Lecture 03 Salt Absorption II Flashcards
What is the ASL?
airway surface liquid made up of the perciliary layer (PCL) and mucous layer
What is the function of the mucous layer?
the first line of defence against infection
the height of the layer is important in terms of clearance
What is the function of the cilia?
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
What is the surface area of the proximal airway?
2m^2
What is the surface area of the distal airway (upper respiratory tract)
50cm^2
How does the surface area of the respiratory tract create a problem?
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
What 2 mechanisms have been proposed to control the ASL? Which one occurs to maintain the ASL height?
passive and active
a mixture of the two
How does the passive mechanism proposed work to control the ASL?
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
How does the active mechanism proposed work to control the ASL?
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
What happens when the active pathway is disrupted?
disease states with multiple symtpoms
What is the optimal height of the liquid layer?
7 micrometers
What did the experiment by Pfleugers Arch in 2003 show?
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
Why did they grow cells in an incubator? How did keeping cells in an incubator compare to cells in culture?
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
How does the liquid layer maintain optimal height? What evidence is there to prove this?
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
Why is ENaC critical at birth?
helps to clear the amniotic fluid from the baby’s lungs
Name the key channels present in the upper respiratory tract epithelia cell - apical and basolateral, and their function
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
What is the pathophysiology of the Respiratory Syncytial Virus (RSV)? What are the symptoms?
common respiratory tract infection seen in children
respiratory pathogens disturb fluid balance in the respiratory tract
nasal congestion, bronchiolitis (children), pneumonia (adults)
What is the effect of RSV on the function of ENaC? What experimental evidence is there?
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
What does inhibition by RSV of ENaC lead to?
inhibition of ENaC results in reduced salt reabsorption
excess liquid in the airway
nasal congestion/runny nose
What are the 3 main pathways viruses can inhibit ENaC?
activation of PKC
bind to glycoproteins
bind to glycolipids
Which pathway does RSV use to inhibit ENaC? How was this shown experimentally?
PKC and glycolipids
- 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
- add neuraminidase (NA) - a glycoprotein inhibitor. When virus and NA added observe a decrease in ENaC function signifying that glycoproteins not important for RSV
- 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
What are other examples of viruses that act via what mechanisms?
Influenza (flu) - PKC and glycoproteins
Parainfluenze - glycolipids and ATP release
What is the pathophysiology of influenza?
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
What does the overexpression of M2 cause?
What is the experimental evidence?
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
How could M2 overexpression by used therapeutically?
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
What downstream effects does M2 have on airway cells? What experimental evidence is there?
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
What is Pseudohypoaldosteronism (PHA)?
inherited condition with a loss-of-function mutation in ENaC
two forms - autosomal dominant and autosomal recessive
What is the pathophysiology of the autosomal dominant form of PHA?
kidney symptoms - kidneys affected salt wasting hypotension hyperkalaemia metabolic acidosis high renin and aldosterone mineralocorticoid receptor gene mutation
What is the pathophysiology of the autosomal recessive form of PHA?
systemic form - multiple organs affected
mutation in all subunits of ENaC
frequent lower respiratory tract illnesses
What is the pathophysiology of PHA1?
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
What is the pathophysiology of PHA2?
no impact on kidney
big impact on systemic system
shows a less negative potential and reduced transepithelial potential
