Lecture 3 Flashcards
Airways Surface Liquid layer
- Clearance mucous
- airways defence against respiratory pathogens
What is the Airways surface layer also known as ??
Periciliary layer
What is most important about the ASL/PCL?
Height is important for mucous clearance
what happens to the volume of the ASL as you go up the respiratory tract ?
As the respiratory structure gets larger the surface area goes down, so the volume decreases as you go up the respiratory tract
What is important about the height of the ASL/PCL?
Height of layer too low or high impacts the ability of lungs to move mucous
What are the to types of control of the ASL ??
- Passive – mucous layer acts as a reservoir
* Active – Active ion transport controlling salt level in pcl
how do you test the effect of height on theASL/PCL?
Culture human airway epithelial cells - made liquid layer higher optimum
- cells move it to the optimum - 7 microns
- optimum in the lab is 14 microns - moving the dish back and forth to imitate movement of breathing in and out
Bumetanide
loop diuretic NKCC1 block CL secretion
Amiloride blocks what
ENac
Why is bumetanide used ?
to see how much Cl secretion is occurring
change in transepithelial potential tell us what
mechanism of epithelial transport changes over time- upreg or down reg
- transport properties change
bumetanide / amiloride experiment
at 48 hours
bumetanide- cl secretion more than double
amiloride- Na secretion halved
When height of layer is high ?
More Na absorption at steady sate more Cl secretion then Na absorption
What happens when height of layer goes up ??
if height of layer goes up cells respond by upregulating ENac function to bring height of layer back down
What happens if height of the layer drops ?
cells upregulate chloride secretion
Draw the cell model
see lecture notes
What is on the apical membrane of an eptihelial channel ?
ENac
CFTR
cilia
What is on the basolateral side of the epithelial cell in the airways?
3NA+ out 2K+ in
NA+ 2Cl- K + in (NKcc1)
K+ out
Epithelial cell function
Na recycles across basolateral membrane
High CL in cell – open cftr secrete cl
Potassium channels sets driving force of NA across the cell
Respiratory Syncitial virus
Respiratory pathogens disturb fluid balance respiratory tract
RSV = nasal congestion
bronchiolitis children
pneumonia adults
Major respiratory pathogen in children
-
RSv inhibits ENac after 1 hour incubation
What animal model is used to show the effect of RSV?
Mouse trachea using SCC
amiloride before exposure to virus
deflection - shift represents function of the epithelial Na channel
exposure to RSV and the amiloride
inhibit ENac - Runny nose liquid
3 main pathways of which viruses have an impact on epithelial N channels ?
Activation of PKC
Bind Glycoproteins
Bind to Glycolipids
M1 model is a cell model for what
Principle cell line
BIM inhibits what
PKC
PPMP inhibits what
Inhibits binding to glycolipids
M1 cell model and trachea experiment to show how RSV disrupts ENac ?
M1 cell
Mouse trachea - addition of BIM so pkc isnt active
control vs virus
if RSV working through activating PKC in the presence of BIM it cant do that.
tells us downstream of the virus and between the virus and ENac is the activation of PKC
- 2nd experiment
NA inhibits Glycoproteins - no change between RSV on its own and with Na - inhibition still seen- Glycoproteins therefore not important.
Final Experiment - M! cells -
PPMP inhibits GLycolipids
- inhibition lost - therefore RSV binds to glycolipids which activates PKC and inhibits ENac.
How does RSV inhibit ENac?
RSV binds to glycolipids which activates PKC and inhibits ENac.
Different viruses
work by inhibiting ENac but through different pathways
influenza
inhibits ENac
Influenza kills ~ 36,000 individuals
influenza virion contains lots of glycoproteins- including Matrix protein M1 -
Haemagglutinin – binds to sialic acid residues – actn PKC & transient inhibition ENaC
M2 in influenza
M2 – forms an acid activated, amantadine inhibited H+ channel, inserted into apical membrane host cell during infection
M2 involved long term regulation
- inhibits ENac
overxpression of M2 and GFP in airway cells
inhibits ENac
- impacts on open probability shown from single channel recordings
- channels open lots in normal
- Open much less in Overexpressio M2 GFP
- M2 protein reducing opening probability
- Reduced amount of ENac in membrane
M2 promotes what process in ENac channels
Endocytosis - when infected
- proven by test on liddles syndrome - dont endocytose well so difference in ENac current not shown
Liddles Mutants
Cant be endocytosed easily from the membrane
Influenza and M2 mode of action
influenza virus- binding to the cell and introducing the M2 protein
What remains down stream of the influenza virus
ROS and PKC shown using RFP control
- Green presence of ROS
- overexpress M2 with RFP - lots of red where green - increasing ROS in cells
- one of the mechanism where M2 inhibits ENac
What is one of the mechanisms M2 uses to inhibit ENac?
Increase ROS
Glutathione
Antioxidant - oxygen scavenger
- reduces some of the inhibition of M2 on ENac
block PKC
block response to M2
How does the virus work ?
virus works by partially inserting M2 in membrane stimulate PKC and generate ROS which inhibits ENac
Mutations in ENac gives rise to what condition
Pseudo hypoaldosteronism
PHA 1
2 forms - autosomal dominant (Renal form) and autosomal recessive
PHA1 autosomal recessive
- frequent lower respiratory tract infections
- ENac gene mutations in all subunits
- multiple organs affected
- permanent runny nose
- LOF in ENac
- Struggle to reabsorb sodium
- Less negative potential
- Height of ALS higher than optimum – difficult to remove mucos by cilia – increased risk of infection
