Dr. Sarah Inglis

Cystic fibrosis (CF) is the most common lethal genetic disorder that affects Caucasians. Sufferers almost invariably die prematurely from lung disease, but the mechanisms by which the basic genetic defect causes the lung pathophysiology characteristic of CF are not yet understood. Our research is focussed on identifying these mechanisms.

Background

The surface of the airway epithelium is lined with a thin layer of fluid, the airway surface liquid (ASL), which plays an important role in defence of the airways and lungs. Crucial to this defensive role is the maintenance of the depth and composition of the ASL in order that the processes that keep the airways clean and free from infection can function normally. This is achieved by the active transport of ions and subsequent osmotically driven movement of water across the airway epithelium. The importance of these processes in maintaining lung health is illustrated by the devastating consequences when the processes malfunction. In particular, the genetic disease cystic fibrosis results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel that normally resides in the luminal membrane of airway epithelia and participates in anion secretion. Secretion of these ions is thus impaired in CF airways, and the subsequent lung disease almost invariably leads to premature death.

HCO3- secretion in the airways

Submucosal Gland

Historically, much CF research has focussed on the role of CFTR in secretion of Cl- into the airway lumen, and development of mechanisms by which Cl- secretion can be restored in CF airways. However, our more recent research has indicated that CFTR may also normally play a role in HCO3- secretion into the airways, and that defects in this secretion may play a role in development of CF lung pathophysiology. In particular, pharmacological inhibition of both Cl- and HCO3- secretion in isolated distal airways results in the ducts of the submucosal glands becoming filled with mucus (Figure 1, (Inglis et al. 1997; Inglis et al. 1998)), one of the earliest signs of CF pathophysiology. This is the first demonstration that inhibition of anion secretion in the submucosal glands, likely to occur in CF glands as a result of abnormal CFTR, results to mucus-occluded gland ducts. This very early step in the development of CF lung pathophysiology may in turn lead to occlusion of the small airways, inhibition of mucociliary transport and other airway defense mechanisms, predisposing the lungs to chronic infections, bronchiectasis and eventual death. This hypothesis is supported by the finding that in addition to occluding the gland ducts, pharmacological inhibition of anion transport in the submucosal glands blocks mucociliary transport (Ballard et al. 2002). These important studies provide a link between the primary genetic defect in CF, mutated CFTR, and key steps in CF lung pathophysiology.

In addition to being essential for gland liquid and mucus secretion, HCO3- secretion may play an important role in regulating the pH of the ASL (pHASL). In fact, despite the secretion of HCO3- by submucosal glands, the pHASL is relatively acidic (Kyle et al. 1990; England et al. 1999; Coakley et al. 2000; Ireson et al. 2001; Jayaraman et al. 2001). We have therefore hypothesised that the HCO3- -rich glandular secretions are acidified, either by surface epithelium or by proximal regions of the gland ducts. Our recent studies have shown that intact distal airways, which comprise both surface and glandular epithelial regions, can secrete both acid and base equivalents into the airway lumen (Inglis et al. 2003).

We use a combination of experimental models including intact, isolated distal and proximal airways and isolated surface and glandular epithelial cells. We apply both electrophysiological, fluorimetric and molecular approaches to answer a number of related questions about secretion in the airways:

Current projects in the lab

It is crucially important to determine how the surface and glandular epithelia work together to regulate the pH of the airway lumen and, to investigate this phenomenon, we are using a pH stat technique to measure changes in the luminal pH of native airways, in which the complex architecture of the surface and glandular epithelium is retained. The aim of this work is to determine how ion transport processes in both the surface and glandular epithelium are co-ordinated to regulate the pH of the airway lumen. Studies of cannulated, perfused distal bronchi indicate that the airways acidify the lumen to approximately pH6.8-6.9, but that stimulation of submucosal gland secretion induces alkalinisation, consistent with HCO3- secretion (Inglis, Wilson et al. 2003).

Using both pH-sensitive fluorescent dyes and molecular techniques, we have recently identified a number of HCO3- transporters in the Calu-3 human submucosal glandular cell line (Inglis et al. 2002). We are now extending those studies to submucosal glandular cells freshly isolated from porcine airways, which closely resemble human airways. These studies will provide valuable information about the identity of HCO3- transporters present in submucosal gland cells. Since abnormal glandular secretion is one of the earliest contributory factors in the development of lung disease in cystic fibrosis, these studies will provide information about glandular secretion that will improve our understanding of cystic fibrosis lung disease. To test whether surface epithelial cells do indeed have the ability to acidify the HCO3- -rich glandular secretions once they are secreted onto the airway surface, we will also study the identity of the pH regulatory transporters present in surface epithelial cells. We will therefore develop methods for isolating surface epithelial cells, and investigate in particular their molecular and functional expression of Na+/H+ exchange, H+-ATPase and K+/H+-ATPase.

Whilst our work stresses the importance of submucosal glands, other workers in the field maintain that CF pathophysiology arises from physiological defects in the surface epithelium. It has been suggested that, as well as acting as an anion channel, CFTR normally tonically inhibits the epithelial Na+ channel (ENaC). According to this model, CF epithelium should display an abnormally high Na+ conductance, which will result in dehydration of the airway surface. We therefore intend to investigate the role of the surface epithelium using a murine model, since murine airways have no distal submucosal glands. In addition, we have access to genetically modified CF knockout mice through collaboration with Dr. W.H. Colledge (University of Cambridge). We will investigate whether rates of Na+ absorption are indeed raised in both distal airway epithelial cells and intact trachea isolated from CF knockout mice and will then investigate whether there are interactions between CFTR and ENaC in these tissues.

Publications

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The University of Dundee is a Scottish Registered Charity, No.SC015096

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