Epithelial Chloride Channels 1&2 Flashcards
Why is epithelial fluid secretion important
Important in lung and intestine function
What happens when epithelial fluid secretion goes wrong
Secretory diarrhoea (too much fluid)
CF (too little fluid)
Why is epithelial fluid secretion important in the gut
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
Where does most fluid reabsorption occur
Ilium
What causes secretory diarrhoea
They use the physiological control mechanisms to induce chloride release and therefore water.
What causes cholera
Infected drinking water with sewage water
How is cholera treated
Salt and sugar rehydration mix as glucose and uptake is sodium dependent in the gut and water follows by osmosis causing water reuptake.
What happens in CF the lungs and GI tract
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
What is the epithelial type in the lungs
Pseudostratified columnar epithelium with cillia and goblet cells.
How do the cilia have a role in the lungs and how does it work
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.
What is the anatomical structure of the microvilli and epithelial cells
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)
How do chloride and water get through the epithelium
Cl - transcellular
H2O - paracellular
What is the difference between the role of crypts and villi
Crypts - secrete but dont absorb
Villi - absorb but dont secrete
How doe chloride enter the cell on the basolateral surface
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.
How does chloride get out of the cell
Via chloride channel in the apical membrane.
How does water enter the lumen
Between cells via osmosis
What are the two pathways of chloride release from the apical membrane
cAMP mediated
Calcium ion mediated
How is the sodium potassium ATPase known to be present
Ouabain (inhibitor of this ATPase) blocks Cl secretion when put on the basolateral surface
How is the Na/K/2Cl co-transporter known to be present
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.
What will vasoactive intestinal polypeptide do
Stimulates cAMP will increase cl and K secretion
What was shown regarding potassium channels via VIP
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.
What are the 3 types of Cl channels identified in T84 cells
- cAMP activated
- Calcium activated
- Cell swelling activated
What Cl channel is important for CF
cAMP-activated channel - Cystic Fibrosis Transmembrane conductance Regulator (CFTR)
How does CF effect
1/2500 Caucasian live births
Give some features of CF
Blockage of pancreatic ducs
Inability to clear mucus in lungs
High NaCl in CF sweat
Who was paul quinton
1983 - key scientist linking salty sweaty and CF and discovered CF was due to Cl channel dsyfunction
How did quinton discover the difference in CF sweat to normal sweat
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
How do sweat cells work normally
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.
How do sweat glands work in CF patients
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.
What did Michael Welsh discover
Broncholavage - found Chloride apical channels in the tracheal epithelium via patch clamp techniques.
The channel type - outward rectifying chloride channels
What was the role of the outward rectifying Cl channels
They are better at bringing chloride into cells than out of cells
What did Frizzell and seperately Welsh and Liedtke discover about outward rectifying chloride channels in CF airway cells
Failure of beta-adrenergic agonists to activate ORCCs in CF airway cells
What did schoumacher et al in 1987 discover about CF phosphorylation
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).
What did Li et al discover in 1988 regarding CF
Same conclusions as schomacher 1987
What did Li et al discover in 1989 regarding CF
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.
What did Gray et al discover in 1989 regarding Cl channels
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
What question was raised by Gray et al regarding Cl channels affected in CF
Could these SLCCs that he discovered be the CL channels affected in CF instead of ORCCs
What was the difference between SLCCs and ORCCs regarding I/V relationship
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.
What was discovered between ORCC and SLCCs when looking at their ability to transport chloride
ORCCs - Iodine>Bromide> chloride (poor Cl channel)
SLCC - Bromide>chrloide>iodine (better Cl channel)
What activates the ORCC and SLCC channels
ORCC - ? Unsure as the PKA and PKC activation seen previously.
SLCC - cAMP and PKA
What pathogen causes cholera
WIDER READING - Broeck 2007
Vibrio cholerae bacteria - cholera toxin (protein enterotoxin secreted by the bacteria)
How does cholera toxin affect the gut epithelial cells?
WIDER READING - Muanprasat and Chatsudthipong 2013
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.
Why is CF believed to be so common
WIDER READING - Hanson 1988
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
What contradicting evidence is there for the CF diarrhoeal advantage
WIDER READING - Cuthbert et al 1995
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.
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
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
What role does CFTR have in the pancreas?
WIDER READING - Wilschanski et al 2013
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
How does CFTR facilitate in pancreatic bicarbonate secretion
WIDER READING - Lee et al 2012
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
How do CF pancreatic secretions cause obstruction
WIDER READING - Kopelman et al 1988
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
How does pancreatic obstruction affect the neonatal pancreas
Wider reading - Meyerholz et al 2010
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.
Is the endocrine pancreas affected?
WIDER READING - Barrio et al 2015
Yes in adulthood - CF induced DM is present in 50% of CF patients
What causes CF diabetes
WIDER READING - Barrio et al 2015
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
How have porcine models been treated for meconium ileus
WIDER READING - Stotlz et al 2013
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.
How does CF gut respond to heat-stable E. coli enterotoxin
WIDER READING - Field and Semrad et al 1993
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.
Does the gut contain calcium-activated chloride channels
WIDER READING - Barrett 1993
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
What use might calcium-dependent chloride channels have therapeutically
WIDER READING - Huang et al 2012
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.
How was the R domain function identified
WIDER READING - Widdicombe and wine 1991
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.
How might thiazolidinone CFTRinhibitor -172 (lab made inhibitor) help in treat cholera
WIDER READING - Thiagarajah et al 2002
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
What is the movement of sodium into the cell and chloride into the cell
An inward current - because positive ion
An outward current - because negative ion so opposite
AND VICE VERSA
