CORRECTING AIRWAY DEHYDRATION IN CYSTIC FIBROSIS

BRETT ROLLINS

March 5, 2009

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

Airway Ion Transport

Cl- / HCO3- Na+

+Proteases

CFTR ENaC

cAMP

-+

Airway Ion Transport in CF

Cl- / HCO3- Na+

cAMP

+

+Proteases

ENaC

Channel Activating Proteases cleanve and activate ENaC

Na+

Channel Activating Protease

CAP

ActiveTrypsin

Na+

CAP Inhibitors Prevent Cleavage and Activation of ENaC

CAP

Channel Activating Protease

CAP-Inhibitor

Inactive

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

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

Palate, LUng & Nasal epithelial Clone

Short PLUNC1 a.k.a. SPLUNC1(256 aa’s)

Bingle & Bingle, 2000

SPLUNC1 is typically found in the upper airways &

glands

Campos et al., 2004

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

Experimental Questions

If Splunc1 is removed will bronchial cultures be able to properly regulate airway surface liquid levels?

Does Splunc1 alone inhibit ENaC conductance?

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

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

Voltage Clamp Set-Up

Xenopus Oocyte

Baseline Trypisin Activated0nA

2000nA

ENaCInhibitor

Wash Buffer

ENaC Inhibitor + Trypsin (protease) Wash Buffer

ENaCInhibitor

Splunc-1 is Sufficient To Inhibit ENaC Current Conductance

* p< 0.05

Splunc1 Activity is pH Sensitive

Per

cent

Inh

ibiti

on

**

*

CF pH Conditions

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

Model of Normal ASL Volume Regulation

CFTR ENaCCAP

(inactive)

HCO3-/ Cl-

ADO

Na+

CFTRENaC

CAP(active)

Cl-