Introduction

Question 1

In epithelia cells what are the two pathways transport can occur through?

Answer 1

Paracellular

Transcellular

Question 2

What are the two types of epithelia?

Answer 2

Tight

Leaky

Question 3

What is the resistance of leaky epithelia?

Answer 3

Less than 200 ohms

Question 4

What is the resistance of tight epithelia?

Answer 4

More than 2000 ohms

Question 5

What is transepithelial resistance?

Answer 5

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

Question 6

Which pathway of transport determines epithelial tightness?

Answer 6

Paracellular

Transcellular resistance is the same

Question 7

Cell to cell contacts between epithelial cells are known as what?

Answer 7

Tight junctions

Question 8

How do tight junctions differ between tight and leaky epithelia?

Answer 8

Tight junctions in leaky epithelia have large gaps which allows more paracellular transport

Question 9

What are the general properties of leaky epithelia?

Answer 9

1) Rte is less than 200 ohms
2) Vte is approx 0mV
3) Flux is large = isosmotic
4) High water permeability

Question 10

What are the general properties of tight epithelia?

Answer 10

1) Rte is greater than 2000 ohms
2) Vte is approx 50mV
3) Flux is small
4) Low water permeability

Question 11

Give examples of leaky epithelia

Answer 11

Proximal Tubule
Gallbladder
Small intestine
Choroid plexus

Question 12

Give examples of tight epithelia

Answer 12

Distal tubule
Stomach
Frog skin

Question 13

What is the transepithelial potential?

Answer 13

Vte is the potential difference across the whole epithelium

Question 14

Why is Vte bigger in tight epithelia?

Answer 14

There is no leak back of ions between cells

Question 15

How is a negative Vte set up?

Answer 15

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

Question 16

How is a positive Vte set up?

Answer 16

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

Question 17

List three types of models we can use in studies of epithelia

Answer 17

Fresh tissues and cells from organs of interest
Cultured cells - primary or cell lines
Whole animal data

Question 18

Why are expression systems not always reliable?

Answer 18

They only express one protein which means other native proteins in a cell are not expressed also which may have important regulatory roles

Question 19

List 5 methodologies used in epithelia research

Answer 19

1) PCR
2) Western blot
3) Immunostaining
4) Flux Radioactive Compounds
5) Electrophysiology

Question 20

What is PCR used for?

Answer 20

Used to look for the presence of mRNA

Question 21

What can electrophysiology be used to measure/calculate?

Answer 21

Currents
Transepithelial potentials
Transepithelial resistance
Short circuit currents

Question 22

List 4 electrophysiological techniques

Answer 22

1) Intracellular microelectrodes
2) Patch clamp
3) Two electrode voltage clamp
4) Ussing chambers

Question 23

What can Ussing chambers be used to measure?

Answer 23

Transepithelial potential
Transepithelial resistance
Short circuit current

Question 24

What is the short circuit current?

Answer 24

Net ion flux across the membrane

Question 25

If lots of K+ channels are open the membrane potential will be driven to what?

Answer 25

-90mV which is the nernst potential for potassium

Question 26

If lots of Na+ channels are open the membrane potential will be driven to what?

Answer 26

+60mV which is the nernst potential for sodium

Question 27

By measuring the membrane potential you can estimate what?

Answer 27

What ion channels are open

Question 28

What is the Vrev?

Answer 28

Where there is no net ion movement i.e. current is zero

Question 29

How is an Ussing chamber set up?

Answer 29

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

Question 30

Why do we inject current in to the Ussing chamber?

Answer 30

It allows us to calculate the resistance of epithelia

This then allows us to calculate the short circuit current

Question 31

How is short circuit current calculated?

Answer 31

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

Question 32

How do Vte traces work?

Answer 32

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

Question 33

What is the role of amiloride?

Answer 33

Blocks ENaC

Question 34

What is the effect of adding amiloride on a Vte trace?

Answer 34

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

Question 35

What is Lub?

Answer 35

Stimulates the release of Cl-

Question 36

Describe how Ussing chamber experiments can show that CFTR is secreting Cl- in the bronchial airway cells

Answer 36

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