Salt secretion Flashcards
2 Different types of pathways across epithelial
- Transcellular= across cells- transport protein
- paracellular= between cells
Polarity of cells
All epithelial cells are polarised (apical and basolateral membrane)
Where are tight and leaky junctions?
Leaky
- proximal tubule
- gall bladder
- small intestine
- choroid plexus
Tight
- distal tubule
- stomach
- frog skin
Difference between tight and leaky
leaky <200 cm^2
tight > 2000 ohms cm^2- high resistance, less transport
What determines a tightness>
paracellular determines resistance numbers= resistance transcellular= same in tight and leaky - depends on DF for absorption and secretion - size of hole= how much can transport
General properties of Tight junction
Rte >2000
Vte~50mv
Flux small
low H20
Genereal properties of leaky junction
rte <200
vte ~0mv
Flux large (isosmotic )
high H20 permeability
Transepithelial potential
Sum of individual membrane potentials
How Is a VT generated?
Use frog skin- sodium resabsorption
epithelium= measure of how much conductance there is
- vte= more anions
+ vte= less anions, more cations
if there is a higher resistance
Less transport
Preparations to make to measure VT
type of cell- fresh tissue/cells, cultured, whole organisms
experimental approach- WT, KO, mutant
pharmacological agents
Overexpression proteins in cells
What method would you use to show protein presence/ location?
presence= western blot location= immunostaining
Method to find mRNA presence?
polymerase chain reaction
Method to find the transport function?
flux radioactive compounds
electrophysiology
Different methods of electrophysiology and what they measure?
intracellular microelectrode- Ic potential
patch clamp- single current/ cell current
2 electrode voltage clamp- cell current
ussing chamber- transepithelial potential and resistance
Ussing chamber- VTE, RTE, ISc method
- inject a known concentration
2. the resulting shift in vte is measured
What happens to Vte if there is more anions/ less cations?
amiloride blocks Na channels
less Na transport
reduced loss pos charge
Vtw decrease due to loss of Na
What is the model tight epithelium
Frog skin
absorb Na from environment
outside= apical
inside= basolateral
Why do we use frog skin?
- robust, easy to get, cheap and useful bc of how it absorbs Na
What can frog epithelium be used for to compare with humans?
- collecting duct distal colon salivary gland sweat duct airway surface epithelium
Short circuit current technique
Amp- 2x electrodes to measure the vte
- kerb solution in chambers either side if looking at Na uptake
- set so no conc gradient - no pot difference
- Add frogs skin- pot difference starts as tight junction
- Na lost from basolateral to apical= neg potential
- validation= uses radioactive tracer (Na )into chamber 1 and measure amount in chamber 2 to work out the amount of Na transport per unit of time
What is the flux of passively and actively transported ions
0- passive- Jnet Cl=0
isc= J net Na = 10.4 mol/s
To calculate the total current
using ussing chamber
did ohms law AND worked out current they would expect to see for Na
Na influx and outflux
in= apical to basolateral Out= basolateral to apical
If you take influx - outflux =
similar to ussing chamber total current net transport
net= almost all Na, apical to basolateral
Active Na reabsorption by
Na/K ATPase- active
K recycling by
passive, K channel- wont be able to find using ussing chamber
What does Na channel on the apical membrane help with and how is this seen?
Move membrane potential to nerst for Na
took electrode and measured Na conc change from outside cell- nerst to Na
If apical membrane is permeable to Na- nerst=Na
What is the effect of amiloride on epithelial Na transport?
Add amiloride, decrease lumen -ve voltage due to decrease to Na absorption
- shifts potential from -1 to 0
- blocks Enac channels
What did they use to measure amiloride on epithelial Na transport?
Human colon biopsy
Ussing chamber work
* note much lower pd than in frog skin = -vte - not as tight, leak back
Human= -1mv not large Vt (Na absorption)
How to get a human nasal biopsy and why is it used?
less invasive than airway epithelium (easy to scrape out )
properties the same
- Na absorption
- Cl secretion
What is Vm
Cell potential
What is the evidence that there are Na selective channels in the apical membrane of the epithelium airway- What does Na entry do?
Tends to depolarise cell
Add amiloride it hyperpolarises cell
change potential- and move nerst away from sodium
What did we know before the patch clamp analysis of Enac?
looked at whole cell epithelium or whole membrane- predict apical Na channel
all indirect evidence add up to this point
predicted opening and closing
What does the patch clamp analysis of Enac (1970) show?
revolutionised ability to look at single ion channels and how they are impacted
What does flickering currents and size deflections show?
single ion channel opening
difficult to read as you get level of noise
What happens to size deflections as you increase the amount of Na?
More Na through ion channel the bigger the current recoreded
molecular approaches
Gene to identify protein
introduce mutation
wanted to clone epithelium ion channel- couldn’t do it
Conventional cloning
Isolate protein and sequence
using amiloride affinity column
1. column with beads with amiloride on
2. run protein down bead and they stick (Enac and amiloride)
Why couldn’t they do normal cloning?
Amount of protein in upper airway/ biopsy is too small
need large amounts
What is the native tissue and what is the native channel properties ?
kidney/ colon
- single channel= spc
- highly amiloride sensitive
- highly Na sensitive
Investigation using tissue source from rat colon
rats fed with low salt diet- fractioned colon mRNA (poly A RNA)
- cDNA cloned in expression vectors
- cDNA clibrary divided into 10 pools (Na sensitive current)
- DNA
- cRNA
- Fractional assay- amiloride sensitive Na current and produces positive pool
- divide into smaller pools until small= no current- chopped off bit that codes for ion channel
- chop into 10 pools- purify more- final pool should be pure sequence that codes ion channel
screening by functional expression
mRNA from cell and chop and inject into oocyte
see which chop of mRNA made Na selective current
What induces Enac
via increased aldosterone
results from functional expression experiment?
Bigger deflection= bigger function
pure clone gives smaller deflection than clone A
- show A subunit can make a channel- need others for full activity A+ B+Y= 10 Fold larger= enhancement
Why might they not have been able to discover this?
If all subunits were needed for channel activity Enac would not have been cloned
What is Enac
3 gene sequence that codes 3 subunits- a,b,y
Mutations in Enac
GOF
- liddles
- hypertension
- Nedd4- channel retrieval
LOF
- PHA
- pseudohypoaldosteronism
- hypotension
- Enac intact
What is barters syndrome and Gitelmans?
loop diuretic NA/CL/K sensitive
Nacl sensitive
What is ASL
Airway surface liquid
How is mucous cleared?
virus gets stuck in mucous and up respiratory tract
airway defence against respiratory pathogens
How does mucous move?
Cilia beat
Epithelial cells
Control amount salt reabsorbed and height of layer
PCL
Percilliary layer= height important mucous clearance
liquid sits above the apical calles
Movement of ASL
Moves from proximal to distal
small airways- upper respiratory tract
* small airways converge in bronchial region ~50 therefore volume load
Control of the ASL
Active- active ion transport controlling salt level in PCL
Passive- mucous layer acts as a reservoir
Experiment to look at control of ASL in cultured human activity
Add liquid to top of cells (make too high)
monitor height overtime using fluorescence
1 day- height decrease
2 day– steady out to optimum height
transepithelial drops from 14 to 5
How to mimic the ASL experiment In labs?
move cells back and forward
liquid moves mimicking breathing
optimum= 14 micrometres
Why are butamide and amiloride used?
b- investigates amount of cl secretion going on
amiloride- plays a role in transport in cell
What are the results after 48hours?
amiloride inhibits 30% but butamine inhibits 55%
shows to get optinmum height you need more Cl secretion and less Na- balance between Na/Cl
What is Cl mediated by?
CFTR
- high cl inside
- open CFTR it secretes Cl
Respiratory syncytial virus (RSV)
Respiratory pathogens disturb fluid balance respiratory tract
- nasal congestion
- bronchiolis in children
- pneumonia in adults
RSV and SSC in mouse trachea
Investigate the response to amiloride
add amiloride before virus- deflection before shift
add after virus- inhibits enac function
What does the bigger deflection bar show?
bigger enac function
virus= transmission decreased
no impact= no virus
RSV inhibits Enac after 1 hour incubation
What happens if you block Enac?
block Na uptake
excess fluid in nose
runny
What are the 3 mechanisms for blocking Enac by the virus?- depends on the virus used
- PKC
- Glycoprotein
- Glycolipid
RSV which pathway does it use
BIM= PKC inhibitor
NA= neuominidase- inhibits binding to glycoprotein
PDMP= inhibits binding to glycolipid
If virus needs one of these pathways you would expect to see a high bar amiloride action (amiloride cant work/ inhibit ENAC) in the presence of the inhibitor and RSV
*RSV uses glycolipid and PKC
approaches of RSV, influenza and parainfluenza
- RSV= PKC, Glycolipid
- Influenza= PKC, glycoprotein
- parainfluenza= Glycolipids, ATP release
What is influenza?
glycoproteins and PKC
influenza kills ~ 36,000 individuals/ year USA
affects young and old
long term
influenza contains many glycoproteins
- MI- matrix protein
- heamagglutinin - binds salic acid residue, PKC and transient inhibition Enac
- M2- forms activated amantadine and H channel which inserts into the apical membrane of most cells
What is M2 glycoprotein?
long term inhibition of Enac in flu
M2 decrease enac currents
normal- channel open lots
influenza- lots of periods where channels closed= decrease open probability, reduces how much there is
How does M2 affect enac?
decreases amount of enac
the ones that are there have a decreased open probability
Endocytosis of enac when M2 is there?
Enac+ m2= enac internalised so the amount of enac decreases
enac current @ 140mv in enac and m2
What is liddles Enac
cant be endocytosed from membrane
mutation in M2
less inhibition
What does influenza bind?
Binds enac and promotes endocytosis
What are ROS and PKC involvement in Enac
ROS- reactive oxygen species
M2 activates PKC and ROS which inhibits Enac
look at ROS using GSH = antioxidant which scavenges mopping up ros
What happens if you inhibits and GO6796
Inhibit PKC which block response to M2
What does PAH do- LOF Enac?
Salt washing hypotension- changes in kidney, low bp hyperkalemia- high plasma K metabolic acidosis high renin and aldosterone
PAH autosomal dominant
- renal form
- problems localised to kidney
- mineralocorticoid receptor gene
PAH 1 autosomal recessive
systemic form
multiorgan affect
Enac gene mutation- all subunits
PAH1 and nasal surface liquid
liquid coming out has higher Na
not function enac
cant be reabsorbed
How does PAH affect transepithelial potential and amiloride % inhibition?
Systemic is less negative
Cl probably still working- epithelial layer increase
cilia cant beat, move mucous= decrease= tract infection
