Pathophysiology of ENaC Flashcards

1
Q

The airway surface layer is divided in to

A

1) The true liquid layer

2) The mucous layer

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2
Q

What is the role of the mucous layer?

A

It is the first line of defence against respiratory pathogens

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3
Q

Describe the problem of volume load in the movement of ASL in the lungs

A

The alveoli have a large surface area, however this decreases up the lungs
This means we have the same amount of liquid over 50cm2 rather than 200cm2 up the respiratory tract
This means the height of the ASL would have to rise which would affect cilia movement if it is higher than the optimum and mucous would not be cleared effectively

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4
Q

Describe two ways it is thought that the ASL can be controlled

A

1) Passive - the mucous layer acts as a reservoir e.g. if the height of the liquid layer is too high then water will move in to the mucous layer
2) Active - Active ion transport where the concentration of salt is controlled in the liquid layer which subsequently controls how much water is in the liquid layer

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5
Q

Describe an in vitro experiment which shows the airway surface layer has an optimum height

A

Fluid was added to the top of cells (epithelia) so that the height was higher than the optimum (30ul)
The height of the layer went down over time to 7 ul where the height remained constant

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6
Q

What is the optimum height in vitro?

A

14 ul

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7
Q

What does amiloride block?

A

ENaC

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8
Q

What does bumetanide block?

A

NKCC1

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9
Q

What is the effect of bumetanide?

A

It blocks NKCC1 on the basolateral membrane which inhibits chloride secretion

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10
Q

Describe an in vitro experiment which shows the mechanisms involved in maintaining the optimum ASL height

A

Amiloride and Bumetanide was used and they looked at how much each blocked the Vte at time zero and at time 48 hours
e.g. on the graph bigger bar = bigger inhibition

Amiloride - at time zero amiloride gave 60% inhibition of the Vte which suggests lots of sodium is being reabsorbed so water follows = decrease in height
At 48 hours this had been down regulated

Bumetanide - at time zero only around 18% if the Vte was generated by chloride secretion but after 48 hours bumetanide inhibited around 50% of the Vte

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11
Q

Describe what the experiment with amiloride and bumetanide shows about the control of the ASL

A

When the ALS height is too high than Na+ reabsorption is greater than Cl- secretion
When the ALS height is too low then C;- secretion is greater than Na+ reabsorption
It also shows that the balance between the two is important for maintenance of the ASL height

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12
Q

Why is ENaC important at birth?

A

It is important for lung clearance at birth i.e. amniotic fluid needs to be cleared

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13
Q

Describe the cell model for the cells in the upper airway respiratory tract

A

Apical - ENaC, cilia and CFTR
Basolateral - Na+/K+ ATPase, NKCC1, potassium channel
1) K+ channel and ATPase set up the driving force for Na, 2Cl and K to move in to the cell through NKCC1
2) Na and K recycle = Cl accumulates
3) CFTR opens = cl secretion

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14
Q

What is RSV?

A

Respiratory syncitial virus

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15
Q

What are the symptoms of RSV?

A

Nasal congestion, bronchiolitis in children and pneumonia in adults
Major respiratory pathogen in children = 20% of hospital admissions

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16
Q

Describe the effect of RSV on short circuit current in mouse trachea

A

Mouse was exposed to RSV for an hour before amiloride was added
Before RSV exposure ENaC had a large function but after exposure this was smaller so it seems that RSV inhibits ENaC
So adding RSV reduced the short circuit current
RSV blocks ENaC so blocks Na+ reabsorption so there is excess fluid in the nose

17
Q

How does RSV cause a runny nose?

A

RSV blocks ENaC so Na+ reabsorption is inhibited = more fluid is in the nose

18
Q

What were the three possible mechanisms that RSV could work through?

A

PKC activation (ie inhibits ENaC)
Glycoproteins
Glycolipids

19
Q

Which cell types were used to investigate RSV mechanisms to inhibit ENaC?

A

mouse trachea cells or principal cells

20
Q

Does RSV work though PKC activation and what is the evidence?

A

Yes
SSC was measured with control conditions, with BIM (PKC inhibitor) or BIM and RSV
Hypothesis: if PKC is needed then when RSV is added with BIM, RSV will have no effect on the short circuit current
Results: There was no change in the SSC when BIM was added with RSV
So PKC is needed to be activated to inhibit ENaC

21
Q

Does RSV work through glycoproteins and what is the evidence?

A

No
SSC was measured in control conditions with or without NA which inhibits binding to glycoproteins
Hypothesis: If glycoproteins are needed then adding RSV with NA will give no change in SSC
Results: When NA is added then the virus is still able to act through ENaC as SSC decreases
So Glycoproteins are not involved in a mechanism through which RSV inhibits ENaC

22
Q

Does RSV work through glycolipids and what is the evidence?

A

Yes
SSC is meausured in control conditions or with PPMP which inhibits binding to glycolipids
Hypothesis: When glycoproteins are needed then adding PPMP with RSV will show no change in SSC
Results: PPMP and RSV = no change in SSC
So glycolipids are involved in ENaC inhibition through RSV

23
Q

RSV works through which mechanisms?

A

PKC and glycolipids

24
Q

Influenza works through which two mechanisms?

A

PKC and glycoproteins

25
Q

Parainfluenza works through which two mechanisms?

A

Glycolipids and ATP release

26
Q

How many people does influenza kill per year in the USA?

A

36,000

27
Q

List glycoproteins the influenza particle contains

A

Matrix protein 1
Haemagglutinin
Matrix protein 2

28
Q

What is the effect of haemagluttinin?

A

It binds to sialic acid residues to activate PKC e.g. = short term inhibition of ENaC

29
Q

What did researchers want to know about influenza ane ENaC inhibition?

A

They wanted to know what mediates the long term inhibition of ENaC

30
Q

Describe M2

A

Gets inserted in to the host membrane
It then forms an acid activated, amantadine inhibited H+ channel
This channel is inserted in to the apical cell membrane during infection

31
Q

What was the effect of overexpressing M2 in airway cells?

A

ENaC was inhibited by reducing its Po and reducing how much ENaC there is e.g. western blot data shows a decrease of all three subunits of ENaC
So overall M2 can be said to increase ENaC endocytosis

32
Q

What happens when Liddle’s syndrome ENaC is expressed with M2?

A

M2 does not have the same effect as in the WT because this mutant can not be endocytosed easily

33
Q

Name another way M2 can inhibit ENaC

A

It can inhibit ENaC through reactive oxygen species

34
Q

Describe an experiment which shows that M2 works though ROS

A

ENaC plus GFP-ROS = decreased SSC due to inhibition of ENaC
Then adding GSH (antioxidant) means that the ROS is mopped up and the inhibition of ENaC is somewhat prevented
PKC inhibitors also block the response to M2 so PKC is also important

35
Q

Loss of function in ENaC can cause what?

A

PHA

36
Q

What are the symptoms of PHA1?

A
Salt wasting 
Hypotension 
Hyperkalaemia 
Metabolic acidosis 
High renin and aldosterone
37
Q

What are the two forms of PHA1?

A

AUTOSOMAL DOMINANT - Renal form, localised to the kidney and is due to a mineralocorticoid receptor mutation
AUTOSOMAL RECESSIVE - Systemic form, multiple organs affected, mutation in ENaC and = respiratory tract illness

38
Q

How is the nasal surface liquid affected in PHA1 patients?

A

Patients have a permanent runny nose

They have a higher wet weight (what comes out the nose) which also has a higher Na+ concentration as ENaC is not working

39
Q

Describe the nasal Vte in PHA1 patients and what this means for the ASL

A

Vte is less negative/ more positive in patients with PHA1
This is because there is less Na+ is being reabsorbed so less positive ions are being lost
This means the height of their PCL will be higher so mucous will be cleared slower = increased risk of incfection