Lecture 2 - ALS and Influenza Flashcards
What is the ASL
Airway surface liquid layer
Where is the ASL
It sits on top of epithelial cells in the respiratory tract
What is the ASL composed of
Mucous layer
Periciliary layer
What is the PCL important in
Height is important in the clearance of mucous
How is the ASL the first line of defence against respiratory pathogens
The mucous moves up and is swallowed
Describe how there is a volume load in the lungs - what is the effect of this on the control of the ASL
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
What are the two control mechanisms of the ASL
ACTIVE and PASSIVE
Describe the active control of the ASL
Active transport of ions and solutes - therefore water
Comlimentary pathways of ENaC and CFTR (when one is active then the other is inactive)
Describe the passive control of the ASL
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
Describe Pfleugers 2003 experiment looking at the changes in the height of the PCL
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)
What is the optimum height of the PCL for cells in culture
7-7.5 um
What happens to the PCL when the height is too high
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
What happens to the PCL when the height is too low
Cl secretion predominates - upregulation of CFTR - inhibition of ENaC
When the height is at optimum what can be said
There is no net movement of solute and so no net movemebt of water
Why is ENaC required at birth?
Lungs full of water at irth
ENaC upregulation required to remove this (Na+ reabsorption and water follows)
What does looking at %inhibition of the Vte show
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
Describe the results seen when looking at the ASL (starting too high) and Vte in the presence of amiloride
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
Why would bumetranide be used?
Blocker of NKCC1 - if this is blocked the DF for Cl secretion is lost - indirectly blocking CFTR
Describe the results seen when looking at the ASL (starting too high) and Vte in the presence of bumetanide
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
When the height is high
Upregulation of ENaC
When the height is low
Upregulation of Cl secretion through CFTR
Describe the upper airway cell model
APICAL
ENaC (na in)
CFTR (Cl out)
BASAL
Na/K ATPase
NKCC1 (Na 2Cl and K in)
K channel (K out)
Most of the water movement is
PARACELLULAR
What does paracellular mean
Between cells
Give some inherited conditions that arise from the competition between CFTR and ENaC
CF
Atypical CF
PHA type 1
Give some causes of acquired conditions that arise from the competition between CFTR and ENaC
Infections
Smoking
Vaping
Death rates from influenza
UK
USA
12-61K individuals/year USA
600-13k individuals/year UK
10% of hospital conditions are due to
Influenza linked bronchopneumonia and oedema
Acquiired respiratory distress syndrome
Why is influenza being studied in the context of this module
Alters ENaC and CFTR function which then impacts on the ASL
MURINE NASAL PDs - RESPONSE TO PR8
What is PR8
What was recorded
What were the different experimental groups
Influenxa strain
Recorded Vte across the nasal epithelium
Control, 5 days infected, 15 days infected
When amiloride was added what does this shift in potential correlate to
The function of ENaC = a large shift in potential= suggestes ENaC makes a large contribution to Vte
Why is forskolin added
How does it worK?
Added to stimulate the CFTR channels
Activation of cAMP production and activation of PK-A phosphorylation of CFTRs R domain
A large shift in Vte when a blocker was added suggests what?
That this has a large function
What is the amiloride sensitive potential
How big the shift when amiloride added - if closer to zero there is less function
Describe the effect of PR8 of ENaC
At day 5 see inhibition of ENaC
But this shift is short lived
TRANSIENT INHIBITION
Describe the effect of PR8 on CFTR function
Same as before - see stimulation leads to less activation cf the wildtype
Sum up the studies in mice infected with influenza
By day 5 inhibition of CFTR and ENaC are seen
Describe the data obtained from HBE Monolayers with Ussing Data?
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
What are the two properties likely to change during the infection
N - number of channels
Po - the open probability
Describe the technique used to investigate effects on Po
Virus tagged w/ GFP so all virus infected cells are green
Patch clamp indiviudal cells that are green
SINGLE CHANNEL RECORDINGS
What is seen when perofrming single channel recordings on H1N1 infected cells
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
Infection is reducing the _____ of ENaC channeles
Po
What technique was used to look to see if the virus was effecting N number of CFTR and ENaC
Look at expression levels
What is the normal subuint strucutre of ENaC
What other subunit is there and where is this found
aby
delta - lungs
Can alpha ENaC form functional channels?
Yes but the currents recorded fro this are very small
Describe the reuslts of looking at the expression levels of a-ENac/b-ENaC and yENaC, CFTR, a-ATPase and b-ATPase
a-ENac/b-ENaC and yENaC, CFTR, a-ATPase - ALL DOWN
b-ATPase unchanged
What is the effect of H1N1 reducing the number of Na/K ATPase
Na/K ATPase sets up the driving force - by inhibiting the cell has a reduced ability to allow Cl and Na movement
What is the effect of the virus on HBE and mouse on the ASL
ASL reduced
The fact that the height of the layer has dropped in these studies suggests …
There is a biggr impact on CFTR than on ENaC
And that there is more inhibition on CFTR than there is on ENaC
What is the effect of the virus of the ciliary beat frequency
Post infection there is a drop in ASL and the CBF goes down
What problem does reduction of CBF cause
Problems clearing liquid and mucous from the lungs
What is unusual about CBF and ASL post infection
ASL remains low but the CBF frequency has reocvered? Unsure about the mechanisms of this
Infection with H1N1 impacts on CFTR in two ways…
Decrease Po of CFTR and decreased abundance of CFTR in the membrane
What is a possible pharmacological correct for infection with H1N1
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
