L2 - ASL, Influence and Ion Channels Flashcards
Where is the ASL found?
Sits on top of epithelia cells in the respiratory tract
Epithelia cells either
- Upper airway cells
- Alveolar cells
What is the structure of the upper airway?
Perciliary layer sits on top of the bronchial epithelia cells
Cilia project from the bronchial epithelial cells
What do cilia do in the upper airway?
They beat constantly - moves ASL and mucus up respiratory tract
First line of defence against infection/respiratory pathogens
What happens if the height of the ASL is disrupted?
Effects your ability to clear pathogens
Pathogens them themselves have an impact on the ASL
Why do the lungs have to be dynamic in how they control the ASL?
Proximally - small airways –> 2m2
Problem - small airways converge in bronchial region –> 50cm2
- As fluid moves up respiratory tract the height of the ASL increases as surface area decreases
- This is why the body needs to have control mechanisms
Distally - upper respiratory tract
- In this region there are mechanisms to control the height of the ASL - allows us to fight infection
- Active and passive mechanisms
What are the passive mechanisms to control the ASL?
Mucous layer acts as a reservoir
Excess fluid moves into the mucous layer or moves out if too little fluid
What are the active mechanisms to control the ASL?
Active ion transport controlling salt level in periciliary layer
- Water follows the ion movement
- Movement of water is mostly between cells –> paracellular
- Where ENaC and CFTR play a role
- When one ion channel pathway is active, the other pathway is inactive
Which pathway is more important the active or passive?
Active
What happened when they grew upper airway epithelial cells in culture and added liquid to the apical surface of the cells to a height of 30 microns?
Cells in culture actively decrease the height of ASL to optimum over time
Most of it happens within the first 24 hours
What is the optimum ASL height in vitro?
7-7.5 microns
What happens when ASL is too high?
Na absorption dominates –> ENac activity upregulated
What happens when the ASL is at steady state?
Cl secretion dominates –> CFTR activity upregulated
Need a balance between Na absorption and Cl secretion
- No net water movement across epithelium
What channel is important in lung fluid clearance at birth?
ENaC
How did they test that ENaC and CFTR are involved in the height of the ASL?
Set high ASL height in cultured cells and monitored over time
What happened when they het high ASL height in cultured cells and monitored over time?
ASL height over time decreased
Transepithelial potential decreased
What happened when they het high ASL height in cultured cells and monitored over time after adding amiloride and bumetanide?
Looked at transepithelial potential shift
The bigger the shift in the potential –> the bigger the function of the blocked channel
Drop in transepithelial potential –> drop in channel function
Amiloride - blocks ENaC
- At 0h - nearly 70% inhibition - shows ENaC important
- At 48h - drop to 30% inhibition - ENaC more important when ASL high
Bumetanide - blocks NKCC1 - blocks Cl secretion
What channels are active when ASL is high?
ENaC function high/upregulated
What channels are active when ASL is steady?
ENaC function drops and NKCC1 upregulated
What channels are active when ASL is low?
NKCC1 function high/upregulated
What inherited mutations are there in ENaC or CFTR?
Cystic Fibrosis - treatments that might target ENaC
Atypical cystic fibrosis - either 1 or no CF gene mutations
PHA type 1 - pseudohypoparathyroidism
What acquired mutations are there in ENaC or CFTR?
Infections
Smoking
Vaping
How many people does influenza kill a year?
12 – 61 thousand a year USA
600 -13 thousand a year UK
10% hospital admissions during flu season are due to?
Influenza linked bronchopneumonia and oedema
Acquired respiratory distress syndrome
- Lung function impaired so much –> can’t get oxygen in –> hospitalised
What channels does influenza impact?
Alters ENaC and CFTR function
This means the ASL is also impacted
What experiment was used to look at the response to the influenza virus PR8?
Measured transepithelial potential across the nasal epithelium in mice
Overall in mice infected with influenza by day 5 both ENaC and CFTR are both inhibited in the upper airways
What happened when they added amiloride to nasal epithelium in mice?
ENaC antagonist
The bigger the shift in transepithelial potential - the bigger the function of ENaC
Small shift in transepithelial potential - only 1 mV (compared to 10 mV in control)
This ENaC inhibition is transient - even if infection is still present
What happened when they added forskolin to nasal epithelium in mice?
CFTR agonist
Shift in transepithelial potential
What happened when they added GLYH-101 to nasal epithelium in mice?
CFTR antagonist
Shift in transepithelial potential
HBE Ussing data after H1N1 infection?
ISC (uA/cm2) against time
Overall in infected cells - reduced ENaC and CFTR function
It has been shown both in mouse and human model in cell culture
HBE Ussing data after H1N1 infection - amiloride
Inhibit ENaC
Control cells – Isc falls - 27 shift
Infected cells – Isc falls - 10 shift
HBE Ussing data after H1N1 infection - forskolin
Activate CFTR
Control cells - has a transient effect - increase in Isc - 40 shift
Infected cells - transient effect gone - increase in Isc - 10 shift
HBE Ussing data after H1N1 infection - CFTR inhibitor
Inhibit CFTR
Control cells - Isc falls - 22 shift
If currents are smaller what could have caused this change?
Channel number
Open probability
How did they test if the changes in ENaC when exposed to Influenza were to do with open probability?
Patch clamp technique to look at single channel recordings of ENaC
- Infected cells glow green (express GFP)
- Able to patch green and non-green cells to allow direct comparison
What did the discover about ENaCs open probability upon Influenza infection?
Transient decrease in open probability
By day 5 there is a further reduction in open probability
Infection reduces likelihood of Na epithelial channels being open
How did they test if the changes in CFTR when exposed to Influenza were to do with open probability?
Patch clamp technique to look at single channel recordings of CFTR
What did the discover about CFTRs open probability upon Influenza infection?
Reduced open probability
How did they test if the changes in ENaC/CFTR when exposed to Influenza were to do with the number of channels?
Densitometry experiments
- How much expressed relative to control
What did the discover about the number of ENaCs/CFTRs upon Influenza infection?
Apical membrane abundance of both ENaC and CFTR reduced
Also impact of Na/K ATPase – only alpha subunit
Overall how does Influenza impact ENaC and CFTR?
Both open probaility and number of channels are reduced upon infection of influenza virus
Virus targets the channels to impact on ASL regulation
When the height of the ASL drops which channel is most affected?
Bigger impact on CFTR than ENaC
Important balance between the two
- If you inhibit CFTR more, ENaC can still absorb some Na so height of layer drops
What happens to the cilia when the height of the ASL drops?
Cilia beat frequency decreased
Height drops so cilia bend
Issues clearing mucous and liquid from lungs
At 72 hours post infection cilia start functioning again even though height of ASL is still low
What is Lumacaftor?
A corrector
What role does Lumacaftor have in controlling the ASL?
Traffics CFTR to the membrane
- Increases number of CFTR channels in the membrane
In infected cells - reverses some of the decrease in the ASL
- Not a full recovery
In non-infected cells – slight decrease in ASl
If a patient has influenza with low ASL what drugs could be tested?
Other drugs/molecules used to treat CF may also have therapeutic potential to treat ill patients with influenza infection
In an in vitro test the function CFTR was determined by measuring the CTFR inhibitor172 sensitive Isc. Isc was measured in the absence and presence of Vape liquid. In the control cells Isc was 25.4 uA. In the presence of the vapour Isc was 7.1 uA. Describe the impact of the vapour on CFTR and explain the impact this has on upper airway cell function.
Vapour reduced Isc either through open
- Evidence that the vapour Inhibits CFTR
- Balance between ENaC and CFTR altered
Reduced CTFR function means less Cl secretion
Less Cl secretion means less water secretion
- Water moves between cells from ASL into basolateral side
Means ASL will reduce in height
Cilia are bent over so cannot beat cilia beat frequency goes down –> reduced mucous clearance –> increased chance of infection
Depending on how often a person vapes more respiratory tract infections seen
As CFTR inhibits ENaC – might lead to over-activation of ENaC exacerbating the symptoms
