EP Flashcards
Why are epithelial cells polarised?
They have different channels on the apical and basolateral membranes so they can allow net transport across. (Transcellular)
Two different transports from one side of epithelial cells to the other?
Transcellular- Net transport through the cells.
Paracellular- Transport between the cells.
What determines the direction of ion movement through epithelia cells?
Driving forces- electrochemical (Both charge and concentration)
Tight vs leaky epithelia differences why?
Resistance much higher in tight ( greater than 2000 ohm cm2 vs less than 200). The difference is the paracellular resistance not transcellular.
The flux is therefore much greater in leaky and due to the free movement the Vte is 0mv, whereas tight is around 50mv.
Resistance of Tight epithelia?
Greater than 2000 ohm cm2. Tight junctions between epithelial cells.
Resistance of Leaky epithelia?
Less than 200 ohm cm2
Tight epithelia example?
Stomach, Frog skin, distal tubule kidney
Leaky epithelia example?
Proximal tubule, gall bladder, small intestine, choroid plexus
What is transepithelial Potential?
Vte- difference between one side of the epithelia voltage and the other side- e.g. the potential difference between voltage on the apical and basolateral side of epithelia.
How can the transepithelial potential be measured?
Two electrode voltage clamp- An electrode is placed either side of the epithelial cell.
One side is clamped to a known voltage e.g. 0mv, and the resultant other side is measured. E.g. If clamped side is zero but measured side is 40 the Vte is 40 or -40 (depending on which direction taken)
In the patch technique why if one side is clamped to a certain voltage does the other side change?
The driving force is changed. Think of an IV curve, depending on what the voltage is, the current through the channel varies (Driving force and some voltage dependant channels). This varying current of ions through will vary the intracellular concentrations and therefore the driving force over the adjacent membrane, so changing the voltage there.
What is Vrev?
When net current through a channel is zero. Equilibrium. On an IV curve, the voltage at which the current is zero (crosses the X axis).
What is current?
a directional flow of ions through a channel. measured in Amps. Usually uA.
What is voltage?
The potential difference in charge generated as a result of movement of ions (current) Measured in Volts, usually mV
What is transepithelial resistance?
Rte- resistance of the epithelia e.g. how easy or hard it is for ions to flow from one side of a membrane to another. E.g. Through the tight junctions paraceullar between epithelial cells. if lots of gap junctions resistance is lower.
Why does paracellular resitance vary?
Occludin/ claudin for tight junctions between epithelial cells. Tight junctions determine the paracellular resistance, and therefore largely the transepithelial resistance. Tight junctions may also serve as leaky pathways by forming selective channels for small cations, anions, or water. Tight junctions are present only in vertebrates. The corresponding junctions that occur in invertebrates are septate junctions.
Equation to work out resistance of an epithelia?
Rte=Change in voltage/ Current injected (Ussing chamber)
Ohms law?
V=IR Voltage=Current x Resistance
If there is a high resistance for a given current the Vte is high or low?
High. Around 50mv
Tight junction- less leaking of current across after addition one side. Therefore, there is a high voltage difference across both sides (transepitethial potential).
Whereas, if leaky, the current is placed one side and leaks across the membrane dispursing the current (large flux) and leaving no difference between the voltages either side (no transepithelial potential- around zero)
Paracellular or transcellular determines tightness of a membrane? Resistance?
Paracellular, transcellular resistance often the same, around 7000ohms
How is Vte generated?
The net movement of ions across the apical and basolateral membranes. Depends on which way the ions move, their charge e.g. cl- or K+, and how many move per ‘cycle’
Model stages used for experiments?
- Fresh tissue/cells- e.g. sharks fin, isolate and if needed enzyme used to break it down.
- cultured cells- Used a few days after taken from patient or frozen e.g. HeLa cells.
- Whole animal data- WT, KD, KO, Pharmacological agent, overexpression data
Method to test protein is there?
PCR is mRNA there? Western blot from gel- for protein
Method to test protein location?
Immunostaining- AB to and secondary with fluorescent marker.
Method to test channel flux?
Radioactive ion one side e.g NaI (iodine) can measure the amount of net transport by its movement.
Patch clamp measures?
Single or whole cell conductance/ current
Two electrode voltage clamp?
One clamps potential the other records the generated potential.
What is a short circuit current?
Net ion flux across the membrane (so individual currents added).
Often small as there is not a lot of net transepithelial movement
Microelectrode measures?
measures potential difference so can create IV curves etc or the single channel recordings.
How can one use a microelectrode to predict whether ion channels are selective for a certain ion?
IC potential (Vm) (Current vs Voltage) Calculate the nernst potential for two ions e.g. K and Na. Clamp the extracellular a potential and then measure the intracellular Vm. If this is near say K nernst, likely to be K channels open at this clamped potential. But confirm by changing the extracellular potential again. This therefore changes the nernst potentials for the ions and if the Vm follows the K nernst, it is likely to be selective to this. Can plug these into an equation to see selectivity ratio using Vrevs.
How are pharmacological inhibitors used for cell channel studies?
Can measure whole cell conductance and then use an inhibitor of certain channels and see if the voltage inside the cell changes.
E.g. add amiloride and the potential moves closer to the nernst for K instead of Na e.g. -40 to -60mv, therefore Na through ENaC was contributing to the current.
Amiloride blocks..
ENaC channels (Sodium)
Barium blocks..
K channels
Current is zero on an IV curve when?
The channel is at its Vrev (reversal potential) where there is no net ion flow, or when a channel is closed.
PCR tells us what info?
mRNA presence
Immunostaining tells us what info?
protein location
Flux radioactive compounds and electrophysiology tells us what info? Experimental evidence.
Ussing & Zerahn, 1951
channel transport function- Put Na24 in chamber one (extracellular- krebs solution only in chamber2) Na will transport across the epithelium to chamber 2. The flux from 1 to 2 is measured. The Na24 is switched to in chamber 2 and the flux 2-1 is measured. Flux(1-2)-Flux(2-1) is used to calculate the Net transport of Na (JNETNa) per unit time.
Based on JNETNa per unit time the expected current was calculated. Use Ussing chamber to work out actual Isc (equations). JNET= Isc therefore the movemrnt of Na is all active (Na/k ATPase). There was a little Na outflux leak. Follows Nernst for Na.
What drug stimulates CFTR?
Lubisterone, IBMX/Forskolin
If JNET=0?
Passive transport only. Using the radioactive Na e.g. Influx=outflux so no net movement.
if JNET not equal zero?
There is a difference between influx and outflux of ions across an epithelial (Radioactive Na e.g.) therefore there must be an active element reabsorbing/secreting the ion.
If =zero likely leaky membrane.
All active components if JNET=?
ISc- if it equals the short circuit current calculated in the ussing chamber, these active ion channels make up all of the net transport across the epithelium.
If influx into cells from lumen is greater than efflux, the ion is being..?
Reabsorbed, if efflux is more- secretion (think kidney- out of tubule lumen into blood)
Apical vs basolateral?
Apical is lumen side- the side facing the ‘tube’ inwards where all the transport is finally decided, e.g. in the airways on the mucous side, and in the GI tract the intestine lumen side, kidney PT lumen side (basolateral-blood side).
For CFTR- CFTR always on the apical (controlling secretion/reabsorption) whereas the Na/K ATPase etc on the basolateral, setting up the concentration gradients.
Aim of the Mall 1999 study?
Do cystic fibrosis patients have higher Na conductance in the colonic epithelium, as is known to be in the airways? (CFTR inhibits ENaC|)
(Yes)
Mall 1999 Model to study?
Human intestinal membrane- 34 non CF biopsies, and 14 CF. Mounted on an Ussing chamber for studies.
Mall 1999 First experiment finding?
The Vte was significantly higher (less negative) and the Rte was significantly higher in CF tissues. Non-CF tissues demonstrated cAMP-dependent Cl− secretion that was absent in biopsies of CF patients. E.g. the Vte was unaffected by IBMX/Fors, but did respond to amiloride.
E.g. CFTR channel disfunction, but EnaC functioning.
Why in Mall 1999 was there a basal negative Vte? How did chemicals affect this?
EnaC is reabsorbing Na+ so there is a negative potential on the apical side.
When Amiloride was added the potential went to zero- the Na was no longer being absorbed.
When Forskolin/IBMX was added in the WT the potential returned negative (CAMP induced CFTR channels) but on CF tissues- no decrease (CFTR faulty). even increase as activates K channels (+ out).
What were different chemicals impacts on the amiloride sensitive Isc?
Non CF- indomethacin increases Na short circuit currents, whereas IBMX/Fors inhbits (CFTR inhibits the channel)
CF-no impact by either chemical but under basal conditions the short circuit current was increased from 6 to 20 (lack of CFTR-dependent regulation of ENaC).
What is JNET?
The net transport of ions
Mall 1999 conclusion?
Amiloride-sensitive Na+ currents are inhibited by activation of CFTR (IBMX/Fors) only in non-CF but not in CF tissues.
What is indomethacin? Why was it used in the Mall 1999 study?
a non-steroidal anti-inflammatory agent (NSAID) that inhibits CYCLOOXYGENASE, which is necessary for the formation of PROSTAGLANDINS. PGE2→CAMP induced CL- secretion, therefore it inhibits CFTR.
Mall 1998 experiment used and found?
Nasal cells- easy to access, scrape out the nose. Found that Na reabsorption causes the depolarisation but the addition of amiloride hyperpolarises the cell (from -40 to -60mv) away from the nernst for Na.Therefore showing this channel is contributing to the voltage.
Before 1995 experiment to isolate the ENac protein?
Found this very hard. Earlier experiments tried to isolate the protein using an Amiloride affinity column, but needed more Enac than in Native cells to extract the sequence.
When/how was Enac finally sequenced?
1995 Canessa-
- Took mRNA from the colons of salt depleted rats (induces ENac expression by aldersterone).
- CDNA of this into an expression vector.
- Chopped up the DNA into 10 groups, cultured to divide.
- Inject the pool back into the Xenopus oocyte.
- See which of the split mRNAs gave a functional amiloride sensitive Na current. “functional expression”
- Further purified this functional pool until purified EnaC.
1995 Canessa- As purified the fraction expected to see increased currents but didnt? Why?
Because ENaC is not just one subunit, and as they were purifying were just getting the alpha subunit, which luckily still formed a functional channel or the protein would not have been isolated. But the addition of Y and B made the full 1800nA current. (if just two around 50-80nA).
What other Na channels bar ENaC?
The Loop diuretic sensitive NKCC1 cotransporter (2 in kidney) (Barters)
Thiazide sensitive NaCl cotransporter. (Gitelman’s syndrome)
Na+/H+ exchangers
Na+/HCO3- cotransporters
How does the ussing chamber work?
Two chambers with the membrane between. One side current is injected which changes the transepithelial potential. The resultant voltage changes are observed (if high resistance epithelia large Vte seen (if low resistance leaks across and disserpated). Resistance and short circuit current can be calculated.
Why do you have to be careful with overexpression data?
have to be careful with as limiting factor could be another protein regulating etc, e.g. if upregulatate CFTR without all the other regulating proteins will it work as well? but if then upregulate those proteins- they could be non-specific and have other functions.
Transepithelial resistance equation?
Rte=change in voltage/ current injected
Rte=AV/I
Equation for short circuit current?
Isc=Vte/Rte
Vte trace?
Vte is the top line, the change in Vte is the difference between top and bottom line (maybe?)
If there is a negative Vte?
More cations moving in this direction than anions
e. g. if vte negative (0 to -40) there will be either anions secreted or cations being reabsorbed, or both.
e. g. CFTR and ENaC.
Frog skin is a model for?
renal collecting duct, distal colon, salivary gland, sweat duct, airway surface epithelium
Why use frog skin?
Oocytes- dissect off. Same mechanisms as humans, robust and easy to use.
Another name for absorptive cells in the small intestine?
enterocytes
Cellular/molecular structure of the upper airways?
Epithelial cells wth cilia.
The periciliary layer of liquid (PCL).
The mucous layer. (With PCL=Airway surface liquid layer ASL).
function of the components of the upper airways?
Epithelial cells with cilia- these secrete ions to maintain the PCL height for optimal beating of the mucous.
PCL: height controls beating ability of cilia- too low and the cilia will bend in on themselves, but too high and the cilia cannot beat the mucous back up to be swallowed (HCL stomach).
Mucous: Traps pathogens so they do not get to lungs
Issue with PCL as you go up the respiratory tract?
In proximal- alevolar and lots of branching so has a higher surface area to cover of 2m2, whereas as rise converge to one oesophagus where the SA distally is only 50cm2. This means that height of the liquid layer will increase as rises as it has less SA to cover. This is an issue.
Control of the ASL height methods?
Active or Passive control.
Passive: Mucous part acts as a reservoir and can passively move into the mucous layer.
Active: Net transport of ions into the PCL controls the osmololity so water will follow/ be reabsorbed.
In vitro the ASL settles at what height? How?
2001 study:
7um- if artifically add water to Cultured human airway epithelial cells to make the layer rise (25um) it will reduce and stabilise at 7um in just over a day.
If also measure Vte, follows the same pattern- the transepithelial potential changes to rescue the height, e.g. upregulation of transporters- reabsorb ions so water decreases so height decreases.
What height is the ASL at in vivo?
around 14um- which can be given in vitro if the layer is mechnically stimulated like the rhythm of breathing.
What inhibits the Vte change as the ASL layer is trying to be rescued after artificially increasing it?
2001 study:
Bumetanide blocked cl- secretion:
- At 0hrs 18% of he Vte is blocked
- At 48hrs 55% of the Vte is blocked (much bigger action at rest)
Amiloride blocks Na absorption:
- At 0hrs 65% inhibition of Vte
- At 48 hrs- 30%
At 0hrs- reabsorbing more and secreting less- osmolality decrease, water reabsorbed, layer decreases.
NKCC1 inhibited by?
bumetanide and Furosemide
bumetanide inhibits?
NKCC1 on basolateral airway epithelial cells.
ASL height determined by which solutes?
Tha balance between Cl- secretion (NKCC1 Baso and apicalCFTR) and Na absorption (apical ENaC).
What are the 5 key channels on the airway epithelial cell?
Apical: ENaC and CFTR.
Basolateral: Na/K (3Na out: 2K in)- sets the driving force for Na in through ENac and NKCC1 (with cl). Now lots of K inside needs a K channel to leak out.
Virus that affects the balance in the respiratory tract?
respiratory syncytial virus. Nasal conjestion (runny nose), bronchiolitis (20% of hospital administrations for children) and Pneumonia in adults.
How can the effect of RSV be measured? Result?
The Enac current is measured before and after the RSV ( by addition of amiloride to see difference in overall voltage change)
Without RSV the voltage change after amiloride is the same 1 hour later (the Enac current therefore is the same). But with the addition of RSV aftert he first measurement, the second had an overall rise in voltage but the same voltage after amiloride addition- meaning that the amount amiloride reduced the voltage was less as there was less Enac to inibit but it inhibited it to the same final amount. short circuit current was reduced.
THEREFORE RSV inhibits ENaC.
What does the inhibition of ENaC by RSV mean?
If block EnaC, less Na is absorbed so more in the airways- the excess fluid is lost through the nose- runny nose.
Through what mechanism does the RSV act?
RSV activates PKC which inhibits ENaC. It binds to glycolipids also.
How do we know how the RSV works?
NA – neuoaminidase, inhibits binding to glycoproteins (no impact- Enac Isc same as RSV alone)
BIM – PKC inhibitor
PPMP – inhibits binding to glycolipids
both inhibited the action of RSV so stopped the virus functioning.
Other viruses use which methods to influence the PCL?
Influenza- PKC and glycoproteins
Parainfluenza- Glycolipids, ATP release- inhibits ENaC.