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BMS346 > Salt secretion > Flashcards

Salt secretion Flashcards

1
Q

2 Different types of pathways across epithelial

A
  • Transcellular= across cells- transport protein

- paracellular= between cells

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2
Q

Polarity of cells

A

All epithelial cells are polarised (apical and basolateral membrane)

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3
Q

Where are tight and leaky junctions?

A

Leaky

  • proximal tubule
  • gall bladder
  • small intestine
  • choroid plexus

Tight

  • distal tubule
  • stomach
  • frog skin
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4
Q

Difference between tight and leaky

A

leaky <200 cm^2

tight > 2000 ohms cm^2- high resistance, less transport

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5
Q

What determines a tightness>

A 
paracellular determines resistance 
numbers= resistance 
transcellular= same in tight and leaky 
- depends on DF for absorption and secretion 
- size of hole= how much can transport
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6
Q

General properties of Tight junction

A

Rte >2000
Vte~50mv
Flux small
low H20

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7
Q

Genereal properties of leaky junction

A

rte <200
vte ~0mv
Flux large (isosmotic )
high H20 permeability

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8
Q

Transepithelial potential

A

Sum of individual membrane potentials

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9
Q

How Is a VT generated?

A

Use frog skin- sodium resabsorption
epithelium= measure of how much conductance there is
- vte= more anions
+ vte= less anions, more cations

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10
Q

if there is a higher resistance

A

Less transport

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11
Q

Preparations to make to measure VT

A

type of cell- fresh tissue/cells, cultured, whole organisms
experimental approach- WT, KO, mutant
pharmacological agents
Overexpression proteins in cells

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12
Q

What method would you use to show protein presence/ location?

A 
presence= western blot 
location= immunostaining
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13
Q

Method to find mRNA presence?

A

polymerase chain reaction

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14
Q

Method to find the transport function?

A

flux radioactive compounds

electrophysiology

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15
Q

Different methods of electrophysiology and what they measure?

A

intracellular microelectrode- Ic potential
patch clamp- single current/ cell current
2 electrode voltage clamp- cell current
ussing chamber- transepithelial potential and resistance

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16
Q

Ussing chamber- VTE, RTE, ISc method

A
  1. inject a known concentration

2. the resulting shift in vte is measured

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17
Q

What happens to Vte if there is more anions/ less cations?

A

amiloride blocks Na channels
less Na transport
reduced loss pos charge
Vtw decrease due to loss of Na

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18
Q

What is the model tight epithelium

A

Frog skin
absorb Na from environment
outside= apical
inside= basolateral

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19
Q

Why do we use frog skin?

A
  • robust, easy to get, cheap and useful bc of how it absorbs Na
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20
Q

What can frog epithelium be used for to compare with humans?

A 
- collecting duct 
distal colon
salivary gland 
sweat duct 
airway surface epithelium
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21
Q

Short circuit current technique

A

Amp- 2x electrodes to measure the vte

  1. kerb solution in chambers either side if looking at Na uptake
  2. set so no conc gradient - no pot difference
  3. Add frogs skin- pot difference starts as tight junction
  4. Na lost from basolateral to apical= neg potential
  5. validation= uses radioactive tracer (Na )into chamber 1 and measure amount in chamber 2 to work out the amount of Na transport per unit of time
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22
Q

What is the flux of passively and actively transported ions

A

0- passive- Jnet Cl=0

isc= J net Na = 10.4 mol/s

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23
Q

To calculate the total current

A

using ussing chamber

did ohms law AND worked out current they would expect to see for Na

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24
Q

Na influx and outflux

A 
in= apical to basolateral 
Out= basolateral to apical
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25
Q

If you take influx - outflux =

A

similar to ussing chamber total current net transport

net= almost all Na, apical to basolateral

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26
Q

Active Na reabsorption by

A

Na/K ATPase- active

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27
Q

K recycling by

A

passive, K channel- wont be able to find using ussing chamber

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28
Q

What does Na channel on the apical membrane help with and how is this seen?

A

Move membrane potential to nerst for Na
took electrode and measured Na conc change from outside cell- nerst to Na
If apical membrane is permeable to Na- nerst=Na

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29
Q

What is the effect of amiloride on epithelial Na transport?

A

Add amiloride, decrease lumen -ve voltage due to decrease to Na absorption

  • shifts potential from -1 to 0
  • blocks Enac channels
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30
Q

What did they use to measure amiloride on epithelial Na transport?

A

Human colon biopsy
Ussing chamber work
* note much lower pd than in frog skin = -vte - not as tight, leak back
Human= -1mv not large Vt (Na absorption)

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31
Q

How to get a human nasal biopsy and why is it used?

A

less invasive than airway epithelium (easy to scrape out )
properties the same
- Na absorption
- Cl secretion

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32
Q

What is Vm

A

Cell potential

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33
Q

What is the evidence that there are Na selective channels in the apical membrane of the epithelium airway- What does Na entry do?

A

Tends to depolarise cell
Add amiloride it hyperpolarises cell
change potential- and move nerst away from sodium

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34
Q

What did we know before the patch clamp analysis of Enac?

A

looked at whole cell epithelium or whole membrane- predict apical Na channel
all indirect evidence add up to this point
predicted opening and closing

35
Q

What does the patch clamp analysis of Enac (1970) show?

A

revolutionised ability to look at single ion channels and how they are impacted

36
Q

What does flickering currents and size deflections show?

A

single ion channel opening

difficult to read as you get level of noise

37
Q

What happens to size deflections as you increase the amount of Na?

A

More Na through ion channel the bigger the current recoreded

38
Q

molecular approaches

A

Gene to identify protein
introduce mutation
wanted to clone epithelium ion channel- couldn’t do it

39
Q

Conventional cloning

A

Isolate protein and sequence
using amiloride affinity column
1. column with beads with amiloride on
2. run protein down bead and they stick (Enac and amiloride)

40
Q

Why couldn’t they do normal cloning?

A

Amount of protein in upper airway/ biopsy is too small

need large amounts

41
Q

What is the native tissue and what is the native channel properties ?

A

kidney/ colon

  • single channel= spc
  • highly amiloride sensitive
  • highly Na sensitive
42
Q

Investigation using tissue source from rat colon

A

rats fed with low salt diet- fractioned colon mRNA (poly A RNA)

  • cDNA cloned in expression vectors
  • cDNA clibrary divided into 10 pools (Na sensitive current)
  • DNA
  • cRNA
  • Fractional assay- amiloride sensitive Na current and produces positive pool
  • divide into smaller pools until small= no current- chopped off bit that codes for ion channel
  • chop into 10 pools- purify more- final pool should be pure sequence that codes ion channel
43
Q

screening by functional expression

A

mRNA from cell and chop and inject into oocyte

see which chop of mRNA made Na selective current

44
Q

What induces Enac

A

via increased aldosterone

45
Q

results from functional expression experiment?

A

Bigger deflection= bigger function
pure clone gives smaller deflection than clone A
- show A subunit can make a channel- need others for full activity A+ B+Y= 10 Fold larger= enhancement

46
Q

Why might they not have been able to discover this?

A

If all subunits were needed for channel activity Enac would not have been cloned

47
Q

What is Enac

A

3 gene sequence that codes 3 subunits- a,b,y

48
Q

Mutations in Enac

A

GOF

  • liddles
  • hypertension
  • Nedd4- channel retrieval

LOF

  • PHA
  • pseudohypoaldosteronism
  • hypotension
  • Enac intact
49
Q

What is barters syndrome and Gitelmans?

A

loop diuretic NA/CL/K sensitive

Nacl sensitive

50
Q

What is ASL

A

Airway surface liquid

51
Q

How is mucous cleared?

A

virus gets stuck in mucous and up respiratory tract

airway defence against respiratory pathogens

52
Q

How does mucous move?

A

Cilia beat

53
Q

Epithelial cells

A

Control amount salt reabsorbed and height of layer

54
Q

PCL

A

Percilliary layer= height important mucous clearance

liquid sits above the apical calles

55
Q

Movement of ASL

A

Moves from proximal to distal
small airways- upper respiratory tract
* small airways converge in bronchial region ~50 therefore volume load

56
Q

Control of the ASL

A

Active- active ion transport controlling salt level in PCL

Passive- mucous layer acts as a reservoir

57
Q

Experiment to look at control of ASL in cultured human activity

A

Add liquid to top of cells (make too high)
monitor height overtime using fluorescence
1 day- height decrease
2 day– steady out to optimum height
transepithelial drops from 14 to 5

58
Q

How to mimic the ASL experiment In labs?

A

move cells back and forward
liquid moves mimicking breathing
optimum= 14 micrometres

59
Q

Why are butamide and amiloride used?

A

b- investigates amount of cl secretion going on

amiloride- plays a role in transport in cell

60
Q

What are the results after 48hours?

A

amiloride inhibits 30% but butamine inhibits 55%

shows to get optinmum height you need more Cl secretion and less Na- balance between Na/Cl

61
Q

What is Cl mediated by?

A

CFTR

  • high cl inside
  • open CFTR it secretes Cl
62
Q

Respiratory syncytial virus (RSV)

A

Respiratory pathogens disturb fluid balance respiratory tract

  • nasal congestion
  • bronchiolis in children
  • pneumonia in adults
63
Q

RSV and SSC in mouse trachea

A

Investigate the response to amiloride
add amiloride before virus- deflection before shift
add after virus- inhibits enac function

64
Q

What does the bigger deflection bar show?

A

bigger enac function
virus= transmission decreased
no impact= no virus
RSV inhibits Enac after 1 hour incubation

65
Q

What happens if you block Enac?

A

block Na uptake
excess fluid in nose
runny

66
Q

What are the 3 mechanisms for blocking Enac by the virus?- depends on the virus used

A
  1. PKC
  2. Glycoprotein
  3. Glycolipid
67
Q

RSV which pathway does it use

A

BIM= PKC inhibitor
NA= neuominidase- inhibits binding to glycoprotein
PDMP= inhibits binding to glycolipid
If virus needs one of these pathways you would expect to see a high bar amiloride action (amiloride cant work/ inhibit ENAC) in the presence of the inhibitor and RSV
*RSV uses glycolipid and PKC

68
Q

approaches of RSV, influenza and parainfluenza

A
  • RSV= PKC, Glycolipid
  • Influenza= PKC, glycoprotein
  • parainfluenza= Glycolipids, ATP release
69
Q

What is influenza?

A

glycoproteins and PKC
influenza kills ~ 36,000 individuals/ year USA
affects young and old
long term

70
Q

influenza contains many glycoproteins

A
  1. MI- matrix protein
  2. heamagglutinin - binds salic acid residue, PKC and transient inhibition Enac
  3. M2- forms activated amantadine and H channel which inserts into the apical membrane of most cells
71
Q

What is M2 glycoprotein?

A

long term inhibition of Enac in flu

72
Q

M2 decrease enac currents

A

normal- channel open lots

influenza- lots of periods where channels closed= decrease open probability, reduces how much there is

73
Q

How does M2 affect enac?

A

decreases amount of enac

the ones that are there have a decreased open probability

74
Q

Endocytosis of enac when M2 is there?

A

Enac+ m2= enac internalised so the amount of enac decreases

enac current @ 140mv in enac and m2

75
Q

What is liddles Enac

A

cant be endocytosed from membrane
mutation in M2
less inhibition

76
Q

What does influenza bind?

A

Binds enac and promotes endocytosis

77
Q

What are ROS and PKC involvement in Enac

A

ROS- reactive oxygen species
M2 activates PKC and ROS which inhibits Enac
look at ROS using GSH = antioxidant which scavenges mopping up ros

78
Q

What happens if you inhibits and GO6796

A

Inhibit PKC which block response to M2

79
Q

What does PAH do- LOF Enac?

A 
Salt washing 
hypotension- changes in kidney, low bp 
hyperkalemia- high plasma K 
metabolic acidosis
high renin and aldosterone
80
Q

PAH autosomal dominant

A
  • renal form
  • problems localised to kidney
  • mineralocorticoid receptor gene
81
Q

PAH 1 autosomal recessive

A

systemic form
multiorgan affect
Enac gene mutation- all subunits

82
Q

PAH1 and nasal surface liquid

A

liquid coming out has higher Na
not function enac
cant be reabsorbed

83
Q

How does PAH affect transepithelial potential and amiloride % inhibition?

A

Systemic is less negative
Cl probably still working- epithelial layer increase
cilia cant beat, move mucous= decrease= tract infection

BMS346 (5 decks)

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