Introduction Flashcards
What does transcellular transport rely on?
The differential expression of transport proteins on the apical and basolateral membrane
Where do you find leaky epithelia?
Proximal tubule
gall bladder
small intestine
choroid plexus
Where do you find tight epithelia?
Distal tubule
stomach
frog skin
What determines the tightness of an epithelium?
The sizes of the gaps between cells
the gaps are mediated by tight junctions
What are the properties of a leaky epithelium?
Resistance of less than 200cm2
Vte of 0mv
large flux- isosmotic
high h20 permeability
What are the properties of a tight epithelium?
Resistance of greater than 2000cm2
Vte 50mV
small flux
low h20 permeability
What’s occurring when there’s a negative vte?
More anions or less cations
loss of positive charge from the basolateral membrane
What’s occurring when there’s a positive vte?
Less anions or more cations
overall- loss of negative charge- every cell model turnover
What are the sort of cells we use in epithelial research?
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
Whats the downfall to overexpression studies?
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
Examples of methodologies used in epithelial research
PCR
Immunostaining
Flux radioactive compounds
Electrophysiology*
What are methods included in electrophysiology?
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
What is the nernst potential for an ion?
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
What does amiloride do?
Blocks ENac channels
stops Na contribution to the vm
What happens in patch clamp/ two electrode voltage clamp?
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
What is the set up in the Ussing chamber?
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
What happens in the Ussing chamber?
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
How do you calculate Rte and short circuit current?
Use ohms law
Rte= change in voltage/ current injected
Isc= Vte/Rte
What pharmalogical tool makes the Vte more positive and why?
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
