Salt Secretion

Question 1

1. Describe the respiratory, exocrine, intestinal defects in cystic fibrosis
2. name 4 epithelia that secrete salt
3. what is an important model tissue of salt secretion?
4. Describe the apical and basolateral proteins in this model
5. How were pharmacological blockers used to determine what proteins are involved in Cl secretion?

Answer 1

1. loss of functional lung tissue due to inflammation and increased risk of infection
   exocrine pancreatic deficieny - blockage of pancreatic ducts
   obstruction of intestine due to thick content (meconium ileus)
2. sweat gland, exocrine gland acini, upper airway, small intestine
3. shark rectal gland
4. basolateral Na/K ATPase, NKCC1 and K channelapical Cl channel
5. ouabain reduced Vte, implicating Na/K ATPaseBarium (K⁺) inhibitor which sets driving force for Cl secretion reduced the amount of Cl secretion

furosemide (NKCC1 inhibitor) also reduced the amount of K+ secretion

Question 2

1. describe the evidence that supports active rather than passive accumulation of Cl in the cell
2. why is it important that Cl is above its electrochemical equilibrium? How has this been shown?

Answer 2

1. the nernst equation at 70mV was used to calculate [Cl]_i that accumulates passively, i.e. in the absence of NKCC1
   in the absence of NKCC1, only 17mM Cl could accumulate in the cell, which would not drive much secretion
   in the presence of NKCC1, 70mM Cl could accumulate in the cell
2. because it generates the driving force for Cl secretion. Furosemide brings E_Cl to equilibrium, and consequently the driving fore for secretion is lost.

Question 3

1. In which membrane is CFTR expressed?
2. give the structure of CFTR
3. How many CF causing mutations are there?
4. where are most of the mutations found?
5. Describe the impact on the channel of the following mutants:
   a) class I
   b) class II
   c) class III
   d) class IV
   e) class V
   f) class VI
6. which classes produce more severe symptoms?
7. what is used as a diagnostic tool? What is the threshold for CF diagnosis?

Answer 3

1. Apical membrane
2. 12 TM domains; 2 nucleotde binding domains (bind cAMP) and a regulatory domain which can be phosphorylated by PKA
3. >1200
4. nucleotide binding domains
   5a) mRNA is unstable therefore no protein is made
   b) trafficking impairments (proteins are sent for degradation because they are misfolded)
   c) impaired channel regulation
   d) impaired conductance (mutations are found in the channel pore)
   e) partial reduction in mRNA (therefore reduction in the amount of protein)
   f) high CFTR turnover - not in the membrane long enough to be functional
5. classes I-III
6. sweat chloride concentration - >60mmol/L

Question 4

1. which cells in the colon secrete Cl?
2. what do CF carriers have certain protection against?
3. describe the cell model of the colon

Answer 4

1. mid to lower crypt cells
2. cholera
3. basolateral NKCC1, Na/K ATPase and a K channelapical Cl channel (CFTR) and K channelCl secretion also drives the paracellular movement of Na and H₂O

Question 5

1. Name 2 factors which stimulate Cl secretion and how
2. describe the action of the following agents in terms of Cl secretion:
   a) carbacol CCH
   b) indomethacin
   c) Forskolin
   d) IBMX
3. what happened when CCH was added to cells?
4. what happened when CCH and indomethacin was added to cells? (why?)
5. what hapeened when all 4 agents were added to cells and why?

Answer 5

1. AcH - causes an increase in IC Ca, which activates K⁺ channels leading to hyperpolarisation which drives secretion

prostaglandins, which when receptors are activated, increases cAMP, leading to PKA activation, which activates CFTR

2a) activates AcHR, therefore drives Cl secretion
b) inhibits prostaglandin production therefore inhibits Cl secretion
c) activates adenylyl cyclase, therefore increases cAMP and stimulates Cl secretion
d) Inhibits phosphodiesterase therefore increases cAMP and stimulates Cl secretion
3. Cl secretion was stimulated
4. there ws no Cl secretion - despite a driving force for Cl secretion provided by CCH, the lack of PG means that CFTR is not activated by PKA, therefore channels are not open to mediate secretion
5. rapid Cl secretion - despite the lack of PG, there is an increase of cAMP independently of PGs; CCH also provides a driving force for Cl secretion.

Question 6

1. describe the cell model for Cl secretion in the upper airway
2. describe the interraction of ENaC and CFTR, and what implications this has in CF

Answer 6

1. apical ENaC and CFTR; basolateral Na/K ATPase, NKCC1 and K channel
2. usually, CFTR inhibits ENaCmutations in CFTR means this interraction is lost, therefore I_ENaC is enhanced, which exacerbates the CF condition

Question 7

1. What is different about the airway model?
2. why is this?
3. Why does alveolar oedema usually occur in CF patients?
4. what other tissue does Cl transport occur in this way and what does this explain?

Answer 7

1. Cl absorption rather than secretion occurs
2. Instead of NKCC1, there is a basolateral K/Cl co-transporter, which means that IC [Ca] is kept low, thus the driving force for chloride movement is for absorption
3. because the driving force for water uptake is lost due to a lack of Cl absorption
4. sweat gland. Causes salty sweat - lack of Cl reabsorption

Question 8

1. name an immune cell in which CFTR is expressed?
2. how do these cells kill bacteria?
3. how was it shown that CFTR is implicated in the way these cells kill P. aeruginosa

Answer 8

1. neutrophils
2. secrete microbial agents including HOCl, which modifies bacterial proteins, lipids and nucleic acids to promote cell death
3. the rate of killing of the bacteira at various [Cl] concentrations were measured. Bacteria were killed better in a physiological (127mM Cl) medium rather than a chloride free medium

neutrophils were isolated from CF patients. The rate of killing was signficantly lowe in the CF neutrophils than WT neutrophils in a Cl free medium and a physiological medium