Epithelial Chloride Channels 1&2

Question 1

Why is epithelial fluid secretion important

Answer 1

Important in lung and intestine function

Question 2

What happens when epithelial fluid secretion goes wrong

Answer 2

Secretory diarrhoea (too much fluid)
CF (too little fluid)

Question 3

Why is epithelial fluid secretion important in the gut

Answer 3

For normal digestive function
Maintains lumenal contents in liquid state (absorption)
Mixes with digestive enzymes
Presents nutrients to absorptive surface
Washes away injurious substances
More than 90% reabsorbed

Question 4

Where does most fluid reabsorption occur

Answer 4

Ilium

Question 5

What causes secretory diarrhoea

Answer 5

They use the physiological control mechanisms to induce chloride release and therefore water.

Question 6

What causes cholera

Answer 6

Infected drinking water with sewage water

Question 7

How is cholera treated

Answer 7

Salt and sugar rehydration mix as glucose and uptake is sodium dependent in the gut and water follows by osmosis causing water reuptake.

Question 8

What happens in CF the lungs and GI tract

Answer 8

Little to no mucus secretion due to chloride channel dysfunction.
This causes the mucus that overlies the epithelium to become sticky and impenetrable - causes malnutrition and intestinal ileus.
Lungs bigger issue - sticky and dehydrated mucus where mucus is essential for gas exchange

Question 9

What is the epithelial type in the lungs

Answer 9

Pseudostratified columnar epithelium with cillia and goblet cells.

Question 10

How do the cilia have a role in the lungs and how does it work

Answer 10

The cilia help move mucus along by a water barrier between the cillia top and the mucus.

Lack of water - mucus too low and thick restricting the cillia movements
Excess of water - mucus too far away from cillia so cant move the mucus along.

Question 11

What is the anatomical structure of the microvilli and epithelial cells

Answer 11

Each epithelial cell has many microvilli on top of them and each cell is seperated by tight junctions. This allows transcellular transport (through the cell) and paracellular transport (between cells via the tight junction)

Question 12

How do chloride and water get through the epithelium

Answer 12

Cl - transcellular
H2O - paracellular

Question 13

What is the difference between the role of crypts and villi

Answer 13

Crypts - secrete but dont absorb
Villi - absorb but dont secrete

Question 14

How doe chloride enter the cell on the basolateral surface

Answer 14

Via the sodium-potassium-chloride cotransporter –> 2 Chlorides, potassium and sodium enter simultaneously (electroneutral).

The sodium leaves via 3 sodium- 2 potassium channel.
The potassium leaves via leaky potassium channels.
The 2 potassium that comes in via the Na-K pump stays which makes it negative inside the cell.

This negative charge is good as it encourages chloride out of the cell, the only way to do this is via the lumen apical membrane.

Question 15

How does chloride get out of the cell

Answer 15

Via chloride channel in the apical membrane.

Question 16

How does water enter the lumen

Answer 16

Between cells via osmosis

Question 17

What are the two pathways of chloride release from the apical membrane

Answer 17

cAMP mediated
Calcium ion mediated

Question 18

How is the sodium potassium ATPase known to be present

Answer 18

Ouabain (inhibitor of this ATPase) blocks Cl secretion when put on the basolateral surface

Question 19

How is the Na/K/2Cl co-transporter known to be present

Answer 19

Furosemide (loop diuretics) known to inhibit chloride secretion when present on the basolateral surface only.

When used oubain and loop diuretic then the co-transporter doesnt work as require all 3 ion types.

Question 20

What will vasoactive intestinal polypeptide do

Answer 20

Stimulates cAMP will increase cl and K secretion

Question 21

What was shown regarding potassium channels via VIP

Answer 21

K secretion was <5% of Cl secretion therefore K must be recycled across the basolateral membrane.
VIP stimulates increased basolateral efflux but had no effect on apical efflux.

Question 22

What are the 3 types of Cl channels identified in T84 cells

Answer 22

• cAMP activated
• Calcium activated
• Cell swelling activated

Question 23

What Cl channel is important for CF

Answer 23

cAMP-activated channel - Cystic Fibrosis Transmembrane conductance Regulator (CFTR)

Question 24

How does CF effect

Answer 24

1/2500 Caucasian live births

Question 25

Give some features of CF

Answer 25

Blockage of pancreatic ducs
Inability to clear mucus in lungs
High NaCl in CF sweat

Question 26

Who was paul quinton

Answer 26

1983 - key scientist linking salty sweaty and CF and discovered CF was due to Cl channel dsyfunction

Question 27

How did quinton discover the difference in CF sweat to normal sweat

Answer 27

Higher potential difference across CF sweat duct epithelia compared to normal.

10 fold higher sodium permeability in CF sweat compared to normal.

Confirmed with SO4 lack of permeability in normal tissue but was just slightly less permeable in CF sweat glands than NaCl suggesting that Cl permeability is near 0 - WR

This reduction in Cl permeability accounts for CF symptoms

Question 28

How do sweat cells work normally

Answer 28

Sodium comes into the sweat duct followed by chloride in a separate channel.

Once sodium enters, it is pumped out of the cell via a sodium-potassium ATPase and the potassium that is pumped into the cell is pumped out via leaky potassium channels.

Chloride that has entered the cell with the sodium is pumped out through chloride channels.

Question 29

How do sweat glands work in CF patients

Answer 29

Sodium enters the cell like before. Once sodium enters, it is pumped out of the cell via a sodium-potassium ATPase and the potassium that is pumped into the cell is pumped out via leaky potassium channels.

However, in CF patients no chloride channels are present therefore no salt is brought into the cell or pumped out the other end. As there is a large negative charged built up in the lumen via the chloride build up, this pushes sodium out through the other side even more. Resulting in excessively sodium-based sweat.

Question 30

What did Michael Welsh discover

Answer 30

Broncholavage - found Chloride apical channels in the tracheal epithelium via patch clamp techniques.

The channel type - outward rectifying chloride channels

Question 31

What was the role of the outward rectifying Cl channels

Answer 31

They are better at bringing chloride into cells than out of cells

Question 32

What did Frizzell and seperately Welsh and Liedtke discover about outward rectifying chloride channels in CF airway cells

Answer 32

Failure of beta-adrenergic agonists to activate ORCCs in CF airway cells

Question 33

What did schoumacher et al in 1987 discover about CF phosphorylation

Answer 33

Failure of protein kinase A to activitie ORCCs in CF epithelial cells.
However, ORCCs were present in CF cells and could be activated by strong depolarisation (was greater than physiological levels).

Question 34

What did Li et al discover in 1988 regarding CF

Answer 34

Same conclusions as schomacher 1987

Question 35

What did Li et al discover in 1989 regarding CF

Answer 35

Isolated ORCCs via excised cells and found that the defect was still present so must be associated with regulatory protein or channel.
PKC activated ORCCs from normal but not CF cells - may also be involved.
Defective transduction from posphorylation sites to channel gating mechanisms.

Question 36

What did Gray et al discover in 1989 regarding Cl channels

Answer 36

Two types of chloride channels - high numbers of small linear conductance Cl channels (SLCCs) in the apical membrane of pancreatic duct cells
ORCCs are present but infrequent.
SLCC activated by secretin and cAMP

Question 37

What question was raised by Gray et al regarding Cl channels affected in CF

Answer 37

Could these SLCCs that he discovered be the CL channels affected in CF instead of ORCCs

Question 38

What was the difference between SLCCs and ORCCs regarding I/V relationship

Answer 38

SLCC - small current but equally linear in the way and out the way. Low conductance - small channel. Really hard to measure due to the small current even at highly positive and negative voltages so were likely to reason for them being missed.

ORCCs - much more transport/current at higher positive depolarising voltages and much less at negative voltages. (outward rectifying - bringing chloride into cells). High conductance - large channel.

Question 39

What was discovered between ORCC and SLCCs when looking at their ability to transport chloride

Answer 39

ORCCs - Iodine>Bromide> chloride (poor Cl channel)

SLCC - Bromide>chrloide>iodine (better Cl channel)

Question 40

What activates the ORCC and SLCC channels

Answer 40

ORCC - ? Unsure as the PKA and PKC activation seen previously.

SLCC - cAMP and PKA

Question 41

What pathogen causes cholera

WIDER READING - Broeck 2007

Answer 41

Vibrio cholerae bacteria - cholera toxin (protein enterotoxin secreted by the bacteria)

Question 42

How does cholera toxin affect the gut epithelial cells?

WIDER READING - Muanprasat and Chatsudthipong 2013

Answer 42

Cholera toxin binds to GM1 ganglioside receptors on the apical membrane of ileal cells and undergoes retrograde vesicular trafficking to the endoplasmic reticulum, where it exploits endoplasmic reticulum-associated protein degradation systems to release a catalytic A1 subunit of cholera toxin into the cytoplasm.
CT A1, in turn, catalyzes ADP ribosylation of α subunits of stimulatory G proteins, leading to persistent activation of adenylate cyclase and an elevation of intracellular cAMP. Increased intracellular cAMP, causes phosphorylation and activation of the cystic fibrosis transmembrane conductance regulator (CFTR protein) leading to the ATP mediated efflux of chloride ions pumped out of the enterocyte followed by water and therefore causing severe diarrhoea.

Question 43

Why is CF believed to be so common

WIDER READING - Hanson 1988

Answer 43

Believed to be genetically advantageous to have 1 of two copies of the gene (heterozygous) because its believed they are more resistant to secretory diarrhoea such as cholera

Question 44

What contradicting evidence is there for the CF diarrhoeal advantage

WIDER READING - Cuthbert et al 1995

Answer 44

Used heterozygous CF mice and normal WT control mice and tested their chloride secretion in gut epithelia to different diarrhoeal compounds - forskolin, vasoactive intestinal polypeptide (VIP), isoprenaline, cholera toxin, heat-stable enterotoxin (STa), guanylin, carbachol and lysylbradykinin

No significant differences in responses of tissues of the two genotypes were found.

Reasosn why - (a) other non-cystic fibrosis transmembrane conductance regulator (non-CFTR) transport processes are involved, (b) prolonged exposure to secretagogues is required, or (c) delta F508 CFTR is responsible for the protective effect.

Question 45

As mice have been known to be difficult CF models due to lack of disease symptoms, why are pigs a good model

WIDER READING - Rogers et al 2008

Answer 45

Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF

Question 46

What role does CFTR have in the pancreas?

WIDER READING - Wilschanski et al 2013

Answer 46

CFTR is expressed on the apical membrane of epithelial cells in the small pancreatic ducts and facilitates the transport of chloride and bicarbonate that produces alkaline fluid in ducts

Question 47

How does CFTR facilitate in pancreatic bicarbonate secretion

WIDER READING - Lee et al 2012

Answer 47

First, secretion of bicarbonate is stimulated in the proximal duct, causing accumulation of bicarbonate within the ductal cell cytoplasm and leading to an osmotic secretion of the anion. This process, coupled with sodium influx, causes the proximal duct to absorb a portion of chloride and secrete bicarbonate, via CFTR, along with a large amount of pancreatic fluid. Within the distal pancreatic duct, CFTR primarily functions as a bicarbonate channel, due to the low chloride content of the pancreatic fluid in this region

Question 48

How do CF pancreatic secretions cause obstruction

WIDER READING - Kopelman et al 1988

Answer 48

In CF, altered composition of pancreatic secretions include lower pH, reduced secretory volume (including water via osmosis) and higher protein content; these factors are thought to alter zymogen secretions, leading to obstruction

Question 49

How does pancreatic obstruction affect the neonatal pancreas
Wider reading - Meyerholz et al 2010

Answer 49

With progression, acinus plugging and dilatation cause epithelial injury and destruction, accompanied by inflammation, fibrosis and fatty infiltration/replacement occur - this was developed using a pig model with induced CF due to CFTR KO.

Question 50

Is the endocrine pancreas affected?

WIDER READING - Barrio et al 2015

Answer 50

Yes in adulthood - CF induced DM is present in 50% of CF patients

Question 51

What causes CF diabetes

WIDER READING - Barrio et al 2015

Answer 51

Has features of type 1 and type 2 - it is characterized by both a loss of functional β cell mass as well as also having varying extents of insulin resistance

The two major mechanisms thought to play a role in the development of CFRD include decreases in islet cell mass and β cell dysfunction - Decreases in islet cell mass have been associated with exocrine destruction, fibrosis and fatty infiltration, as well as islet amyloid deposition

Question 52

How have porcine models been treated for meconium ileus

WIDER READING - Stotlz et al 2013

Answer 52

Intestinal obstruction occurs in 100% of CF pigs at the time of birth, but transgenic expression of CFTR cDNA under the control of the intestinal fatty acid-binding protein (iFABP) by placing porcine CFTR in the iFABP promoter, this allows for the alleviation of meconium ileus. However, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time.

These data indicate that expressing CFTR in the intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus.

Question 53

How does CF gut respond to heat-stable E. coli enterotoxin

WIDER READING - Field and Semrad et al 1993

Answer 53

ST enterotoxins are a known stimulator of intestinal guanylate cyclase which is known to phsophroylate CFTR inducing Cl channel opening and secretion of CL and water, however as CF patients dont have CFTR - no effects of enterotoxin in vivo shown in CF patients.

Question 54

Does the gut contain calcium-activated chloride channels

WIDER READING - Barrett 1993

Answer 54

Likely - chloride secretion to be essentially unaffected in cystic fibrosis when T84 cells are exposed to calcium agonists.
Believed that this may be due to opening of potassium channels when grown on permeable supports -The resulting fall in intracellular potassium is proposed to provide the driving force for chloride uptake and its subsequent exit across the small proportion of apical chloride channels
that are constitutively open

However - jury still out on this. Possible some chloride channels exist and can have theruptic use

Question 55

What use might calcium-dependent chloride channels have therapeutically

WIDER READING - Huang et al 2012

Answer 55

Known to be in lungs but unsure of intestines - in lungs the CFTR maintains the airway surface liquid height (distance between cillia and mucus via water) but no significant role of CDCC of ASL height in CFTR KO.
Believed CDCC may have more of an acute role of raising ASL height via histamines, inflammation and asthma as TMEM16A expression (key component of CDCC) is upregulated when cytokines are raised.

Question 56

How was the R domain function identified

WIDER READING - Widdicombe and wine 1991

Answer 56

Deletion of the R-domain causes CFTR-generated CI channels to be open even in the absence of increased intracellular cAMP.This supports the suggestion that the R-domain acts as a gating particle, which in its unphosphorylated state closes the CFrR to the passage of CI-. Once the R-domain is phosphorylated, however, one role of ATP hydrolysis may be to transport it out of the channel mouth.

Question 57

How might thiazolidinone CFTRinhibitor -172 (lab made inhibitor) help in treat cholera

WIDER READING - Thiagarajah et al 2002

Answer 57

In a mouse closed-ileal loop model, injected fluid was rapidly cleared and remained unaffected by CFTRinh-172 administration (Figure 4a), an important prerequisite for antidiarrheal application of a CFTR inhibitor. Injection of cholera toxin into loops produced fluid secretion over six hours after a slow onset (Figure 4b). In a dose-response study, a single intraperitoneal injection of CFTRinh-172 (just after cholera toxin infusion) reduced fluid accumulation by ∼90%, with an IC50 of ∼5 μg CFTRinh-172 (Figure 4c). Inhibition of fluid accumulation was seen when CFTRinh-172 was administered three hours before or after cholera toxin

Question 58

What is the movement of sodium into the cell and chloride into the cell

Answer 58

An inward current - because positive ion

An outward current - because negative ion so opposite

AND VICE VERSA