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CORRECTING AIRWAY DEHYDRATION IN CYSTIC FIBROSIS
BRETT ROLLINS
March 5, 2009
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Context of Study: Cystic Fibrosis
Airways
Liver
Pancreas
Small Intestine
Reproductive Tract
Skin
AirwaysNormal
Cystic Fibrosis
Cell Surface
Collapsed Cilia
Cell Surface
Extended Cilia
Airway Surface Liquid (ASL)
Mucus Layer
Dehydrated ASL
Thick/Static Mucus Layer
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Airway Ion Transport
Cl- / HCO3- Na+
+Proteases
CFTR ENaC
cAMP
-+
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Airway Ion Transport in CF
Cl- / HCO3- Na+
cAMP
+
+Proteases
ENaC
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Channel Activating Proteases cleanve and activate ENaC
Na+
Channel Activating Protease
CAP
ActiveTrypsin
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Na+
CAP Inhibitors Prevent Cleavage and Activation of ENaC
CAP
Channel Activating Protease
CAP-Inhibitor
Inactive
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ENaC is Spontaneously Inhibited over Time in Bronchial Cultures
V
ENaC is Active
ENaC is Inactive
NormalLung
CF Lung
Tarran et al 2006
Cultured Human Lung Cells
Cilia
Cell
Apical Side
Basolateral Side
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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
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Palate, LUng & Nasal epithelial Clone
Short PLUNC1 a.k.a. SPLUNC1(256 aa’s)
Bingle & Bingle, 2000
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SPLUNC1 is typically found in the upper airways &
glands
Campos et al., 2004
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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
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Experimental Questions
If Splunc1 is removed will bronchial cultures be able to properly regulate airway surface liquid levels?
Does Splunc1 alone inhibit ENaC conductance?
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SPLUNC1 Is Necessary for ASL Regulation in Bronchial Cultures
time (hours)
He
igh
t o
f L
iqu
id L
aye
r (
m)
0
5
10
15
20
25
30
35
0 12 24
Control ± rSPLUNC1
0
5
10
15
20
25
30
35
0 12 24
SPLUNC1 knockout
0
5
10
15
20
25
30
35
0 12 24
SPLUNC1 KO + rSPLUNC1
t (h)
AS
L h
eig
ht
(m
)
CellsCiliaLiquid Layer
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Xenopus Oocyte Injection and Voltage Clamp Recording
cRNA Injection
24 hours
Perfusion Solution
Waste
Reference Electrode
Xenopus Oocyte Xenopus Oocyte
Voltage Electrode
Current Electrode
-
+
Voltage Clamp
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Voltage Clamp Set-Up
Xenopus Oocyte
Baseline Trypisin Activated0nA
2000nA
ENaCInhibitor
Wash Buffer
ENaC Inhibitor + Trypsin (protease) Wash Buffer
ENaCInhibitor
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Splunc-1 is Sufficient To Inhibit ENaC Current Conductance
* p< 0.05
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Splunc1 Activity is pH Sensitive
Per
cent
Inh
ibiti
on
**
*
CF pH Conditions
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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
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Model of Normal ASL Volume Regulation
CFTR ENaCCAP
(inactive)
HCO3-/ Cl-
ADO
Na+
CFTRENaC
CAP(active)
Cl-