Jeffrey J. Wine - US grants

The aim of this project is to study the functional significance of additional transmitter substances in motoneurons. We have shown that, in insects and crustacea, the pentapeptide neurotransmitter candidate proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is present in individually identifiable skeletal motoneurons. Our studies will focus on a neuromuscular system in crayfish which contains just five identified excitatory motoneurons. We have shown that three of these neurons contain the neuropeptide proctolin, in addition to their 'conventional' transmitter, which is thought to be L-glutamate. We propose to examine the relative contributions of the two transmitter substances to generating tension in the muscle. We shall demonstrate release of proctolin and shall examine the factors that lead to the release. We shall measure tension and correlate transmembrane potential and conductance changes produced by application of proctolin and L-glutamate, alone and in combination. The results will be compared to the effects of selective stimulation of the motoneurons that contain proctolin and those that do not contain proctolin. We will further elucidate the role of proctolin by eliminating it's effect(s) with either a proctolin-specific antiserum or by selectively depleting the peptide from terminals by removing the motoneuron's cell body. If time permits, we will examine the ultrastructure of nerve terminals depleted of the peptide and compare them and terminals of non-proctolin containing axons to normal proctolin-containing terminals. This could provide insight into the packaging of multiple transmitters at the neuromuscular junction. Finally, we will examine biochemically and physiologically the levels of the two transmitters in these neurons during the course of two behavioral changes, which involve an altered use of these muscles, in order to examine, at a cellular level, potential regulatory roles of additional transmitters. This research has considerable health relevance to the extent that specific neuromuscular disorders result from abnormal levels and functioning of 'extra' transmitters.

Three hypotheses will be tested. The first hypothesis is that all symptoms of cystic fibrosis (CF) stem from the absence or malfunction of a chloride channel that is gated by cAMP. The second hypothesis is that the defect resides in a kinase which regulates chloride channels, and which also might regulate other cellular properties and so contribute to the pathophysiology of CF in ways unrelated to its effects on chloride permeability. A third hypothesis is that the decrease in chloride permeability occurs in the paracellular pathway. Our specific aim is to compare ion channels in the apical membranes of endocervical columnar epithelium from control and CF subjects, using patch clamp recording techniques. We will also test comparable tissue from rabbits to see if it is a suitable model, at the single channel level, for human cervical secretion. Because fluid secretion in this tissue is under hormonal control, a cellular understanding of secretion here may eventually help clarify the persistently lower survival rate of CF women relative to men. The experiments involve 5 main steps: (1) maintain the tissue in culture; (2) obtain single channel recordings from it; (3) identify apical (and possibly basolateral) channels; (4) describe in more detail the chloride channels, including their gating characteristics; (5) repeat these steps with CF tissue and rabbit tissue.

Cystic fibrosis (CF) is a recessive genetic disease caused by a single defective gene located on the long arm of chromosome 7. The carrier frequency in the U.S. Caucasian population is about 1 in 23. The clinical syndrome includes elevated sweat electrolytes, pancreatic insufficiency, thickened cervical mucus in females and blockage and eventual degeneration of the vas deferens in males, susceptibility to meconium ileus in newborns and an equivalent form of intestinal blockage later, and chronic lung infection, usually by gram negative bacteria. Eventually complications from the lung infections lead to death; the median survival age in the U.S. is now about 21 years. We propose to help understand CF by using electrophysiological techniques to localize the relevant gene product, clarify how it works, and determine where it is expressed. We take as our starting point that the gene product helps regulate a C1- ion channel complex that is a portion of cAMP-regulated ion transport. The defective gene product is expressed in a variety of exocrine cells, where it results in reduced fluid secretion and reduced salt absorption. The proposed experiments are designed to fill in some of the many gaps between the defective gene and the clinical syndrome. The experiments are designed to decide between two possibilities: Is the defective gene product inextricably linked to C1- ion conductance, or is C1- ion conductance simply one of several mechanisms affected by a defect in a more general regulatory product? Our specific aims are (1) to analyze apical chloride channel gating in cultured cells using patch-clamp techniques; and to determine: (2) if the CF gene is expressed by heterozygotes; (3) if the reabsorption of NaC1 by sweat ducts is regulated by a cAMP-dependent increase in chloride conductance; and (4) if beta-adrenergically stimulated sweating depends upon regulation of chloride channels by a cAMP-dependent pathway. We also propose to establish the CF phenotype in cultured cell types not presently studied. For each of the systems we study, we plan to begin to trace the consequences of decreased C1- conductance on cell and organ system function. In addition to on-cell, whole-cell and inside-out excised patch-clamp techniques, we will use intracellular microelectrode recording and microperfusion of sweat ducts.

Cystic fibrosis (CF) is a recessive, single gene disease affecting about 1/2000 Caucasians-, exocrine secretion is affected, and the most serious consequence is a compromise of mucosal defenses in the lung, leading to chronic respiratory infections. Median survival age is about 23 years. The CF gene codes for a 1480 amino acid putative membrane protein (CFTR) with 2 nucleotide binding domains. The two defective alleles identified so far are a single amino acid deletion (68%) or substitution (4%) in the N-terminal nucleotide binding domain. The most throughly described cellular abnormality in CF is a marked reduction of chloride permeability in several cells types, leading to hyposecretion of fluid and altered salt and water reabsorption. The general goal of this proposal is to enable the visiting researcher to gain direct experience with a set of methods not currently available in his laboratory while attempting to answer important questions about the molecular basis of the transport defect in CF. Specific research goals include mapping the cellular and subcellular distribution of the CFTR as a prelude to expression studies. Proposed studies require the visiting researcher to learn RNA preparation, Northern and Western blotting, PCR techniques, immunoprecipitation, and immunohistochemistry, production of polyclonal and monoclonal antibodies, immunogold labeling, analysis of electromicrographs, and possibly pulse labeling techniques.

Cystic fibrosis (CF) is caused by mutation in a single gene that codes for a 1480 amino acid protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The mutated CFTR results in a marked decrease in Cl- permeability in several cell types, an increase in Na+ transport in airway cells, and differences in mucus composition. The best present evidence indicates that CFTR is a 6-9 pS (at 22-37 degrees C respectively), linear, cAMP-regulated Cl- channel that requires PKA- dependent phosphorylation and ATP hydrolysis to function. The possibility that CFTR has additional functions remains to be assessed. Most mutations in the CFTR gene that give rise to cystic fibrosis also lead to abnormal processing of CFTR. However, at least when overexpressed in certain non-epithelial cells, the deltaF508 mutant form of CFTR retains partial function. Therapies for CF could attempt to add a normal CF gene to affected cells (gene therapy); add normal CFTR protein (protein therapy), or stabilize mutant CFTR and facilitate its trafficking to the membrane. These methods share the advantage that they could be therapeutically successful regardless of what CFTR normally does. If decreased Cl- conductance is a major factor producing CF airway dysfunction, therapies might also be based upon pharmacological strategies to increase the open probability of any existing mutant CFTR in the membrane, or therapies might seek to activate other Cl- channels in the membrane to bypass the CFTR cl- channel. To help provide a grounding for therapies, we propose experiments in four areas. Studies of CFTR expression will test the hypothesis that levels of CFTR mRNA, protein and cAMP-activated GCl- are directly related. Studies of the single channel kinetics of CFTR-associated channels will determine how channel kinetics are altered by PKA levels, ATP levels, temperature, PKC, and various pharmacological treatments. Experiments with deltaF508 and other mutant forms of CFTR will determine if residual function can be detected in native epithelial tissues, especially with the use of xanthines and related compounds; we also propose to study the single channel kinetics of selected mutant forms of CFTR. Finally, we have identified other species of epithelial Cl- channels and propose experiments to characterize these channels, some of which might serve as potential pathways for bypassing defective CFTR.

DESCRIPTION (Taken directly from the application) The central hypothesis of our research is that the loss of CFTR-mediated, apical membrane chloride conductance is the fundamental physiological defect that leads to airways disease in cystic fibrosis. The general goal of our research is to confirm or negate that hypothesis. The most puzzling aspect of CF lung disease is how it begins. A direct attack on that question is not possible at present because no adequate animal model of human CF lung disease exists. Elsewhere, we propose a strategy to produce such a model. Here, we propose experiments that are feasible with available model tissues and that deal with two of most basic questions in CF research: how does CFTR operate as an ion channel, and what other chloride ion channels are important players in the lung. The proposal has 4 specific aims. Aim 1 is to understand the mechanism and functional significance of natural "lock-open" kinetics that we have discovered in CFTR channels of human airway cells. Aim 2 is to test the hypothesis that some mutations in CFTR lead to disease in whole or in part because they interfere with CFTR's ability to display locked-open kinetics. Aim 3 is to identify other chloride channels and determine their role in airway cell function. Aim 4 is to study changes in channel populations that occur with epithelial cell polarization.

The genetic disease cystic fibrosis (CF), which causes death primarily by predisposing the lungs to chronic infections, is presently a leading candidate for somatic gene therapy. However, the mouse model of CF does not develop lung disease, and no other animal model for CF exists. Other animal models could be of considerable usefulness for testing therapies. This proposal will evaluate a general approach for finding such models. Genetic screening, based on polymerase chain reaction single strand conformation polymorphism and heteroduplex analysis (PCR-SSCP/HA), will be used to test two hypotheses: (1) that DNA sequence variation (mutations + polymorphisms) within the CFTR gene is approximately equivalent in non- human primate and human populations, and (2) that across human populations, the aggregate frequency of disease causing mutations in CFTR (after subtraction of DeltaF508), provides a rough estimate of expected mutation frequencies in non-human primates. Confirmation of the second hypothesis will require the detection of one or more primates who are heterozygous for a disease-causing mutation in the CFTR gene. In humans, the combined carrier frequency rate in various populations for mutations other than DeltaF508 (about 500 mutations identified to date) is estimated to be about 1/85 to 1/150 equivalent to disease frequencies of about 1/30,000 to 1/90,000. The proposed research program is designed to have an about 95% chance of detecting a mutation at carrier frequencies corresponding to a disease rate of 1/1,000,000. When primates with candidate CF mutations on one chromosome are identified, we assess the effects of the mutation on CFTR trafficking and function. If the mutation disrupts CFTR function, a breeding program will be initiated to produce a renewable population of CF homozygous primates.

respiratory system; inborn metabolism disorder; congenital disorders; biotechnology; animal tissue;

Most people with cystic fibrosis (CF) die from chronic lung infections, but it is unknown how CFTR mutations compromise mucosal defenses. The hypothesis to be tested is that CF lung disease begins because CFTR mutations disrupt serous cell secretion, thus depriving CF airways of the antibiotic-rich fluid secreted by serous cells, which express the highest levels of CFTR among airway cells. Preliminary data indicate that serous cells require CFTR for fluid secretion. The serous cell malfunction hypothesis will be directly tested by measuring antibiotic levels in secretions. This project is made possible by a model serous cell line (Calu-3 cells) and especially by improve human airway 1 degree cultures that retain a "pure" serous cell phenotype. Aim 1 test the hypothesis that serous cell 1 degree cultures and Calu-3 cells express abundant antimicrobials, including defensins and collectins. Semi-quantitative RT- PCR will be used to measure expression of mRNA for lysozyme, lactoferrin, secretory component, serum leukocyte protease inhibitor (SLPI), the human defensin molecules hBD-1 and hBD-2, and the collectin SP-A. Aim 2 will test the hypothesis that human airway serous cells do not secrete antibiotics in CF. ELISA will be used to quantify the release of antimicrobials from Calu-3 cells, from serous cell 1 degree cultures from control and CF subjects, and in nasal lavages from normal and CF subjects. Aim 3 tests the hypothesis that CF human airway serous cells do not secrete fluid. A double-sided capacitance probe method will be used to measure fluid secretion across the 1 degree serous cell monolayers from controls and CF individuals. Gland secretions will also be measured with constant bore capillaries in freshly excised trachea and bronchi from controls and CF individuals. Aim 4 will test the hypothesis that serous cells require CFTR to secrete in response to increase [Ca/2+]/i, and that they secrete a variable mixture of HCO/3 and Cl-. This will be tested by shot circuit current measurements, isotope fluxes and patch-clamping. The results expected for this project are that Ca/2+ dependent secretion of Cl and fluid by serous cells will be greatly diminished in CF, resulting in reduced volumes of antibiotics reaching the airway surface from the glands.

Cystic fibrosis is caused by mutations in the gene for the anion channel CFTR. A link between CFTR, altered fluid transport and the persistent infections that cause most CF deaths has not been established. We will test the hypothesis that CF airways disease arises, at least in part, because of diminished or altered serous cell secretion. Our evidence is that CFTR is the only functional anion channel in the apical membranes of airway serous cells, so its loss will reduce rich serous cell secretion. We will directly test the hypothesis by comparing single gland secretions in airway tissues from CF and control humans. The series of experiments we propose have sufficient power to allow us to support or reject the serous cell dysfunction hypothesis. The proposal has 4 specific aims. The purpose of Aim 1 is to quantify and characterize airway submucosal gland secretions by using single gland monitoring to test hypotheses of gland function in pigs, sheep and cats. For individual glands, we measure the rate of mucus secretion, ion content, pH, and transepithelial potential difference and collect uncontaminated secretions to measure viscoelasticity, solids content, and secreted compounds such as lysozyme. Individual glands are labeled via dye injection and subsequent staining to provide single-gland, structure-function correlations. The goal of Aim 2 is to determine if gland secretions of CF subjects are diminished or altered by using the above methods to compare secretions from trachea and bronchi of normal control, disease control, and CF subjects, obtained following lung transplants. We project approximately 194 tissues will be acquired during the proposed 5-year grant period, consisting of 37 CF, 60 disease control, and 97 normal donor tissues. The design and interpretation of experiments in Aims 1 and 2 require a model of gland secretion, and the experimental results test the model. The purpose of Aim 3 is to develop a comprehensive model of ion transport for serous cells, using the Calu-3 cell model studied with open circuit Ussing chamber and pH stat studies. To link electrophysiological and fluid secretion studies, we have developed a novel method to study fluid secretion by epithelial cell sheets. In Aim 4, we will use this 'Virtual Gland' to study fluid secretion by Calu-3 cells and primary cultures of gland serous and gland mucous cells. The virtual gland operates in open circuit and can identify electrically silent mechanisms of fluid transport. The combination of methods will allow us to test the main hypothesis of altered gland function in CF, and will provide new information on airway gland function of use in other airway diseases.

DESCRIPTION (provided by applicant): The proposed research is part of program that seeks to determine why people with cystic fibrosis (CF) have chronic airway infections. The general hypothesis is that defective airway submucosal glands produce mucus that is under-hydrated, compromising mucus clearance and the bio-availability of antimicrobial and anti-inflammatory compounds. In previous work we showed a profound defect in the response of CF glands to agents that elevate cAMP. To progress from those observations we propose 4 specific aims. Aim 1: To determine how local (intrinsic) neurons, especially those containing VIP, control gland secretion directly and via interaction with cholinergic input. Aim 2: To study secretory mechanisms at the single cell level within four anatomical compartments of the glands: the serous acini, mucous tubules, collecting duct and ciliated duct. Cellular responses are quantified using differential interference contrast (DIC) time-lapse digital imaging with which we optically section isolated, functioning glands from control and CF subjects. We will study how each of the four compartments respond to agents that increase [cAMP]i, [Ca2+]i or both, in the presence of control solution (Krebs-bicarbonate buffer) or solutions in which ion substitutions or transport inhibitors are used to dissect the transport processes within the glands. We will also clarify mechanisms for fluid and protein secretion by serous and mucous cells: our imaging methods allow us to quantify individual exocytotic events. Aim 3: To determine the pH of luminal mucus within the 4 gland compartments by using ratiometric imaging of fluorescent indicators (BCECF) injected into the gland lumen. Aim 4: To patch clamp partially dissociated gland serous and mucous cells, to obtain evidence for against the hypothesis that CFTR in the only apical anion channel in serous cells and is absent from mucous cells. Successful completion of these aims will clarify the contribution of glands, which produce approximately 95% of airway mucus, to the mucus clearance and chemical shield components of airway innate defenses.