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Correcting Airway dehydration in Cystic Fibrosis BRETT ROLLINS

Correcting Airway dehydration in Cystic Fibrosis BRETT ROLLINS. March 5, 2009. Context of Study: Cystic Fibrosis. Normal. Mucus Layer. Airways. Airways. Airway Surface Liquid (ASL). Liver. Pancreas. Extended Cilia. Small Intestine. Cell Surface. Reproductive Tract. Cystic Fibrosis.

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Correcting Airway dehydration in Cystic Fibrosis BRETT ROLLINS

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  1. Correcting Airway dehydration in Cystic FibrosisBRETT ROLLINS March 5, 2009

  2. Context of Study: Cystic Fibrosis Normal Mucus Layer Airways Airways Airway Surface Liquid (ASL) Liver Pancreas Extended Cilia Small Intestine Cell Surface Reproductive Tract Cystic Fibrosis Thick/Static Mucus Layer Skin Dehydrated ASL Collapsed Cilia Cell Surface

  3. Airway Ion Transport Proteases + ENaC CFTR + - cAMP Na+ Cl- / HCO3-

  4. Airway Ion Transport in CF Proteases + ENaC + cAMP Na+ Cl- / HCO3-

  5. Channel Activating Proteases cleanve and activate ENaC Na+ Channel Activating Protease CAP Trypsin Active a g b a

  6. CAP Inhibitors Prevent Cleavage and Activation of ENaC Na+ Channel Activating Protease CAP a CAP-Inhibitor g Inactive b a

  7. ENaC is Spontaneously Inhibited over Time in Bronchial Cultures CF Lung Normal Lung Cultured Human Lung Cells V Apical Side Cilia Cell Basolateral Side Tarran et al 2006 ENaC is Active ENaC is Inactive

  8. How To Identify This CAP Inhibitor • Add Trypsin-coated beads to the apical side of Normal and CF cells • Identify bound proteins by mass spectrometry

  9. Palate, LUng & Nasal epithelial Clone Short PLUNC1 a.k.a. SPLUNC1(256 aa’s) Bingle & Bingle, 2000

  10. SPLUNC1 is typically found in the upper airways & glands Campos et al., 2004

  11. Goals of Project • Further Understand Protease Regulation of ENaC • Characterize Defective Protease Regulation in CF • Use These Findings to Develop Strategies to Treat Overactive ENaC in CF

  12. Experimental Questions • If Splunc1 is removed will bronchial cultures be able to properly regulate airway surface liquid levels? • Does Splunc1 alone inhibit ENaC conductance?

  13. SPLUNC1 knockout SPLUNC1 KO + rSPLUNC1 SPLUNC1 Is Necessary for ASL Regulation in Bronchial Cultures Liquid Layer Cilia Cells Height of Liquid Layer (mm) ASL height (mm) Control ± rSPLUNC1 time (hours) t (h)

  14. Xenopus Oocyte Injection and Voltage Clamp Recording Voltage Clamp cRNA Injection Voltage Electrode Current Electrode - Reference Electrode + Perfusion Solution Waste 24 hours Xenopus Oocyte Xenopus Oocyte

  15. Voltage Clamp Set-Up Xenopus Oocyte Wash Buffer ENaC Inhibitor ENaC Inhibitor Wash Buffer ENaC Inhibitor + Trypsin (protease) 0nA Baseline Trypisin Activated 2000nA

  16. Splunc-1 is Sufficient To Inhibit ENaC Current Conductance * p< 0.05

  17. Splunc1 Activity is pH Sensitive CF pH Conditions * Percent Inhibition * *

  18. Key Points • Channel Activating Proteases Regulate ENaC Activity in Human Bronchial Cultures • Splunc1 Is Necessary For ASL Regulation, As Shown By Knockdown Techniques • Splunc1 Is Sufficient For ENaC Current Conductance Inhibition, As Shown By Voltage Clamping Techniques

  19. Model of Normal ASL Volume Regulation Na+ CAP (active) ADO CFTR CAP (inactive) ENaC CFTR Cl- ENaC HCO3-/ Cl-

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