L3

Question 1

what does ASL stand for?

Answer 1

airway surface liquid

- made up of periciliary layer and mucous layer

Question 2

what is the PL and why is this important?

Answer 2

periciliary layer - the layer of ASL which the cilia project into
- the height of the PL is important as it is required to be at a certain height for cilia to beat and clear mucous efficiently

Question 3

what is the convergance issue?

Answer 3

As you move up the airways SA decreases - is a mucb larger SA in alveoli than the bronchus

• the height of the PL should be consistent in all cells - so when move u the airways get and accumulation of ASL as more mucous/liquid is required lower in the airways to cover large SA
• this excess liquid needs to be reabsorbed as move up airways, as otherwise ASL/PL would be too high

Question 4

how can ASL be controlled?

Answer 4

Passive - musouc layer acts as a resovoir

Active - active ion transport controlling salt level in PCL

Question 5

what role does ENaC have in newborn babies?

Answer 5

Newborn baby lung clearance - Absorb liquid with Na+ (ENaC stimulates Na+ absorption

Question 6

what is the optimum PCL height in the lungs and in cultured h epithelial cells?

Answer 6

• in culture - 7um (2 exp showed this)
• in vivo/lungs - 14um
  lungs are not static which stimulates extra secretion due to shear stress
• cells in culture are static so do not undergo sheer stress

Question 7

what transport / channels function to determine the height of the liquid layer?

Answer 7

• at high ASL levels, ENaC has high function to absorb Na which stimulates H2O absorption
• as optimum height, Cl- secretion predominates effect of ENaC - via NKCC1

Question 8

what balance determines the height of the liquid layer??

Answer 8

The balance between Na+ absorption and Cl- secretion in the epithelial cells of the airway
- anything that disrupts Na+ abs or Cl- secretion disrupts the height of the ASL as it disrupts water movement

Question 9

What does bumetanide do?

Answer 9

blocks NKCC1

Question 10

what does NKCC1 do and where is it?

Answer 10

NKCC1 transports Na, 2Cl and K into the cell via the basolateral membrane
- NKCC1 is on the basolateral membrane of most epithelial cells - e.g. upper airways

Question 11

What is the main channel where Cl- secretion occurs through?

Answer 11

CFTR (on apical membrane of cell)

Question 12

what is the cell model of the upper airways?

Answer 12

• apical - CFTR (Cl- secretion)
• apical - ENaC (Na+ absorption)
• basolateral - Na/K ATPase (3Na out of cell, 2K into cell)
• basolateral - NKCC1 (Na, 2Cl and K into cell)
• basolateral - K+ secretion

Question 13

What happens to Cl secretion when bumetanide is added?

Answer 13

Bumetanide inhibits NKCC1 - this creates driving force for Cl- secretion as it brings Cl into the cell
- When bumetanide added Cl- secretion is inhibited

Question 14

how do ENaC and Cl- drive water?

Answer 14

They drive water movement in opposite directions

- water moves paracellularly

Question 15

what does RSV stand for?

Answer 15

respiratory syncytial virus

- major resp pathogen - especially in children

Question 16

What channel does RSV affect and how was this shown?

Answer 16

Measuring SSC(Vte) and I amiloride in mouse trachea

• RSV inhibits ENaC - shown as response to amiloride much smaller when RSV present and is a shift in Vte - becomes more positive as less Na+ is being absorbed into cell
• This means less Na+ is absorbed by cell in presence of RSV - less water uptake from liquid layer - more liquid in airway - why get a running nose

Question 17

what components are important for RSVs effect on ENaC and how was this shown?

Answer 17

PKC and glyolipids are important

• If PKC is blocked (by bim), inhibition of ENaC doesn’t occur
• when virus is inhibited from binding to gycolipids (PPMP) - lose inhibition of ENaC
• likely that RSV binds to glycolipids on cells, and PKC is activated downstream

Question 18

How was if shown that glycoproteins ARE NOT important in RSVs effect on ENaC?

Answer 18

When gycoprotiens are inhibited (NA), still is inhibition of ENaC by RSV- RSV doesn’t need glycoprotein to have an effect

Question 19

What are inhibitors of PKC and glycolipids?

Answer 19

PKC inhibitor - BIM

glycolipid inhibitor - PPMP

Question 20

what components does influenza need to have an effect on cells?

Answer 20

glyoproteins

PKC

Question 21

what glyoproteins does the infuenza virion contain?

Answer 21

• M1
• M2
• haemagglutinin
• neuraminidase

Question 22

what does haemagglutinin do in influenza?

Answer 22

• activates PKC and transiently inhibits ENaC

Question 23

what does M2 do in influenza?

Answer 23

forms acid activated, amantadine inhibited, H+ channel in the apical membrane - long term regulation of ENaC

Question 24

How does M2 contribute to the inhibition of ENaC and it’s current?

Answer 24

• M2 promoting the endocytosis of ENaC
• when M2 present is less ENaC in the membrane - shown by western blot compared to when M2 is present
• fewer openings of ENaC as less ENaC in membrane

Question 25

what happens when the lid ENaC mutant is used?

Answer 25

lid ENaC mutant - ENaC stuck in the membrane ( cannot be endocytosed) - there is no difference between IENaC when M2 is present or absent (M2 cannot endocytosis the lid mutant of ENaC)

Question 26

What does the endocytosis of ENaC by M2 require?

Answer 26

PKC and ROS - When add antioxidant (GSH) inhibition of ENaC is smaller - When PKC inhibitors added e.g. BIM - inhibition of ENaC is less

Question 27

what can be caused by loss of function of ENaC?

Answer 27

PHA - pseudohypaldosteronism

Question 28

what are the 2 forms of PHA1?

Answer 28

- systemic - autosomal recessive form - effects multiple organs - all ENaC subunits mutated - renal - autosomal dominant - effects kidneys

Question 29

Give some features/symptoms of PHA

Answer 29

- salt wasting (inactive ENaC) - hypotension - increased urine flow rate - hyperkalaemia - acidosis - high renin and aldosterone

Question 30

What was shown by monitoring PHA1 patients nasal surface liquid?

Answer 30

- in systemic form is more wet weight and an increase in [Na+] in the liquid from nose. - is a decrease in Vte in systemic patients - % inhibition with amiloride decreased in systemic patients as ENaC function is reduced - less functional ENaC in membrane of PHA1 cellsq

Question 31

why does ENaC not reach the membrane in PHA1 cells?

Answer 31

- ENaC is mutated so this is recognised and ENaC is degraded via ENaC - why doesn't reach membrane - less ENaC in membrane - less Na+ reabsorption - less H2O reabsorption - water moves into lumen through cells - higher liquid layer - higher risk of infection