L1 - Introduction Flashcards
What is the importance of epithelial cells being polarised?
Can get net transport
- Transcellular
- Paracellular
Different transport proteins on apical and basolateral membranes
What is transcellular transport?
Move across apical and basolateral membrane
Move across cell
What is paracellular transport?
Move between cells
What are the two different structures of epithelia?
Sheet
Tubular - upper airway
What is the main difference between tight and leaky epithelia?
Transepithelial resistance
Where are leaky epithelia found?
Proximal tubule
Gallbladder
Small intestine
Choroid plexus
Where are tight epithelia found?
Distal tubule
Stomach
Frog skin
What is the transepithelial resistance of leaky and tight epithelia?
Leaky - <200 ohms/cm2
Tight - >2000 ohms/cm2
What is the transepithelial potential of leaky and tight epithelia?
Leaky - 0mV
Tight - 50mV
What is the flux of leaky and tight epithelia?
Leaky - large - isomotic
Tight - small
What is transepithelial resistance?
Resistance across the epithelium to movement
High transepithelial resistance = not a lot of net transport across the epithelium
What is transepithelial resistance determined by?
Paracelluar permeability
Transcellular net transport is very similar between all epithelia
- What makes them tight or leaky is there paracellular transport
What is the flux of epithelia?
Amount of transport
Tight - small as only transcellular transport
Leaky - large due to transcellular and paracellular transport
– Ions are followed by water - isosmotic
What is the transepithelial potential?
Net transport of ions and charge generates a potential that exists across the epithelium
Sum of the individual (apical and basolateral) membrane potentials
Measured using electrode on either side of the epithelium
Why is the transepithelial potential of leaky epithelia low?
Low value/0
Can’t sustain potentials as some ions leak back so you get lots of paracellular transport
How is transepithelial potential generated in the principal cells of the collecting duct?
Na/K ATPase and basolateral K channel
- Set up electrochemical driving force for Na uptake across apical membrane
- Set up negative membrane potential
ENac
- Na uptake into intracellular fluid across apical membrane
- Na then pumped by Na/K ATPase across basolateral membrane
Net movement of positive charge from apical to basolateral membrane
- Leaves behind a negative charge negative Vte
How is transepithelial potential generated in the thick ascending limb?
Na/K ATPase and basolateral K channel
- Set up electrochemical driving force for Na uptake across apical membrane
- Set up negative membrane potential
- Drives NKCC2
NKCC2 and apical K channel
- NKCC2 electroneutral transport protein no net movement of charge
- Na and Cl and reabsorbed
- K is recycled through apical K channel
Net movement of 1 positive charge from apical to basolateral membrane
- Net loss of negative charge from apical solution
- Leaves behind a positive charge - positive Vte
What are the four methods to look at electrophysiology?
Intracellular microelectrode
Patch clamp
Two electrode voltage clamp
Ussing chamber
What can intracellular micro electrode measure?
IC potential
What can the patch clamp technique measure?
Vhannel/whole cell current/Po of channel
Tends to be used for smaller cells
What can the two electrode voltage clamp technique be used for?
Whole cell current
What can the Ussing chamber technique be used for?
Vte, Rte and Isc
What does the Ussing chamber technique look at?
How a whole epithelium behaves - looking at all the cells that makes up the epithelium
How does the Ussing chamber calculate the short circuit current?
Use potential and resistance values we record to calculate short circuit current
- Can’t measure direct net current flow across an epithelium
- Isc – indirect measurement of net ion flux across membrane
- Equivalent short circuit current
What does transepithlial potential allow us to identify?
If epithelium is tight or leaky
Can also identify is tissues are viable
How can we use pharmacology to look at Na ion channels?
Use specific ion channel blockers to see if membrane potential changes
- Get a negative or positive shift in membrane potential
E.g. Amiloride block Na channels
- Membrane potential moves away from Nernst of Na
How can we use pharmacology to look at K ion channels?
Used Ba - K channel blocker
Currents go down –> K channels mediate K current
How can you tell which is the dominant open ion in a membrane?
Look at dominant ion channels open by comparing Nernst values with membrane potential
Vrev is closer to the Nernst of the dominant ion channel
Ussing chamber method
Between two blocks of Perspex you clamp the epithelium you are interested in
- Can grow culutres cells on an insert
Epithelium then perfused with Krebs or ringer solution
Bicarbonate as a buffer so pH remains at a constant
What are the 4 electrodes in an Ussing chamber?
1st set
- Measure transepithelium potential
- 1 is reference, 1 is to record
2nd set
- Inject a known current (connected to a current injection box)
- The resulting shift in Vte is measured
- This shift is dependent on the Rte of the epithelium
How do you calculate Isc from an Ussing chamber experiment?
- Every minute you inject 10 mA of current
- Shift in Vte when you inject 10 uA is shown by the red bar
- Calculate Rte
= ΔV / I injected - Calculate Isc
= Vte / Rte
What is the drug Lub?
Exposed epithelia cells to Lub – a prostaglandin activator
Lub –> cAMP –> PKA –> CFTR Cl channels
Increase in Vte due to stimulated secretion of Cl ion
When CFTR inhibiter added this increase in Vte is reversed
How did they validate the Ussing chamber technique?
Used Ussing chamber to record
- Isc
- Na24 influx - apical to basolateral
- Na24 outflux - basolateral to apical
They knew 10.4pmol/s = 1µA
- Able to work out how much current the Na would generate
Used radioactive studies to validate that in frog skin Na is the main actively transported ion through ENaC
Worked out the total current –> similar to short circuit current measured in Ussing chamber
How is Na and Cl handled in the upper airway?
Na/K ATPase and K channel on basolateral membrane
- Set driving force for Na influx through basolateral and apical membrane through NKCC1 and ENac
- Na is recycled across basolateral membrane
- K is recycled across basolateral membrane
- Cl accumulates in cell - above electrochemical equilibrium
If we open Cl channel in apical membrane –> Cl leave through CFTR Cl channel
- Net secretion of Cl
What has to be carefully controlled in the upper airway?
Balance between Cl secretion and Na absorption (through ENaC)
- Sets the peri cilia layer that cilia project into
- First line of defence against infection
A researcher is interested in the Po of an ion channel. What techniques would they use to measure this?
Single channel patch clamp
In an Ussing chamber experiment you record a Vte of 6.5mV. On injection of 10uA a current of Vte changes to 21.9mV. Using these data work out the equivalent Isc in uA.
Work out the shift in Vte o 21.9 – 6.5 = 15.4mV Work out Rte o 15.4mV / 10uA = 1540 ohms Use the Rte to work out Isc o 6.5mV / 1540 = 4.22uA
