Introduction Flashcards
In epithelia cells what are the two pathways transport can occur through?
Paracellular
Transcellular
What are the two types of epithelia?
Tight
Leaky
What is the resistance of leaky epithelia?
Less than 200 ohms
What is the resistance of tight epithelia?
More than 2000 ohms
What is transepithelial resistance?
The measurement of how conductive the epithelia is
For example if it is leaky lots of transport of ions and solutes across the epithelia can occur
Which pathway of transport determines epithelial tightness?
Paracellular
Transcellular resistance is the same
Cell to cell contacts between epithelial cells are known as what?
Tight junctions
How do tight junctions differ between tight and leaky epithelia?
Tight junctions in leaky epithelia have large gaps which allows more paracellular transport
What are the general properties of leaky epithelia?
1) Rte is less than 200 ohms
2) Vte is approx 0mV
3) Flux is large = isosmotic
4) High water permeability
What are the general properties of tight epithelia?
1) Rte is greater than 2000 ohms
2) Vte is approx 50mV
3) Flux is small
4) Low water permeability
Give examples of leaky epithelia
Proximal Tubule
Gallbladder
Small intestine
Choroid plexus
Give examples of tight epithelia
Distal tubule
Stomach
Frog skin
What is the transepithelial potential?
Vte is the potential difference across the whole epithelium
Why is Vte bigger in tight epithelia?
There is no leak back of ions between cells
How is a negative Vte set up?
Negative Vte = MORE ANIONS OR LESS CATIONS
1) Low intracellular Na+ = Na+ moves in to the cell (set up by basolateral channels)
2) Na+ is a positive ion so we have the loss of apical positive charge
3) This means a negative charge is left behind
4) Tight epithelia = Na+ can not leak back
How is a positive Vte set up?
Positive Vte = LESS ANIONS OR MORE CATIONS
1) Low intracellular Na+ = driving force for Na+ influx
2) This is through NKCC2 which brings in 2 Cl- ions
3) More negative ions (Cl-) are being lost on the apical membrane than positive
4) So there is an overall loss of one negative charge when the cell turns over = positive Vte
List three types of models we can use in studies of epithelia
Fresh tissues and cells from organs of interest
Cultured cells - primary or cell lines
Whole animal data
Why are expression systems not always reliable?
They only express one protein which means other native proteins in a cell are not expressed also which may have important regulatory roles
List 5 methodologies used in epithelia research
1) PCR
2) Western blot
3) Immunostaining
4) Flux Radioactive Compounds
5) Electrophysiology
What is PCR used for?
Used to look for the presence of mRNA
What can electrophysiology be used to measure/calculate?
Currents
Transepithelial potentials
Transepithelial resistance
Short circuit currents
List 4 electrophysiological techniques
1) Intracellular microelectrodes
2) Patch clamp
3) Two electrode voltage clamp
4) Ussing chambers
What can Ussing chambers be used to measure?
Transepithelial potential
Transepithelial resistance
Short circuit current
What is the short circuit current?
Net ion flux across the membrane
If lots of K+ channels are open the membrane potential will be driven to what?
-90mV which is the nernst potential for potassium
If lots of Na+ channels are open the membrane potential will be driven to what?
+60mV which is the nernst potential for sodium
By measuring the membrane potential you can estimate what?
What ion channels are open
What is the Vrev?
Where there is no net ion movement i.e. current is zero
How is an Ussing chamber set up?
An epithelial sheet is clamped between two blocks filled with solution (usually Krebs)
There are 4 electrodes where two measure the Vte and the other two inject current
Why do we inject current in to the Ussing chamber?
It allows us to calculate the resistance of epithelia
This then allows us to calculate the short circuit current
How is short circuit current calculated?
We inject a known current which causes a change in Vte
How much the Vte changes is set by the epithelial resistance (V=I*R)
High resistance means there is a big shift
Short circuit current is then calculated as Vte/Rte
How do Vte traces work?
Deflections in a Vte trace show how much the potential changes when the current is injected
From this we can work out the short circuit current
What is the role of amiloride?
Blocks ENaC
What is the effect of adding amiloride on a Vte trace?
When amiloride is added there is a shift in the Vte from -1 to 0
Adding amiloride-
1) The initial Vte is negative as a positive charge is being lost
2) However, adding amiloride = less positive charge lost so Vte increases
3) This shows that ENaC contributes to the resting potential of the cell
What is Lub?
Stimulates the release of Cl-
Describe how Ussing chamber experiments can show that CFTR is secreting Cl- in the bronchial airway cells
Injecting a current of 10 amps causes a shift in Vte
Adding lub which increases Cl secretion = Vte becomes more positive
This can be reversed using a CFTR inhibitor which makes the Vte more negative