Signal Transduction and the Regulation of Ion Transport in Airway Epithelia
It has recently been shown that a critical component of airways innate defense is the thin (7 µm) liquid layer lining airway surfaces, the periciliary liquid (PCL), that provides a low viscosity solution for ciliary beating and acts a lubricant layer for mucus transport. Normal airways appear to be able to sense the PCL volume and adjust ion channel activity accordingly by unknown mechanisms. A proposed model for ASL volume regulation is shown in Figure 1.
Apical membrane ion channel activity controls the amount of salt (and water) on airway surfaces and hence, PCL volume and mucus hydration levels. It has recently been proposed that the initiating event in CF lung disease is depletion of the PCL due to abnormal ion channel activity (i.e. a lack of CFTR), which causes dehydrated mucus to adhere to airway surfaces, preventing it from being cleared (Fig. 2), causing increased bacterial infections.
Fig 1. Role of the A2b signaling pathway in ASL hydration. ATP is secreted from cells basally or following shear stress and is hydrolyzed by ecto-enzymes to AMP. The 5’ ecto-nucleotidase (5’NT; 1) then degrades AMP to ADO, which activates A2b receptors (A2b-R, 2) that are coupled to G-proteins (Gs) and adenylyl cyclase (AC) to raise local concentrations of intracellular cAMP, resulting in activation of CFTR and inactivation of ENaC. ADO is then degraded to inosine by adenosine deaminase (ADA, 3) and taken up into the cell by nucleotide transporters (eNT1, 4).
Fig 2. Schematic model of early pathogenic events in CF airways. Left, on normal airway epithelia, a thin mucus layer resides atop the periciliary liquid layer (PCL), which is maintained by active ion transport (e.g. Na+ & Cl- channels). The presence of the low viscosity PCL facilitates efficient mucociliary clearance and allows free movement of neutrophils which engulf inhaled bacteria. Right, excessive CF volume depletion caused by abnormal ion transport removes the PCL, mucus becomes adherent to epithelial surfaces, and mucus transport slows/stops. Due to the concentration of mucus, neutrophil movement also becomes impaired and natural antibactierial agents such as lactoferrin and lysozyme are insufficient to kill bacteria.
The long term goal of this laboratory is to understand how homeostasis of PCL volume occurs in airway epithelia under normal and pathophysiological conditions. Currently, research in the Tarran lab is focused on three main areas, listed below, and we utilize cell biological and biochemical techniques coupled with in vivo translational approaches to address these questions:
- Regulation of epithelial cell function by the extracellular environment: We have hypothesized that nucleotides, proteases and other molecules contained in the ASL (ATP for example) can regulate airway ion transport. Fig. 3 depicts cystic fibrosis airway cells that have lost the ability to regulate ASL volume following infection with GFP-labeled viruses that inhibit intracellular Ca2+ signaling by depleting extracellular ATP.
- Gender differences in cystic fibrosis lung disease: Females suffer more severely with cystic fibrosis than their male counterparts, resulting in a significantly shorter lifespan. This gender difference becomes apparent after puberty. Accordingly, we are currently investigating whether estrogens affect Ca2+ signaling and ASL homeostasis. Figure 4 shows an example of simultaneous Fura-2 imaging (as a marker of intracellular Ca2+) and a fusion protein of Estrogen Receptor α (ER α) conjugated to orange fluorescent protein (mOr).
- The effects of cigarette smoke on epithelial airway ion transport: Similar to CF, long term tobacco exposure also results in chronic mucus accumulation. We have found that acute tobacco exposure causes a loss of CFTR from the plasma membrane (Fig. 5) and are currently investigating this phenomenon.
- Ca2+ imaging
- Confocal microscopy
- Electrophysiology (In vivo nasal potential difference measurements, microelectrodes & Ussing chambers)
- Fluorescence resonance energy transfer (FRET)
- Mass Spectrometry
- Molecular Biology & Real Time (q)PCR
- Tissue culture
- Western Blot
Fig 3. RSV infections inhibit PCL homeostasis in CF airways. XZ confocal image of PCL (red) covering RSV-gfp-infected CF ciliated cells (green). This culture has lost the ability to regulate PCL volume due to RSV-induced upregulation of ecto-ATPases, which deplete the ASL of ATP, a vital signaling molecule in CF ASL.
Fig 4. Simultaneous imaging of ERα and Fura-2, as an indicator of intracellular Ca2+ in BHK cells. Images of Fura-2-loaded BHK cells (green) and BHK cells expressing ERα linked to orange fluorescent protein (mOrange).
Fig 5. Cigarette smoke induces removal of CFTR from the plasma membrane. BHK cells constitutively expressing CFTR are labeled with a green antibody against an extracellular portion of CFTR. After cigarette smoke exposure, the amount of CFTR in the plasma membrane is markedly diminished.
|Actin staining in BHK cells||JME cell transfected with STIM1yfp (green) and ERα (red)|
Robert Tarran, PhD, Associate Professor of Medicine
(1998) University of Newcastle-upon-Tyne. Awarded a Ph.D. in Cellular Physiology supervised by Dr. M.A. Gray and Prof. B.E. Argent.
(1994) University of Leeds. Awarded an upper second class B.Sc.(hons) in Physiology.
(2003- present) University of North Carolina at Chapel Hill. Associate Professor. Homeostasis of airway surface liquid in healthy, cystic fibrosis and chronic obstructive pulmonary disease airways using both biophysical and cell biology-based approaches.
(2002-2003) University of California at Berkeley. Assistant Research Scientist affiliated with Prof. T.E. Machen. Employed two photon and epifluoresence microscopy to study the non-genomic effects of estrogens in airway epithelia.
(1997-2002) University of North Carolina at Chapel Hill. Postdoctoral Research Associate supervised by Prof. R.C. Boucher. Used both confocal and epifluoresence microscopy in parallel with conventional electrophysiology (ion-selective microelectrodes and Ussing chambers) to study the structure/function of airway surface liquid in human normal and cystic fibrosis airway epithelia.
Honors and Awards
(1994) Research scholarship from the University of Newcastle-upon-Tyne.
(1995) Recipient of the “Phil Hearne Memorial Prize” which is awarded to the best first year Ph.D. student in Physiological Sciences at the University of Newcastle-upon-Tyne.
(2003) Recipient of the Adolf Windorfer Prize which is awarded for outstanding research in the field of cystic fibrosis.
Please see Pubmed feed in the righthand column for links to current publications.
|Rob Tarran, PhD, Assistant Professor of Medicine||Chong Da Tan, PhD, Postdoctoral Fellow
||Kaitlyn Dykstra, PhD, Postdoctoral Fellow
||Alaina L. Garland, PhD, Postdoctoral Fellow||
Christine Kim, Graduate Student
Jean Tyrrell, PhD, Postdoctoral Fellow
Contact Information7102 Marsico Hall
The University of North Carolina at Chapel Hill
Campus Box #7248
Fax: (919) 966-5178
Email: Robert Tarran