 channel. Differences in kinase levels or phosphorylation patterns in CF would provide information as to the basic metabolic defect in this disease.

The research proposed here concentrates on the regulation of apical membrane Cl channels in airways. In addition a novel genetic approach to the localization of the defective gene on chromosome 7 will take advantage of the screening methods used in the ion transport studies. Cultured cells will be used throughout this proposal. In Project 1 (Widdicombe), levels of second messengers, protein kinases and their target phosphoproteins in normal and CF cells will be compared. Changes in protein phosphorylation will be compared with changes in Cl secretion. In Project 2 (Wine), regulation of Cl channels will be studied using a variety of techniques including patch-clamping. Emphasis will be placed on determining if the same Cl channel defect found in airways can be demonstrated in other affected epithelia, and on whether more than one channel type is affected. In Project 3 (Verkman), regulation of the Cl channel will be studied following reconstitution into liposomes or planar lipid bilayers. Apical membrane vesicles from airway cultures and other tissues will be used as sources of Cl channels. Attempts will be made to purify the Cl channel, though this is not necessary for successful reconstitution. In reconstitution studies, the Cl channel is effectively separated from other apical membrane proteins, providing a direct means of testing whether it is defective in CF. Clinical research will be performed at a clinical/cell acquisition CORE in stanford and human cell culture CORE at each university. The clinical/cell acquisition CORE (Lewiston) will provide airway tissues for the USCF Culture CORE (Finkbeiner). Attempts will be made to transform the cells and to improve the level of differentiation of the cultures as revealed by electrophysiological studies. Ultimately, the clinical/cell acquisition CORE will apply information obtained in the basic science projects to the treatment of patients with CF.

The proposed research in this renewal application seeks to understand what roles CFTR plays in the function of human airway epithelium, and how its malfunction in CF leads to the airway pathology characteristic of this disease. Five interrelated projects will study aspects of CFTR ranging from the molecular to the clinical. Dr. Lingappa (Project 1) will study how CFTR is inserted into cell membranes and is trafficked within the cell. Dr. Verkman (Project 2) will study the function of CFTR in intracellular organelles. In particular, using newly developed techniques, he will test the conclusions of others that CFTR is involved in exocytosis and in regulating the pH of endosomes. Using the patch-clamp technique, Dr. Wine (Project 3) will characterize in detail the anion channel properties of wild-type and mutant CFTR. Recent work from our Center has demonstrated that mediator-induced Cl secretion by cultures of CF airway submucosal gland cells is less than 5% of normal. Therefore, the aim of Project 4 (Widdicombe & Finkbeiner) will be to test the hypothesis that the initial accumulation of airway mucus in CF reflects predominantly dehydration or other alterations in gland mucous secretions. Finally, Dr Szoka (Project 5) will test a non-viral approach towards gene therapy of CF. Attempts will be made to transfect airway epithelium in vivo by both aerosol and intravenous routes. The latter route increases the likelihood of correcting defects in submucosal gland cells. All projects will utilize the highly differentiated primary cultures of surface and gland epithelial cells provided by a cell culture CORE directed by Dr Finkbeiner.

Microscopy has shown the fluid lining the airways to consist of two layers. A mucous blanket of variable depth lies on top of the periciliary sol, a clear layer which bathes the cilia. The sources and regulation of these layers are incompletely understood. However, in cystic fibrosis, it is believed that abnormal ion transport by the surface epithelium alters the relationship or hydration of these layers in such a way as to impair mucociliary clearance. In this proposal we will test two hypotheses. Firstly, we suggest that ion transport by the surface epithelium is of little importance in regulating the depth of the periciliary sol, which is set precisely at the length of the cilia by forces of capillarity generated by the large combined circumferential length (600 m per cm2 of epithelial surface) of the cilia. Secondly, we propose that defective Cl and fluid secretion by the glands is mainly responsible for the initial accumulation of mucus in cystic fibrosis. These defects result in dehydrated mucus which will not detach fully from the gland openings and/or will not be effectively transported by the cilia. In addition malfunction of CFTR in glands may alter the sulfation or other properties of gland mucus, again impairing mucociliary clearance. To test these hypotheses, we will investigate how osmotic and active fluid flows across the surface epithelium alter the depth of the mucus layers, and how these changes affect mucus transport. The fluid layers will be visualized in transverse sections of rapidly frozen tissues in the Scanning electron microscope. We will measure mediator-induced fluid secretion across CF and non-CF cell cultures using a capacitance probe technique, and from intact glands by micropipette sampling from duct openings. We will determine if the sulfation and rheology of mucins from CF gland and surface cultures is abnormal. Finally, we will determine the localization and total levels of CFTR in our cultures, and correlate hormonally-induced alterations in these parameters with changes in function. Most of these proposed experiments will be performed on our recently developed primary cultures of surface and gland epithelia, which show high levels of ultrastructural and functional differentiation.

The proposed SCOR will test two basic hypothesis to explain the CF-related modifications in airway surface liquid (ASL) that lead to colonization by Pseudomonas. The serous cell malfunction hypothesis proposes that serous cells in the submucosal glands of large airways and on the surface of small airways show reduced secretion of fluid and antibiotics in CF. The resulting decreased levels of serous cell antibiotics and increased mucin concentrations in ASL favor pathogen colonization. The high salt hypothesis proposes that human airway epithelium absorbs Na and Cl, that a substantial fraction of Cl absorption occurs by an transcellular route, and that block of this route by malfunction of CFTR promotes higher than normal NaCl content of ASL in CF. A less likely possibility is that elevated NaCl levels in CF ASL are caused by saltier than normal gland secretions resulting from failure of ductal salt absorption. High salt content of ASL may encourage Pseudomonas colonization by inhibiting the action of natural antibiotics. Project 1 (Widdicombe/Bastacky) will use low-temperature scanning electron microscopy and X-ray microanalysis of rapidly frozen tissues to determine how the regulation of ASL depth is altered in CF. Salt and mucin content of ASL and gland changes in depth, composition and viscosity of ASL in living tissues using novel fluorescence microscopy. Project 3 (Wine) will study the secretory mechanisms contributing to the ASL, and test specifically the serous cell malfunction hypothesis. Project 4 (Miller) will determine the routes and mechanisms by which Na and Cl are absorbed across airway surface epithelium. Nasal PD measurements will be used to verify in vitro findings. A Cell Culture Core (Finkbeiner) will provide intact human airways and cultures of human gland and surface epithelial cells.

This project has two objectives. First, in normal tissues we will characterize how epithelial processes (gland secretion, surface epithelial ion transport, goblet cell discharge) affect the depth of airway surface liquid. We will then determine how the regulation of ASL depth is altered in cystic fibrosis (CF). Second, we will determine how the ion and mucin concentrations of ASL and gland secretions are altered in CF. The depth of ASL will be measured by low temperature scanning electron microscopy (LTSEM) of frozen hydrated tissues fractured perpendicular to the plane of the surface epithelium. Ion contents will be measured by X-ray microanalysis of specimens in the LTSEM. Mucin concentrations will be estimated from the sulfur peak in the X-ray microanalysis spectrum. As an alternative to X-ray microanalysis we will estimate Na and Cl levels in ASL of surface epithelial cultures by adding tritiated water (or mannitol), /36 Cl and /22 Na to the basolateral side of cell cultures. Na and Cl concentrations are then estimated from the ration of equilibrium uptake for 22/Na or 36/C1 to the equilibrium uptakes of 3/H/2/0 (or /3 H- mannitol). To determine concentrations of Na, Cl and mucin in gland secretions, frozen hydrated sections will be fractured in a plane parallel to, and just below the surface epithelium. This will result in profiles of gland ducts of approximately 50 mum diameter and 1 per mm/2, over which the electron beam will be centered. Finally, we will estimate the forces of surface tension holding the periciliary sol in place. When solute is added to the basolateral medium, there should come a point at which the osmotic grandient exceeds the forces of surface tension. At this point, the sol should disappear and the cilia collapse onto the epithelium. The information on depth and composition of ASL obtained in this project will serve as guidelines for the other projects.

cystic fibrosis; bactericidal immunity; pathologic process; antibiotics; lactoferrin; secretion; lysozyme; clinical research; human subject;