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A BMS346 Epithelial Physiology > Overview and review of basic concepts > Flashcards

Overview and review of basic concepts Flashcards

1
Q

What is a key property of epithelial cells?

What does this allow?

A

They are POLARISED - have different transport proteins on their apical and basolateral surfaces

Allows NET transport of ions and water

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2
Q

How can epithelial cells be arranged?

A

In sheets or in a tubular shape

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3
Q

How are epithelial cells arranged in the upper airways?

A

In tubular structures

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4
Q

What are the 2 ways that net transport can occur?

A

TRANSCELLULAR - across the cell (through ion channels in the apical and basolateral membrane)

PARACELLULAR - between the cells

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5
Q

What is a key difference between transcellular and paracellular transport?

A

Transcellular transport –> CONTROLLED/REGULATED transport of ions, solutes and water

Paracellular - no channels –> no regulation

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6
Q

What determines if an epithelium is tight or leaky?

What is this?

A

The transepithelial resistance (Rte)

The resistance across the epithelium to movement

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7
Q

What does the type of epithelium determine?

A

Which DIRECTION different ions and solutes move

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8
Q

What does a high Rte mean?

A

Harder to move ions across the epithelium - not a lot of transport occurs across the epithelium (in terms of paracellular transport)

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9
Q

What is the difference between the Rte of a leaky and a tight epithelium?

A

Leaky Rte < 200 Ohmscm2 (low resistance - lots of transport across the epithelium)

Tight Rte >2000 Ohmscm2 (high resistance - not a lot of transport across the epithelium)

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10
Q

What are 4 examples of leaky epithelia?

A
  • Proximal tubule (kidney)
  • Gallbladder
  • Small intestine
  • Choroid plexus
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11
Q

What are 3 examples of tight epithelia?

A
  • Distal tubule (nephron)
  • Stomach
  • Frog skin
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12
Q

What is Rte determined by?

Why?

A

PARACELLULAR permeability

As the net transport in terms of TRANSCELLULAR permeability is similar for all different types of epithelium

SO, what makes cells tight or leaky –> how much transport is occurring BETWEEN the cells

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13
Q

What are the contacts between the cells in the epithelium called?

How are they different in tight and leaky epithelia?

A

Tight junctions

Tight epithelia - TIGHT tight junctions (not a lot of paracellular transport)

Leaky epithelia - LEAKY tight junctions (a lot of paracellular transport occurring)

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14
Q

What is Vte?

A

The transepithelial potential - potential that exists ACROSS the epithelium

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15
Q

What determines the transepithelial potential?

A

The SUM of the membrane potentials across the 2 epithelial membranes (apical and basolateral)

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16
Q

How is the Vte generated?

A

Net ion/charge flow across the membranes

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17
Q

What is the difference between the Vte in tight and leaky epithelium?

A

Leaky:
- Have a very small (1/2mV) or absent transepithelial potential

Tight:
- Vte ~50mV (LARGE)

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18
Q

Why is the transepithelial potential for leaky epithelial absent/low?

A

Vte is generated by TRANSCELLUAR transport (across the cell)

But in a LEAKY epithelium - leaks back again (paracellularly) as soon as transported across the cell –> cannot sustain the potentials

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19
Q

What is the difference between flux in a tight and leaky epithelium?

A

Leaky:

  • Large IOSMOTIC (followed by water PARACELLULARY)
  • Transcellular and paracellular transport

Tight:

  • Small flux
  • Only TRANSCELLULAR
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20
Q

How can the Vte be measured?

When is Vte measured?

A
  • Reference electrode at 0mV
  • Electrode in the APICAL surface of the epithelium

Vte measured - TIGHT epithelium

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21
Q

Is Vte +ve or -ve?

A

Can be +ve OR -ve depending on the net movement of charge across the membrane:

  • More anions or less cations (at the APICAL membrane)–> -ve Vte
  • More cations or less anions (at the APICAL membrane) –> +Vte
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22
Q

Describe the epithelium of the principle cell of the kidney

A
  • ve and large Vte:

- Tight epithelium

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23
Q

Describe the principle cell model in the kidney in terms of Vte

A

Basolateral membrane:

  • NaKATPase
  • K+ channel

Apical membrane:
- ENaC (Na channel)

Na through apical and basolateral membrane (caries a +ve charge)

  • -> loss of +ve charge from the apical side to the basolateral side
  • -> leaves behind -ve charge at the apical membrane

–> -Vte

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24
Q

What does the NaKATPase pump and the K channel in the basolateral membrane do?

How?

A

Sets up and maintains the electrochemical driving force for Na uptake across the APICAL membrane:

1) -ve intracellular potential
Net loss of 1 +ve charge

2) Low intracellular Na
Pumps 2 x Na out and 1 x K into the cell

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25
Q

In which direction does Na travel through ENaC?

Where does this then go?

A

INTO the cell from the APICAL environment

Na then PUMPED by the NaKATPase channel across the basolateral membrane

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26
Q

Describe the epithelium of the thick ascending limb of the kidney

A

Basolateral:

  • NaKATPase
  • K channel
  • Cl channel (Cl out)

Apical:

  • NKCC2
  • ROMK (K out)
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27
Q

What is the function of the NKCC2 channel?

A 
Transport:
1 x Na
1 x K
2 x Cl 
INTO the cell
28
Q

What is the drives the function of the apical NKCC2 channel?

A
  • Low intracellular Na
  • -ve intracellular potential

Both of which are set up and maintained by the NaKATPase pump in basolateral membrane

29
Q

What is the net charge through the NKCC2 channel?

A

ElectroNEUTRAL (no net movement of charge)

30
Q

What happens to the ions once they have been absorbed into the cell through the apical NKCC2 channel?

What does this mean for the Vte of the cell?

A
  • Na and 2 x Cl are reabsorbed at the basolateral membrane (through the NaKATPase and the Cl)
  • K ions are RECYCLED through the apical ROMK channels

Therefore only 1 net +ve charge across the basolateral membrane but 2 net -ve

Vte = +ve (due to loss of more -ve charge from the apical membrane)

31
Q

How can we determine changes in epithelial function?

A

Monitor changes in Vte - change in response to manipulation

32
Q

What is electrophysiology used for?

A

To look at the FUNCTION and PHYSIOLOGY of epithelia through measurements of:

1) Current
2) Potential

33
Q

How is a current generated?

A

Ion movement through a channel

34
Q

What are 4 electrophysiology techniques and what do they measure?

A

1) Intracellular micro electrode - measure IC membrane potentials (Vm)
2) Patch clamp - single channel or cell current, Po
3) Two electrode voltage clamp - total cell current
4) Ussing chamber - WHOLE epithelial function (Vte, Rte and single cell current (SCC))

35
Q

What are similarities/differences between the patch clamp and the two electrode voltage clamp method?

A

Similarities:

  • Clamp the potential and measure the CURRENT
  • Measure total cell current

Differences:

  • Patch clamp - can measure single cell currents?
  • Patch - 1 electrode, smaller cells (mammalian)
  • 2 electrode - 2 electrodes, larger cells (xenopus)
36
Q

What can the Ussing chamber technique used for?

A
  • Looking at WHOLE epithelial function/behaviour (not single cell)
  • POPULATION of cells
37
Q

As well as identifying in the epithelium is tight or leaky, what does the Rte identify?

A

If the tissue is VIABLE - if the Rte is not in the range you would expect –> tissue is dead

38
Q

What is the SCC?

A

Short circuit current:
- Indirect measurement of the net flux of ions across the membrane

  • Amount of current needed drive Vte –> 0mV
  • EQUAL and OPPOSITE to the net current flowing across the epithelium in the ussing tecnique
39
Q

How is SCC calculated?

Why calculated in this way?

A

Using the Vte and Rte values recorded from the Ussing chamber technique

Cannot directly measure the net current flow across an epithelium (have to work out the equivalent)

40
Q

What is the Nernst of K?

41
Q

What is the Nernst of Na?

42
Q

If measure Vm close to the Nernst of ion X what is this an indication of?

Why in reality is the Nernst not equal to the Vm of ion X?

A

Indication that the membrane has a high selectivity for the ion X (dominant channels open in the membrane are X channels)

In reality:
- More than one ion channel open in the membrane –> drives the Vm towards the Nernst of that ion

43
Q

How can the protein channels in the membrane be determined?

A

1) Measuring the Vm and seeing what Nernst it is close to

2) Adding ion channel blockers and identifying the shift in the Vm

44
Q

If Na channels are open in the membrane, what happens to the Vm when add amiloride?

A

Amiloride blocks Na channels:

  • No longer contribute to Vm
  • Vm moves AWAY from the Nernst of the ion moving through the blocked channel (Na)
  • Vm moves TOWARDS the Nernst of the ion moving through other channels in the membrane (membrane becomes more selective for these ions)
  • Eg. Ek
45
Q

What is the selectivity of an ion channel?

46
Q

What is Vrev?

47
Q

What does it mean if the Vrev is close to the Nernst of an ion?

48
Q

If add barium to a cell and it decreases the size of the currents, what does this show?

A

Shows K channels mediate the current as barium block the K channels

49
Q

Describe the Ussing chamber technique

A
  • 2 chambers with sheet of epithelium in the middle
  • Experimental solutions either side of the sheet of epithelium
4 electrodes (2 each side):
- First pair (reference and recording) - measure the Vte
  • Second pair - injects the current across the epithelium (magnitude of this current is determined (SET/KNOWN) by a current injection box)
50
Q

What happens when inject the current in the Ussing chamber experiment?

A

The Vte shifts

51
Q

What is the shift of Vte when a known current is injected (in the Ussing chamber) dependant on?

Why?

A

How much the Vte shifts is dependant the RESISTANCE

Ohms law:
Vte = IR (I is known)

52
Q

How work out the Vte of the epithelium from ussing chamber technique?

A

Directly measured from the electrodes

53
Q

How can the Rte of a the epithelium from ussing chamber technique?

A

Rte = change in V/ I injected

Rearrangement of Ohms law Vte = IR

54
Q

How can Isc be calculated from the ussing chamber technique?

A

Isc = Vte (in the absence of current injection) / Rte

55
Q

Describe the graph that is recorded from the ussing chamber technique

A

Line at the top - Absolute Vte (when no current injected)

Deflections downwards - Change in Vte when there is an injection of a known current

(change/known current = Rte)
(Isc = Absoulte Vte (line @ the top) / Rte)

56
Q

What happens to Vte in the presence of Lub?

Why?

A

Vte increase:

  • Lub is a prostaglandin mimic –> activates receptors
  • Stimulates cAMP
  • Activates PKA
  • Activates the CFTR Cl receptors
57
Q

What happens to Vte in the presence of CFTR inhibitor after Lub addition?

Why?

A

Increase in Vte (with Lub) is REVERSED

Due to a blockage of Cl secretion

58
Q

What are the ion channels present in the upper airway cell?

A

Basolateral:

  • NaKATPase
  • K channel
  • NKCC1

Apical:

  • CFTR
  • ENaC
59
Q

What happens to the ions that come through the NKCC1 channel in the basolateral membrane of the upper epithelial cell?

A
  • Na recycle across the basolateral membrane
  • K recycles across the basolateral membrane
  • BUT the Cl ACCUMULATES inside the cell
60
Q

What happens in the upper airway epithelium cell when CFTR channel opens? Why?

A

Cl LEAVES the cells (net Cl secretion) as Cl has accumulated inside the cell from the NKCC1

61
Q

What happens in the upper airway epithelium cell when the ENaC channel opens? Why?

A

Na influx through ENaC

Due to the electrochemical potential (low Na and -ve IC0 set up by NaKATPase and K channels in the basolateral membrane

62
Q

What is the function of CFTR and ENaC in the upper airway epithelium cell?

A

BALANCE between Cl secretion (through CFTR) and Na absorption (through ENaC)

Sets the HEIGHT of the periciliary layer (PCL) that is SPECIFIC and OPTIMUM

63
Q

What is the function of the PCL?

A
  • Layer which the cilia (from the bronchial epithelial cells) project into

First line of defence against infection:
- Cilia beat and move the PCL and the mucus on the top of the PCL up the respiratory tract

  • Swallow this mucus (contains trapped viruses and bacteria and anything else breathed in)
64
Q

Where is the PCL present?

A

Sits on top of the epithelial cells of the respiratory tract (upper airway and alveolar cells)

65
Q

What feature of the PCL is important in mucous clearance?

What happens if this is disrupted?

A

The HEIGHT

Disruption of the height - impacts on the ability to clear pathogens from the respiratory tract

A BMS346 Epithelial Physiology (13 decks)

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