Lecture 3

Question 1

Airways Surface Liquid layer

Answer 1

• Clearance mucous

- airways defence against respiratory pathogens

Question 2

What is the Airways surface layer also known as ??

Answer 2

Periciliary layer

Question 3

What is most important about the ASL/PCL?

Answer 3

Height is important for mucous clearance

Question 4

what happens to the volume of the ASL as you go up the respiratory tract ?

Answer 4

As the respiratory structure gets larger the surface area goes down, so the volume decreases as you go up the respiratory tract

Question 5

What is important about the height of the ASL/PCL?

Answer 5

Height of layer too low or high impacts the ability of lungs to move mucous

Question 6

What are the to types of control of the ASL ??

Answer 6

• Passive – mucous layer acts as a reservoir

* Active – Active ion transport controlling salt level in pcl

Question 7

how do you test the effect of height on theASL/PCL?

Answer 7

Culture human airway epithelial cells - made liquid layer higher optimum

• cells move it to the optimum - 7 microns
• optimum in the lab is 14 microns - moving the dish back and forth to imitate movement of breathing in and out

Question 8

Bumetanide

Answer 8

loop diuretic NKCC1 block CL secretion

Question 9

Amiloride blocks what

Answer 9

ENac

Question 10

Why is bumetanide used ?

Answer 10

to see how much Cl secretion is occurring

Question 11

change in transepithelial potential tell us what

Answer 11

mechanism of epithelial transport changes over time- upreg or down reg
- transport properties change

Question 12

bumetanide / amiloride experiment

Answer 12

at 48 hours
bumetanide- cl secretion more than double
amiloride- Na secretion halved

Question 13

When height of layer is high ?

Answer 13

More Na absorption at steady sate more Cl secretion then Na absorption

Question 14

What happens when height of layer goes up ??

Answer 14

if height of layer goes up cells respond by upregulating ENac function to bring height of layer back down

Question 15

What happens if height of the layer drops ?

Answer 15

cells upregulate chloride secretion

Question 16

Draw the cell model

Answer 16

see lecture notes

Question 17

What is on the apical membrane of an eptihelial channel ?

Answer 17

ENac
CFTR
cilia

Question 18

What is on the basolateral side of the epithelial cell in the airways?

Answer 18

3NA+ out 2K+ in
NA+ 2Cl- K + in (NKcc1)
K+ out

Question 19

Epithelial cell function

Answer 19

Na recycles across basolateral membrane
High CL in cell – open cftr secrete cl
Potassium channels sets driving force of NA across the cell

Question 20

Respiratory Syncitial virus

Answer 20

Respiratory pathogens disturb fluid balance respiratory tract
RSV = nasal congestion
bronchiolitis children
pneumonia adults
Major respiratory pathogen in children
-
RSv inhibits ENac after 1 hour incubation

Question 21

What animal model is used to show the effect of RSV?

Answer 21

Mouse trachea using SCC

Question 22

amiloride before exposure to virus

Answer 22

deflection - shift represents function of the epithelial Na channel

Question 23

exposure to RSV and the amiloride

Answer 23

inhibit ENac - Runny nose liquid

Question 24

3 main pathways of which viruses have an impact on epithelial N channels ?

Answer 24

Activation of PKC
Bind Glycoproteins
Bind to Glycolipids

Question 25

M1 model is a cell model for what

Answer 25

Principle cell line

Question 26

BIM inhibits what

Answer 26

PKC

Question 27

PPMP inhibits what

Answer 27

Inhibits binding to glycolipids

Question 28

M1 cell model and trachea experiment to show how RSV disrupts ENac ?

Answer 28

M1 cell Mouse trachea - addition of BIM so pkc isnt active control vs virus if RSV working through activating PKC in the presence of BIM it cant do that. tells us downstream of the virus and between the virus and ENac is the activation of PKC - 2nd experiment NA inhibits Glycoproteins - no change between RSV on its own and with Na - inhibition still seen- Glycoproteins therefore not important. Final Experiment - M! cells - PPMP inhibits GLycolipids - inhibition lost - therefore RSV binds to glycolipids which activates PKC and inhibits ENac.

Question 29

How does RSV inhibit ENac?

Answer 29

RSV binds to glycolipids which activates PKC and inhibits ENac.

Question 30

Different viruses

Answer 30

work by inhibiting ENac but through different pathways

Question 31

influenza

Answer 31

inhibits ENac Influenza kills ~ 36,000 individuals influenza virion contains lots of glycoproteins- including Matrix protein M1 - Haemagglutinin – binds to sialic acid residues – actn PKC & transient inhibition ENaC

Question 32

M2 in influenza

Answer 32

M2 – forms an acid activated, amantadine inhibited H+ channel, inserted into apical membrane host cell during infection M2 involved long term regulation - inhibits ENac

Question 33

overxpression of M2 and GFP in airway cells

Answer 33

inhibits ENac - impacts on open probability shown from single channel recordings - channels open lots in normal - Open much less in Overexpressio M2 GFP - M2 protein reducing opening probability - Reduced amount of ENac in membrane

Question 34

M2 promotes what process in ENac channels

Answer 34

Endocytosis - when infected | - proven by test on liddles syndrome - dont endocytose well so difference in ENac current not shown

Question 35

Liddles Mutants

Answer 35

Cant be endocytosed easily from the membrane

Question 36

Influenza and M2 mode of action

Answer 36

influenza virus- binding to the cell and introducing the M2 protein

Question 37

What remains down stream of the influenza virus

Answer 37

ROS and PKC shown using RFP control - Green presence of ROS - overexpress M2 with RFP - lots of red where green - increasing ROS in cells - one of the mechanism where M2 inhibits ENac

Question 38

What is one of the mechanisms M2 uses to inhibit ENac?

Answer 38

Increase ROS

Question 39

Glutathione

Answer 39

Antioxidant - oxygen scavenger | - reduces some of the inhibition of M2 on ENac

Question 40

block PKC

Answer 40

block response to M2

Question 41

How does the virus work ?

Answer 41

virus works by partially inserting M2 in membrane stimulate PKC and generate ROS which inhibits ENac

Question 42

Mutations in ENac gives rise to what condition

Answer 42

Pseudo hypoaldosteronism

Question 43

PHA 1

Answer 43

2 forms - autosomal dominant (Renal form) and autosomal recessive

Question 44

PHA1 autosomal recessive

Answer 44

- frequent lower respiratory tract infections - ENac gene mutations in all subunits - multiple organs affected - permanent runny nose - LOF in ENac - Struggle to reabsorb sodium - Less negative potential - Height of ALS higher than optimum – difficult to remove mucos by cilia – increased risk of infection