Epithelial cells Flashcards
what is the main thing that makes epithelial cells different to other cells? and why is this so?
they are polarised
they are polarised due to the apical membrane and basolateral membrane which have expression of different transport proteins which is what allows for the net transport across the epithelia
what are the 2 different transport pathways that happen in epithelial cells
transcellular and paracellular
transcellular is across the cell and is dependent on this differential expression of the 2 membranes
paracellular pathway -between the cells
what are the 2 different classifications of epithelial cells? give some examples of them
tight = distal tubule, stomach, frog skin leaky = proximal tubule, gall bladder, small intestine and choroid plexus
what is the resistance in tight epithelia vs leaky epithelia?
leaky = Rte < 200 .cm2 Vte ~0mv Flux large and isosmotic high H2O permeability
Tight = Rte > 2000 .cm2 Vte ~ 50mV Flux small Low H2O permeability
what is the transepithalial potential (Vte) measuring?
Sum of the individual membrane potentials
what determines the tightness of epithelia?
its actually paracellular transport
in leaky epithelia, the gaps in the tight junctions means that you get a lot of transport if you have a driving force
in tight junctions, there is cell-cell contact in leaky and tight epithelia, here they have transport proteins
these gaps are mediated by tight junctions and
how much transport will occur depends on how big the gaps are
in a tight epithelia, tight junction gaps are much smaller so you get loess transport
resistance in terms of transport across the cell = the same in leaky and tight epithelia
what is the difference in the transport across cels in tight vs leaky epithelia?
differences = how much ion and fluid movement occurs between cells
leaky has more movement between the cells - tight has not a lot of movement
how can you measure the trans-epithelial potential in a cell?
sum of the individual membrane potentials
- if you put a reference electrode on 1 side of the epithelium and set it to 0mV and then put the reference electrode on the other side, if it is a tight epithelia, you will get a positive or negative 40mV
how is a potential difference generated? (-Vte and +Vte)
-Vte = more anions, less cations in frog skin and salt reabsorption Na in from apical membrane Na leaves across BLM K comes in at BLM K is recycled across BLM there is net movement of + ions (Na) from the apical membrane to the interstitial fluid therefore the apical side = losing the -Vte trans-epithelial so, Na cant come back (tight)
\+Vte = less anions, more cations driving force for Na uptake Na, K, 2 Cl- in through NKCC Na in through the apical side and out through BLM K is recycled across the BLM Cl is lost through the BLM have a charge on the apical side this is important for Ca reabsorption
what methodology would you use for finding mRNA/protein function/location?
PCR - mRNA presence
western blotting - protein presence
immunostaining - protein location
flux radioactive compounds - transport function (put radioactive compound on 1 side then sample from the other side of the epithelium
if the radioactive levels go up then you have transport
helps to validate electrophysiological approaches
electrophysiology - transport function
allows us to measure membrane potentials, currents, transepithelial potentials/resistance and short circuit currents
describe the different electrophysiology techniques to measure membrane potential
intracellular microelectrode - IC potential (Vm)
Patch clamp - single channel/cell current
2 electrode voltage clamp - cell current
ussing chamber - transepithelial potential resistance, short circuit current
– measurement of the net ion flux across the membrane. cant measure this directly so we do it indirectly using the ussing chamber technique
what would a big short circuit current mean?
big - lots of movement across the epithelium
what would a small short circuit current mean?
small - don’t have a lot of net transport
how can you measure membrane potential using intracellular electrodes?
clamp cell, take electrode and stick it into the cell and measure the membrane potential
membrane potential of a cell is determined by which ion channels are open and the selectivity of these ion channels
i.e. if a K channel is open, nernst moves to that of K y K channel driving this
what molecule can be used to detect whether or not Na channels are contributing to the resting membrane potential
can use amiloride to block the ENaC and so if the membrane potential changes to (for example) go closer to the membrane potential for K, this means that the channel has Na partially contributing to its membrane potential
if you add amiloride and the membrane potential stays at the same point, then Na was not contributing to the resting membrane potential and that ENaC is not active
what does the patch clamp/2 electrode voltage clamp tell you about voltage?
does the same thing as the intracellular electrodes
clamp membrane potential to a value that you decide and measure the net current flowing across the cell membrane - take whole cell recordings
what is the net current flow set by?
what ion channels are open?
how many ion channels there are
how often the ion channels are there
what happens to the driving force when you change the potential?
you change the driving force and also the magnitude of the current
describe the set up of an ussing chamber
works by having a solution where you have 2 electrodes measuring the voltage difference across the 2 sides of the epithelia
have another 2 electrodes that are injecting a set current (that you know) into the solution
you have the current and the voltage as when you inject the current, the voltage will change
use the equation V=IR and rearrange so that you can work out R. want to get the resistance so that you can work out the short circuit current
what kind of shift does a highly resistance epithelia cause?
big shift in potential
what kind of shift does a low resistance epithelia cause?
small shift in potential
how can the shift in potential work out the resistance?
can use ohms law
what do each of the electrodes in ussing chambers do?
2 electrodes to measure the trans-epithelial potential
2 additional electrons injected with current to cause a shift in trans-epithelial potential so you can calculate resistance of epithelium therefore you can calculate the short circuit current
what is the model tight epithelium?
frog skin
what part of the frog skin describes what part of the epithelium?
outside surface is the apical surface
inside surface is the basolateral surface
why is frog skin a good model for tight epithelium?
it uses the same mechanism that we utilise in upper airways
the molecular components are almost identical
what can frog skin be a relevant model for?
renal collecting duct distal colon salivary gland sweat gland airway surface epithelium
what is involved in the short circuit current technique?
2 electrodes measure transepithelial potential
2 additional electrodes with current to cause a shift in transepithelial potential so that you can calculate resistance of epithelium therefore calculate short circuit current
describe experiment 1 for Ussing chambers. what kind of potential would you expect to generate?
have the 2 chambers with krebs solution inside. when looking at Na uptake, put the same solution on both sides so that when the experiment starts, there is no potential difference between the 2 sides. this value changes when Na starts to be transported across the frog skin. lose Na from apical to basolateral side so you’d expect to record a negative transepithelial potential.
describe the validation experiment done after the first Ussing chambers experiment
used radioactive Na24 and put it into chamber 1 then checked how much Na24 there was in chamber 2 by taking a sample. you’d know that any Na24 here could only have come from chamber 1 and therefore proving the presence of a Na channel in the frog skin. (can also be done in reverse
how can the radioactive Na24 be used to make a calibration curve?
by testing how much Na24 is transported over a set time period, this allows you to measure the net amount of Na24 is being transported per unit/time in each of the directions. you should initially have the same amount of Na on both sides but then you can subtract he Na on one side from the other side and this tells you how much has moved at each time point you select.
if you know the no. of moles that has moved per unit time, you can do some calculations of what would be expected from a short circuit current
describe experiment 2 - the evidence for net transport of Na including who conducted the study
once you’ve made a graph and found the mol/s of Na influx using values from Na24 moving from chamber 1->2 you have a bar chart for Na influx. can make another bar for Na movement from chamber 2-> 1 which would be the bar for Na outflux. the ussing chamber was used to make another graph of the total current. these calculations found that there was 140() Na influx, 15() Na outflux and the total current was ~125(). since the total Na current from the ussing chamber recorded was similar to Na influx - Na outflux, this must mean that the current acquired is mostly due to the Na influx
what did Ussing conclude about the components of frog skin and Na
he concluded that since they found there was no passive transport of ions (ion flux) through the frog skin and the fact that there is net Na transport from the pond into the frog, then there must be a Na channel in the apical membrane of the frogs skin
what do deflections down mean when looking at potential?
downwards deflections mean that there is a change in potential upon the injection of a current
what is the role of amiloride?
to block Na channels
what could account for the difference in membrane potential in the frog skin and the real colon?
in frog skin, the membrane potential is very negative whereas in the colon, it is -1mV. the reason for this difference could be because the colon is doing many other jobs that are different to contribute to its potential. whereas in the frog skin, it isn’t doing much more than transporting the Na across the 2 sides.
what is teh affect of adding amiloride to an epithelial channel that is -1mV?
it would go from -1mV to 0mV because the Na re-absorption is being inhibited due to the inhibition of the Na channels.
what is the benefit of doing a human nasal biopsy?
it is a less invasive way of obtaining airway epithelium because the properties of the cells here are essentially the same as in the upper airways
why is it suitable to use human nasal cells as models for upper airways?
because the cells in these 2 places have essentially the same properties and they both do Na reabsorption and Cl secretion which are the essential processes we want to study in the airways
what experiment did Mall et al do in terms of nasal biopsy?
took cells in the nose as a model for the upper airways and instead of changing their Na concentration like Ussing did, they they added amiloride and observed.
what was observed in the nasal cells after adding amiloride? - the evidence of a presence of Na channels
they observed a hyperpolarisation (becoming very negative). this moved the membrane potential away from the nernst potential for Na which provides evidence for the presence of Na selective channels in the apical membrane in a human nasal biopsy
what had not been done before the first patch clamp study?
they had looked at the whole epithelium/whole membrane behaviour but they saw that there was absolutely no direct evidence for an ENAC channel
describe the first direct evidence of ENAC channels and what technique was able to find this?
a study of patch from renal collecting duct provided direct demonstration of Na channels. the patch clamp has many flickerings which signify the opening and closing of a channel. the size of the deflections increase as you increase the Na concentration. the most obvious difference was at 100-145 concentration of Na. this suggests that in ion channels, the more Na you make available to move through the channels, the bigger the current read.this gives direct evidence for ENAC in these channels and so verified their presence
describe the conventional expression cloning techniques
isolate the protein and sequence e.g. using an amiloride affinity column (for enac)
leave the column with beads that has something to stick to the protein that you want. take the poretin taht you gpt from the kidenys and run it down the columnthen your protein of interest sticks to the beads and you harvest the protein and send it for sequencing
how was the first attempt at expression cloning for ENAC done and what were the problems?
had beads with amiloride n and they ran through a solution but they found that the protein abundance was too low in native cells
how was expression cloning of ENAC eventually successfully done?
a different group decided to screen by fucntional expression. they decided to instead of taking protein from cells and running them through an infinity column, they decided to tae the mRNA from cells of the kidney colon in salt depleted rats, chop the RNA into pool and take 1 of these pools then inject this into a xenopus oocyte and then identify which section of mRNA made Na selective amiloride inhibitable currents (functional expression)
how was the process of having 10 pools of RNA to getting functional ENAC?
looked at the pools and divided them again. got smaller and smaller amount of mRNA till they got to the point that they couldnt get it any smaller. then they used this and identified the sequence that coded for the ENAC.
what was found when they thought they found the full functional ENAC?
they identified 3 different channels cause when you do the main isolation, you have polyA RNA and as you have to break it down, you get smaller and smaller which is the purification process. in the final pool, it would be expected that you have the pure mRNA that codes for the Na channel but actually, the currents werent as big as it should be if it ere pure
what was the conclusion on what the ‘purified’ polyA mRNA actually was?
the alpha subunit was what they had but they realised that they needed the other subunits in order to have full activity
if all the subunits had been needed for channel activity, ENAC wouldnt have been cloned. the deflection shown in the graph tells you about the function of the Na channel. - the bigger the deflection, the bigger the current. they found that clone a can act as a Na channel and sits in the membrane but they identified 2 more sequencesB and Y that when these 3 come together, it enhances the ENAC function and physiologically can have a, b and y which creates maximum function
are all 3 subunits of ENAC needed for any function?
no. there is function if it is the alpha subunit alone and even the A+B or A+Y but when thee are all togther, you can achieve maximum function
how much of a difference is seen when it is all 3 subunuts of ENAC coming together? (A, B and Y)
there is a 10 fold larger scale bar when measuring the activity when A,B and Y is put together. this signifies how much larger it is
what disease happens when there is a gain of function of ENAC? what does it cause? how is ENAC affected?
Liddles’s syndrome. causes hypertension. upregulation of ENAC
what disease happens when there is a loss of function of ENAC? what does it cause? how is ENAC affected?
psuedohypoaldosteronism. causes hypotension. causes ENAC inactivation.
why did the rats in the expression cloning experiments need to be salt depleted?
because salt depletion stimulates aldosterone release which upregulates mRNA. so, you will have more of it for the experiment to be injected into expression plasmids.
what are the most important roles for the airway surface liquid?
- clearance of mucous
- airways defence against respiratory pathogens
what is the difference between the airway surface liquid (ASL) and the periciliary layer (PCL)?
the difference is that the ASL is the liquid + basement membrane whereas the PCL is the liquid alone
what acts as the first line of defence to protect people from getting pathogens?
there being a mucus layer on top of the ASL/PCL
how does the mucus act as the first line of defence against pathogens?
idea is that a pathogen comes in and gets stuck in the mucus and this mucus would move up the respiratory tract. this would be swallowed with the saliva
what is it that allows mucus to move? what can happen if this goes wrong?
there is apical cilia that beat and move the PCL - this has mucus on top of it. anything disrupting this process can cause major respiratory issues
describe the volume load and surace area relation and how this links to mucus clearance
as you go up the airways, the surface area decreases and the volume load increases. the mucus goes up the airways and when it gets from an area with a large surface area to an area with a small surface area, the volume load through this change would remain the same. through this, the mucus layer is still getting higher. this is bad because there is an optimum height for the mucus layer and this allows for the appropriate movement of mucus out of the respiratory tract
what would result from the mucus layer going up or down?
anything increasing or decreasing the the mucus layer impacts on the ability of the lungs to clear mucus
what is working to control the ASL?
there are 2 possible mechanisms. the passive mechanism and the active mechanism. it is thought that they are working together - passive laying a small role and active transport playing the lead role in controlling the height of the layer
describe the passive way of controlling the ASL
this follows the idea that the mucus layer acts as a reservoir and so if the height of the liquid layer is too high, fluid then moves into the mucus slayer and leaves just the cilia which would be at the right height and so be able to move and direct everything to the respiratory tract for you to swallow
describe the active mechanism when controlling the ASL
involved active ion transport controlling salt levels in the PCL
it is how net transport is by epithelial cells that sit beneath the PCL to control how much salt and water is in the layer which then maintains the right height.
- if the height is too low, ions are secreted into the layer, water follows and so the height of the layer goes up
- if the height is too high, ions are reabsorbed by the cells, water follows and so the height of the layer would go down
