1 / 19

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.

Télécharger la présentation

Correcting Airway dehydration in Cystic Fibrosis BRETT ROLLINS

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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-

More Related