Introduction

Question 1

What does transcellular transport rely on?

Answer 1

The differential expression of transport proteins on the apical and basolateral membrane

Question 2

Where do you find leaky epithelia?

Answer 2

Proximal tubule
gall bladder
small intestine
choroid plexus

Question 3

Where do you find tight epithelia?

Answer 3

Distal tubule
stomach
frog skin

Question 4

What determines the tightness of an epithelium?

Answer 4

The sizes of the gaps between cells

the gaps are mediated by tight junctions

Question 5

What are the properties of a leaky epithelium?

Answer 5

Resistance of less than 200cm2
Vte of 0mv
large flux- isosmotic
high h20 permeability

Question 6

What are the properties of a tight epithelium?

Answer 6

Resistance of greater than 2000cm2
Vte 50mV
small flux
low h20 permeability

Question 7

What’s occurring when there’s a negative vte?

Answer 7

More anions or less cations

loss of positive charge from the basolateral membrane

Question 8

What’s occurring when there’s a positive vte?

Answer 8

Less anions or more cations

overall- loss of negative charge- every cell model turnover

Question 9

What are the sort of cells we use in epithelial research?

Answer 9

Fresh tissue/cells- dissect relevant sections of organs via enzymatic breakdown or manual dissection
cultured cells- airway cultured etc, or primary cultured cells taken from a patient/animal and used for a few days
or commercial cells- HeLa cells etc- been available for a long time

Question 10

Whats the downfall to overexpression studies?

Answer 10

Problems with expressing a protein in an environment other than its natural one
doesn’t have its usual interactions
loss of regulators found in natural environment

Question 11

Examples of methodologies used in epithelial research

Answer 11

PCR
Immunostaining
Flux radioactive compounds
Electrophysiology*

Question 12

What are methods included in electrophysiology?

Answer 12

Intracellular microelectrodes - Vm
Patch clamp- single channel current or total current
Two electrode voltage clamp- cell current
Ussing chamber- Vte, Rte and short circuit current

Question 13

What is the nernst potential for an ion?

Answer 13

The potential when all the ion channels are open and there is no net transport
E.g- K channels will try and drive the membrane potential to it’s nernst potential- which is very negative

Question 14

What does amiloride do?

Answer 14

Blocks ENac channels

stops Na contribution to the vm

Question 15

What happens in patch clamp/ two electrode voltage clamp?

Answer 15

Clamp the potential and measure what the net current is
the current is dependent on:
what channels are open
how many channels there are
how long theyre open for

Question 16

What is the set up in the Ussing chamber?

Answer 16

Piece of epithelium (frog skin etc). is clamped by two perspex blocks
solutions either side of the epithelium
4 electrodes- 2 measure the Vte
the other 2 inject current into the solutions

Question 17

What happens in the Ussing chamber?

Answer 17

A known amount of current is injected
the resulting shift in the Vte is measured
How much the Vte shifts is set by the resistance of the epithelium

Question 18

How do you calculate Rte and short circuit current?

Answer 18

Use ohms law
Rte= change in voltage/ current injected
Isc= Vte/Rte

Question 19

What pharmalogical tool makes the Vte more positive and why?

Answer 19

Amiloride sets the Vte to zero
blocks na channels-> less na transport-> reduces loss of positive charge-> less negative Vte
Negative Vte was due to the loss of Na