Lecture 2 - ALS and Influenza

Question 1

What is the ASL

Answer 1

Airway surface liquid layer

Question 2

Where is the ASL

Answer 2

It sits on top of epithelial cells in the respiratory tract

Question 3

What is the ASL composed of

Answer 3

Mucous layer

Periciliary layer

Question 4

What is the PCL important in

Answer 4

Height is important in the clearance of mucous

Question 5

How is the ASL the first line of defence against respiratory pathogens

Answer 5

The mucous moves up and is swallowed

Question 6

Describe how there is a volume load in the lungs - what is the effect of this on the control of the ASL

Answer 6

Proximal airways have diameter of 2m^2
Distal airways converge in the bronchial region at only 50cm2
So there has to be a way to regulate this height to maintain an optimum level

Question 7

What are the two control mechanisms of the ASL

Answer 7

ACTIVE and PASSIVE

Question 8

Describe the active control of the ASL

Answer 8

Active transport of ions and solutes - therefore water

Comlimentary pathways of ENaC and CFTR (when one is active then the other is inactive)

Question 9

Describe the passive control of the ASL

Answer 9

Mucous is able to act as as reservoir - if the height of the PCL is too high then water is able to move into the mucous layer

Question 10

Describe Pfleugers 2003 experiment looking at the changes in the height of the PCL

Answer 10

Grow epithelial cells in culutre
Addition of liquid to the apical surface to 30um (much too hight)
Over time (in the first 24 hrs or so) the PCL is brought down to the optimum level (7-7.5um)

Question 11

What is the optimum height of the PCL for cells in culture

Answer 11

7-7.5 um

Question 12

What happens to the PCL when the height is too high

Answer 12

Na re absorption predominates - upregulation of ENaC (inhibition of Cl secretion through CFTR)
Net movement of Na+ into the cell (transcellular) leads to water movement out of the ASL by paracellular transport reduciing the height of the airway surface liquid layer

Question 13

What happens to the PCL when the height is too low

Answer 13

Cl secretion predominates - upregulation of CFTR - inhibition of ENaC

Question 14

When the height is at optimum what can be said

Answer 14

There is no net movement of solute and so no net movemebt of water

Question 15

Why is ENaC required at birth?

Answer 15

Lungs full of water at irth

ENaC upregulation required to remove this (Na+ reabsorption and water follows)

Question 16

What does looking at %inhibition of the Vte show

Answer 16

How much an ion channel contributes to Vte

E.g if add amiloride and see a 50% reduction in the Vte then can say that ENaC is responsible for 50% of the Vte

Question 17

Describe the results seen when looking at the ASL (starting too high) and Vte in the presence of amiloride

Answer 17

T=0 then 65% (majority) of Vte from ENaC
Since ENaC must be high to drive Na re absorption to the bring down the height of the ASL
At T=48 height is back to optimum so ENac activity decreases so is balanced with CFTR function

Question 18

Why would bumetranide be used?

Answer 18

Blocker of NKCC1 - if this is blocked the DF for Cl secretion is lost - indirectly blocking CFTR

Question 19

Describe the results seen when looking at the ASL (starting too high) and Vte in the presence of bumetanide

Answer 19

At t=0 activity of CFTR is low - around 20% contribution to Vte
At t=48 this value is higher as balance to maintain optimum height

Question 20

When the height is high

Answer 20

Upregulation of ENaC

Question 21

When the height is low

Answer 21

Upregulation of Cl secretion through CFTR

Question 22

Describe the upper airway cell model

Answer 22

APICAL
ENaC (na in)
CFTR (Cl out)

BASAL
Na/K ATPase
NKCC1 (Na 2Cl and K in)
K channel (K out)

Question 23

Most of the water movement is

Answer 23

PARACELLULAR

Question 24

What does paracellular mean

Answer 24

Between cells

Question 25

Give some inherited conditions that arise from the competition between CFTR and ENaC

Answer 25

CF Atypical CF PHA type 1

Question 26

Give some causes of acquired conditions that arise from the competition between CFTR and ENaC

Answer 26

Infections Smoking Vaping

Question 27

Death rates from influenza UK USA

Answer 27

12-61K individuals/year USA 600-13k individuals/year UK

Question 28

10% of hospital conditions are due to

Answer 28

Influenza linked bronchopneumonia and oedema | Acquiired respiratory distress syndrome

Question 29

Why is influenza being studied in the context of this module

Answer 29

Alters ENaC and CFTR function which then impacts on the ASL

Question 30

MURINE NASAL PDs - RESPONSE TO PR8 What is PR8 What was recorded What were the different experimental groups

Answer 30

Influenxa strain Recorded Vte across the nasal epithelium Control, 5 days infected, 15 days infected

Question 31

When amiloride was added what does this shift in potential correlate to

Answer 31

The function of ENaC = a large shift in potential= suggestes ENaC makes a large contribution to Vte

Question 32

Why is forskolin added How does it worK?

Answer 32

Added to stimulate the CFTR channels Activation of cAMP production and activation of PK-A phosphorylation of CFTRs R domain

Question 33

A large shift in Vte when a blocker was added suggests what?

Answer 33

That this has a large function

Question 34

What is the amiloride sensitive potential

Answer 34

How big the shift when amiloride added - if closer to zero there is less function

Question 35

Describe the effect of PR8 of ENaC

Answer 35

At day 5 see inhibition of ENaC But this shift is short lived TRANSIENT INHIBITION

Question 36

Describe the effect of PR8 on CFTR function

Answer 36

Same as before - see stimulation leads to less activation cf the wildtype

Question 37

Sum up the studies in mice infected with influenza

Answer 37

By day 5 inhibition of CFTR and ENaC are seen

Question 38

Describe the data obtained from HBE Monolayers with Ussing Data?

Answer 38

Two traces one control and one 48 hours post infection CONTROL Drop in SSC when amiloiride is added (shows contribition from ENaC). FSK added and SSC peaks shortly after that then plateaus. Large reduction in SCC when a CFTR blocker is applied 48H INFECTED Initial SSC much lower - reduced drop when amiloiride added, reduced activation of CFTR by FSK and reduced inhibition when inhibitor added

Question 39

What are the two properties likely to change during the infection

Answer 39

N - number of channels | Po - the open probability

Question 40

Describe the technique used to investigate effects on Po

Answer 40

Virus tagged w/ GFP so all virus infected cells are green Patch clamp indiviudal cells that are green SINGLE CHANNEL RECORDINGS

Question 41

What is seen when perofrming single channel recordings on H1N1 infected cells

Answer 41

Non-infected - frequent channel openings Mean data Po around 0.3 for the control Infected - reduced Po and a reduction in the number of opening events REDUCTION IN PO

Question 42

Infection is reducing the _____ of ENaC channeles

Answer 42

Po

Question 43

What technique was used to look to see if the virus was effecting N number of CFTR and ENaC

Answer 43

Look at expression levels

Question 44

What is the normal subuint strucutre of ENaC What other subunit is there and where is this found

Answer 44

aby delta - lungs

Question 45

Can alpha ENaC form functional channels?

Answer 45

Yes but the currents recorded fro this are very small

Question 46

Describe the reuslts of looking at the expression levels of a-ENac/b-ENaC and yENaC, CFTR, a-ATPase and b-ATPase

Answer 46

a-ENac/b-ENaC and yENaC, CFTR, a-ATPase - ALL DOWN b-ATPase unchanged

Question 47

What is the effect of H1N1 reducing the number of Na/K ATPase

Answer 47

Na/K ATPase sets up the driving force - by inhibiting the cell has a reduced ability to allow Cl and Na movement

Question 48

What is the effect of the virus on HBE and mouse on the ASL

Answer 48

ASL reduced

Question 49

The fact that the height of the layer has dropped in these studies suggests ...

Answer 49

There is a biggr impact on CFTR than on ENaC And that there is more inhibition on CFTR than there is on ENaC

Question 50

What is the effect of the virus of the ciliary beat frequency

Answer 50

Post infection there is a drop in ASL and the CBF goes down

Question 51

What problem does reduction of CBF cause

Answer 51

Problems clearing liquid and mucous from the lungs

Question 52

What is unusual about CBF and ASL post infection

Answer 52

ASL remains low but the CBF frequency has reocvered? Unsure about the mechanisms of this

Question 53

Infection with H1N1 impacts on CFTR in two ways...

Answer 53

Decrease Po of CFTR and decreased abundance of CFTR in the membrane

Question 54

What is a possible pharmacological correct for infection with H1N1

Answer 54

Idea to get more channels to the membrane to reverse the drop of the ASL Lumacaftor - correct - causes trafficking of CFTR to the membrane (increases N) Treat infected cells and see that height of the ASL has made somewhat of a recovery Could moleucles that are used to treat CF be used in this case