Lecture 03 Salt Absorption II

Question 1

What is the ASL?

Answer 1

airway surface liquid made up of the perciliary layer (PCL) and mucous layer

Question 2

What is the function of the mucous layer?

Answer 2

the first line of defence against infection

the height of the layer is important in terms of clearance

Question 3

What is the function of the cilia?

Answer 3

cilia beat to move the mucous up the airway into the upper tract where is can be swallowed (or coughed)
dependent on the thickness of the liquid layer

Question 4

What is the surface area of the proximal airway?

Answer 4

2m^2

Question 5

What is the surface area of the distal airway (upper respiratory tract)

Answer 5

50cm^2

Question 6

How does the surface area of the respiratory tract create a problem?

Answer 6

the decreasing surface area as you move up the airway but the total volume stays the same the whole way
the thickness of the liquid layer must compensate

Question 7

What 2 mechanisms have been proposed to control the ASL? Which one occurs to maintain the ASL height?

Answer 7

passive and active

a mixture of the two

Question 8

How does the passive mechanism proposed work to control the ASL?

Answer 8

mucous layer acts as a reservoir
when layer is too high, water moves into the mucous layer
when layer is too low, water moves into the PCL
allows optimum ASL height and cilia function to be maintained

Question 9

How does the active mechanism proposed work to control the ASL?

Answer 9

active ion transport controlling the salt level in the PCL
when the layer is too high, ions are reabsorbed by the cells, water follows decreasing the height of the layer
when the layer is too low, ions are secreted into the PCL, water follows increasing the height of the layer

Question 10

What happens when the active pathway is disrupted?

Answer 10

disease states with multiple symtpoms

Question 11

What is the optimal height of the liquid layer?

Answer 11

7 micrometers

Question 12

What did the experiment by Pfleugers Arch in 2003 show?

Answer 12

human-airway epithelium cells
added too much fluid to the cells (30 microns) and added fluorescent indicator to monitor the height
within 24 hours the height of the layer had dropped dramatically
within 48 hours the height of the layer had evened out and maintained

Question 13

Why did they grow cells in an incubator? How did keeping cells in an incubator compare to cells in culture?

Answer 13

moving cells backwards and forwards to simulate physical breathing causing the liquid to move
optimal height cells in culture 7 microns whereas in incubator was 14 microns

Question 14

How does the liquid layer maintain optimal height? What evidence is there to prove this?

Answer 14

to decrease the liquid layer there is increased sodium absorption and decreased levels of chloride secretion
to increase the liquid layer there is increased chloride secretion and decreased levels of sodium reabsorption
use amiloride to block ENaC
use bumetanide (loop diuretic) to block NKCC1
look at the percentage inhibition of the transepithelial potential

Question 15

Why is ENaC critical at birth?

Answer 15

helps to clear the amniotic fluid from the baby’s lungs

Question 16

Name the key channels present in the upper respiratory tract epithelia cell - apical and basolateral, and their function

Answer 16

apical
ENaC - sodium reabsorption
CFTR - chloride secretion

basolateral
Na/K-ATPase - 3Na+ out/2K+ in
NKCC1 - 1Na+:1K+:2Cl- in, uses sodium gradient to drive chloride uptake into the cell
K+ channel - recycle potassium across the BL membrane

Question 17

What is the pathophysiology of the Respiratory Syncytial Virus (RSV)? What are the symptoms?

Answer 17

common respiratory tract infection seen in children
respiratory pathogens disturb fluid balance in the respiratory tract
nasal congestion, bronchiolitis (children), pneumonia (adults)

Question 18

What is the effect of RSV on the function of ENaC? What experimental evidence is there?

Answer 18

RSV inhibits the function of ENaC through a smaller shift in the transepithelial potential
measured transepithelial potential and SCC of mouse trachea under normal conditions
expose trachea to RSV for an hour then retest
measured by looking at the impact of amiloride
smaller size in shift after exposure to the virus
control shows minimal effect on the second addition of amiloride

Question 19

What does inhibition by RSV of ENaC lead to?

Answer 19

inhibition of ENaC results in reduced salt reabsorption
excess liquid in the airway
nasal congestion/runny nose

Question 20

What are the 3 main pathways viruses can inhibit ENaC?

Answer 20

activation of PKC
bind to glycoproteins
bind to glycolipids

Question 21

Which pathway does RSV use to inhibit ENaC? How was this shown experimentally?

Answer 21

PKC and glycolipids

1. BIM - a PKC inhibitor was added. Virus and BIM added causing no effect as the virus cannot activate PKC, therefore PKC is important for RSV
2. add neuraminidase (NA) - a glycoprotein inhibitor. When virus and NA added observe a decrease in ENaC function signifying that glycoproteins not important for RSV
3. M1 cells (model cell line of principle cells) added PPMP - a glycolipid inhibitor. No effect observed when PPMP and RSV added therefore glycolipids are important for RSV

Question 22

What are other examples of viruses that act via what mechanisms?

Answer 22

Influenza (flu) - PKC and glycoproteins

Parainfluenze - glycolipids and ATP release

Question 23

What is the pathophysiology of influenza?

Answer 23

influenza viro contains many glycoproteins including
M1 (matrix protein 1)
haemagglutin - activates PKC and inhibits ENaC
M2 - forms an acid activated, amantadine inhibited H+ channel that inserts into the apical membrane host cell

Question 24

What does the overexpression of M2 cause?

What is the experimental evidence?

Answer 24

inhibition of ENaC through decreased open probability and decreased amount of ENaC protein present

patch clamp technique
control ENaC - one channel open almost all the time but channels open lots
overexpression M2 - many points where no channels are open

western blot technique
overexpression M2 both alpha and beta ENaC subunit bands much fainter compared to control

Question 25

How could M2 overexpression by used therapeutically?

Answer 25

M2 decreases the amount of ENaC in the membrane through endocytosis
Liddle’s patients have a mutation causing too much ENaC to be present and be stuck in the membrane
overexpressing M2 in Liddle’s patients decreases the amount of ENaC in the membrane and decreases the open probability of the channel

Question 26

What downstream effects does M2 have on airway cells? What experimental evidence is there?

Answer 26

Presence of M2 causes high levels of ROS and PKC

Immunofluorescence - GFP tagging ROS and RFP tagging M2 showed the two proteins to be expressed together

ENaC currents with different pharmacological inhibitors - inhibiting ROS (GSH - glutathione) prevented some of the M2 inhibition of ENaC
inhibiting PKC (Go6796) prevented some of the ENaC inhibition by blocking the response of PKC to the M2 protein

Question 27

What is Pseudohypoaldosteronism (PHA)?

Answer 27

inherited condition with a loss-of-function mutation in ENaC

two forms - autosomal dominant and autosomal recessive

Question 28

What is the pathophysiology of the autosomal dominant form of PHA?

Answer 28

kidney symptoms - kidneys affected
salt wasting 
hypotension
hyperkalaemia 
metabolic acidosis 
high renin and aldosterone 
mineralocorticoid receptor gene mutation

Question 29

What is the pathophysiology of the autosomal recessive form of PHA?

Answer 29

systemic form - multiple organs affected
mutation in all subunits of ENaC
frequent lower respiratory tract illnesses

Question 30

What is the pathophysiology of PHA1?

Answer 30

small impact on kidney small increase in liquid layer
big impact on systemic system liquid layer and increased salt present in liquid layer
permanent running nose
nasal passages blocked and infected
high liquid layer with high sodium content
reduced ENaC function causes reduced sodium uptake and increasing water content of liquid layer

Question 31

What is the pathophysiology of PHA2?

Answer 31

no impact on kidney
big impact on systemic system
shows a less negative potential and reduced transepithelial potential