ASL, influenza and ion channels Flashcards
What impacts on the ASL?
1) Normally physiology - lungs are dynamic in how they deal with ASL
2) Pathogens
What is the problem with the movement of the ASL?
What is needed to overcome this problem?
As the fluid moves up the respiratory tract - small airways converge in the bronchial region and the surface area decreases
–> get a volume load
If lungs don’t do anything, as the fluid moves up the respiratory tract:
- The height of the ASL gets bigger because the surface area is smaller
- Height is no longer optimum
To overcome this problem:
- Have active and passive mechanism under normal physiological conditions to regulate the height of the liquid layer
Describe the height of the ASL in the distal airways?
Same height
What are the proximal airways?
Closer to the external environment (bronchi)
What are the distal airways?
Further away from the external environment (in the alveoli)
Describe the PASSIVE control of the ASL
Mucous layer acts as a RESERVOIR:
- Height of the layer too heigh - excess fluid into mucus layer –> height drop
- Height of the layer too low - liquid from the mucus layer
- -> height increases
Describe the ACTIVE control of the ASL
Active ion transport (and following water) controls the salt level in the PCL:
- Via complimentary ENaC and CFTR
How do CFTR and ENaC interact in the upper airway?
They are COMPLIMENTARY:
- When one is active the other is inactive
What is the optimum of the ASL in VITRO?
7-7.5 microns
What happens in VITRO when increase the height of the ASL to 30 microns?
When does this occur?
The layer of the cells in culture actively DECREASE the height of the layer down the optimum layer
Happens in the first 24hrs
What happens when the height of the ASL is HIGH?
ENaC (Na absorption) PREDOMINATES/upregulated:
- Na out of the PCL
- Water follows (PARACELLUARLY)
- Heigh goes down
What happens when the height of the ASL is LOW/STEADY STATE?
CFTR (Cl secretion) PREDOMINATES/is upregulated:
- Balance between Cl secretion and a little bit of Na absorption maintains the height of the ASL
- No net water movement paracellularly across the epithelium
What happens to the Vte when experimentally increase the height of the ASL too high?
- Increases with the increase of the height of the ASL
- Decreases as the height of the ASL decreases (due to the active mechanisms)
What happens to the Vte of cells when add Amiloride 0h after experimentally increasing the height of the ASL?
48hrs?
At 0hr - 65% decrease in Vte (65% inhibition of Vte)
At 48hr - 30% decrease in Vte (30% inhibition of Vte)
What happens to the Vte of cells when add Bumetanide 0h after experimentally increasing the height of the ASL?
48hrs?
At 0hr - 18% decrease in Vte (18% inhibition of the Vte)
At 48hr - 52% decrease in Vte (52% inhibition of Vte)
What is the function of Amiloride?
Blocks ENaC
What is the function of Bumetanide?
Blocks NKCC1 (on the basolateral membrane of the upper airway epithelial cells)
When add a blocker the the cell and there is a X% decrease/inhibition of Vte what does this show?
That the ion channel that is blocked contributes to X% of the Vte
The bigger the shift in the Vte with the potential - the bigger the function of the blocked protein
What disrupts the balance between ENaC and CFTR function?
Inherited conditions and acquired conditions
What are the inherited conditions that disrupt the balance between ENaC and CFTR function?
- Cystic fibrosis
- Atypical CF
- PHA type 1
What are the acquired conditions that disrupt the balance between ENaC and CFTR function?
- Infections (influenza)
- Smoking
- Vaping?
What are the effects of influenza?
Kills ~ 12K to 61K individuals / year USA
~ 600 to 13K individuals / year UK
10% hospital admissions during flu season are due to:
1) Influenza linked bronchopneumonia & oedema
2) Acquired respiratory distress syndrome
Lung function is compromised so much you need to be hospitalised to enable to get treatment to get oxygen in
How does influenza cause damage to the body?
Alters ENaC and CFTR function –> impacts on ASL level
What is the contradictory data regarding influenza and ASL height?
Different studies show that the height of the ASL in influenza to go up or down
Dependant on many different factors
What was the mice study that displayed the evidence that the influenza viruses impact on ENaC and CFTR
Measurement of murine nasal potential differences in response to the PR8 virus:
- Measure the Vte across the nasal epithelium with a references and recording electrode up the nose of the mouse
Then, add different drugs to see the shift in the Vte:
1) Amiloride
2) Forskkolin
3) GLyH-101
What is the PR8 virus?
An influenza virus that expresses a GFP-NS1 protein
What is the affect on the nasal Vte in mice when Amiloride is added (not in the presence of the PR8 virus) What does this show?
Shift in Vte - indication that the ENaC channel is important in the membrane potential
What does the shift in the Vte when block a channel show?
What does a big shift in this value show?
Shows the function of that channel
- The bigger the shift the bigger the function the channel that is blocked
What is the Na+ dependant Amiloride inhibited NPD?
Describe this value and how it changes over time with infection with PR8
What does this show about the virus?
The shift in Vte when amiloride is added (showing the function of the Na channel)
What is the Na+ dependant Amiloride inhibited NPD at no infection?
Day 5 infection?
Day 10/15 of infection?
What does this show?
No infection (control): - Around -12mV
Day 5 infection:
- Very close to 0mV
- Shows hardly any change in ENaC inhibition when Amiloride is added
- ENaC already inhibited (by the virus)
Day 10/15 of infection:
- Amiloride sensitive NPD increases
- Shows more of the channels to be inhibited by Amiloride (ENaC no longer inhibited by the virus - recovery of function)
–> TRANSIENT inhibition of ENaC with the virus
If add Amiloride to a channel and the Amiloride sensitive NPD is 0, what information does this show about the channel prior to addition of Amiloride?
Shows that the channel is inhibited prior to the addition of Amiloride
What is the affect in the nasal Vte in mice when Forskolin is added (following Amiloride addition)
What does this show?
Opposite shift in the Vte
Shows the activation of CFTR
What is the affect in the nasal Vte in mice when GlyH-101 (CFTR inhibitor) is added (following Forskolin and Amiloride addition)
What does this show?
Shift in Vte
Shows inactivation of CFTR - contributes the the Vm?
What is the Cl dependant GlyH-101 inhibited NPD at no infection?
Day 5 infection?
Day 10/15 of infection?
What does this show?
No infection (control): - About -17mV
Day 5 infection:
- Closer to 0mV
- Shows hardly any change in CFTR inhibition when GlyH-101 is added
- CFTR already inhibited (by the virus)
Day 10/15 of infection:
- Sustained inhibition
What is the effect of of PR8 (influenza virus) on ENaC and CFTR?
By day 5 BOTH ENaC and CFTR channels are inhibited in the upper airway
What other cell is the nasal epithelium a model for?
Bronchial epithelial cells
What is H1N1?
Another influenza virus
What happens to the SCC in HBE cells when Amiloride is added?
SCC falls
What does a decrease in SCC when a channel blocker is added determine?
Determines the function of the ion channel in the cell:
The bigger the drop in SCC - the bigger the function in the cell
What is the function of Forskolin?
What impact does this have on the SCC in the cell?
Stimulates Cl secretion
Increases the SCC with a transient effect - increases to a peak and then reaches a lower steady state
How does Vte relate to SCC?
If the resistance stays the same:
- If Vte increases SCC increases??
- If Vte decreases SCC decreases??
What happens to SCC when CFTR inhibitor is added?
SCC reduces
What is the effect on the SCC mediated by ENaC in infected cells compared to in uninfected cells?
How is this seen?
SCC mediated by ENaC is less than half than what is seen in uninfected cells
Smaller amiloride sensitive SCC
–> shows a reduced function of ENaC
What is Forskolin a measure of?
Cl ion secretion through the CFTR channel
What happens to Forskolin function in the presence of H1N1?
What does this show?
Smaller increase in SCC and loss of transient behaviour is lost compared to in non-infected cells
Shows a reduces function in CFTR (as Forks is a measure of Cl secretion through CFTR)
What does the influenza data show so far?
Evidence that the ENaC and CFTR function are inhibited in the presence of influenza in both:
1) In VIVO - in the mouse
2) In VITRO human cell culture
What is the SCC a measure of?
The sum of:
- Number of channel
- Open probability
- V-Vrev (driving force)
- g
For EACH of the channels looking at?
What are two things that can change SCC in terms of infection?
- Number of channels
- Open probability
How can we distinguish between PR8 infected and P8 non-infected cells?
PR8 with GFP:
- Infected (green - GFP)
- Non-infected (not green)
Describe the Po of ENaC in non-infected cells prior to infection?
Following infection?
- High Po (many openings and closings of the channel
- ENaC channels are open 1/3 (Po is 0.3)
- Following infection - Po is constant?????
What is the conductance of the ENaC channel?
Has 2 different conductances depending on the subunit concentration of ENaC?
- Small conductance (4pS channel)
- Large conductance (18pS channel)
Describe the Po of the infected cells
Is this a transient or permanent change?
Reduced number of opening events –> reduced Po
Transient reduction - increases again after peak low at 7 days
Describe the Po of CFTR in infected and non-infected cells?
In infected cells - Po is REDUCED (from 0.6 –> 0.1)
What are the different subunits of ENaC?
Alpha, Beta and Gamma (also Delta in the lungs)
What is the function of the ENaC alpha subunit?
What is required for the proper function of ENaC?
Alph subunit can form a channel that functions BUT the size of the currents through this subunit alone are very small
So only get normal function of the channel with alpha, beta and gamma subunits to form the channel
What do densitometry recordings of the subunits of ENaC show?
What does this mean?
Compared to the control, there is a REDUCED protein level of the subunits in the membrane
Shows that the number of the channels in the membrane is reduced
What do densitometry recordings of the subunits of CFTR show?
Number of CFTR in the membrane are reduced
What do densitometry recordings of the subunits of the alpha subunit of the ATPase show?
What does this cause?Why?
Reduced protein level in the membrane
Reduces the ability to set up the driving force to allow Na and Cl movement (affects Na and Cl handling)
What ion channels are affected in the membrane by influenza?
- ENaC (Po, number of channels, Rte, SCC)
- CFTR (Po, number of channels, Rte, SCC)
- NaKATPase (number of channels)
What is the impact on ASL in HBE cells in culture when infected with H1N1?
48hr post-infection - height of ASL drops
What is the impact on ASL in murine trachea when infected with H1N1?
4 days post-infection - height of the ASL drops
What does the reduction in the ASL in HBE cells in culture and in the murine trachea suggest?
Why?
Suggest a bigger impact of influenza on CFTR over ENaC (more CFTR inhibition, less ENaC inhibition)
Why?
- As normally, there is a balance between CFTR and ENaC function
- Inhibit CFTR more, ENaC can still function, absorption can still occur and water follows
How do the height of the ASL impact on ciliary beat frequency in HBE at 48hrs?
At 72hrs?
Why?
48hrs:
- Drop in ciliary beat frequency
- Due to the cilia being bent over –> cannot beat
72hrs:
- Height of the layer remains low but the CBF recovered
- Compensation of the cells –> allow cilia to function (even though the height of the layer is down)
What is the impact of a reduced ciliary beat frequency?
Problem clearing liquid and mucus from the lungs
What type of drug is Lumacaftor?
What is the impact on the CFTR channel in H1N1 infected cells?ASL?
A corrector compound
Traffics CFTR to the membrane –> more channels in the membrane –> reduce the decrease of the height of the ASL layer –> increase height –> increase CBF
What is the affect of Lumacaftor in H1N1 uninfected cells?
No effect
