Lecture 6

Question 1

use of liposome complexes in alternative treatments in cf

Answer 1

pGM169/GL67A nebulised – contains wt coding for CFTR
liposomes contain wt genetic material
stabilised lung function
more chloride secretion

Question 2

why is saline a bad control ?

Answer 2

Want placebo patients to have liposomes with scrambled sequences – might have negative effect - ethical reasons against it

Question 3

ENaC function in CF in upper airways

Answer 3

ENaC function is enhanced
CFTR usually inhibits ENaC - CFTR mutated so no inhibition of ENaC 
Lose chloride secretion 
enhances Na absorption 
ASL lowered

Question 4

could amiloride be used as a treatment?

Answer 4

Amiloride blocks ENaC - meaning less Na absorption so ASL height should increase
however, no improvement to lung function

Question 5

what is SPLUNC1?

Answer 5

• a naturally occurring ENaC antagonist – blocks ENaC

* Secreted protein short palate lung & nasal epithelial clone 1

Question 6

What is the action of SPLUNC1

Answer 6

• SPLUNC1 binds to ENaC and prevents cleavage.
o Increase number of channels, or increase open probability
o Suppress Na uptake

Question 7

why does cleavage of ENaC need to occur?

Answer 7

• ENaC undergoes proteolytic cleavage – via Trypsin
o When you cleave ENaC  Po (open probability)– enhanced Na absorption
• Trypsin – cleaves ENaC – open probability increases - current mediated by ENaC increased – increased Na absorption across apical membrane of airway cell

Question 8

what is S18?

Answer 8

• ENaC inhibitory domain of SPLUNC1

* S18 binds to HBE cells & blocks pH-dependent binding of SPLUNC1

Question 9

What is the effect of pH on the activity S18?

Answer 9

o pH 6 = binding low, s18 wasn’t shown to make a big difference – acidification inhibits binding
o pH 7.5 = SPLUNC1 alone = binding to ENaC goes up. fluorescence goes down with s18 – binds where SPLUNC1 binds – prevents proteolytic cleavage

Question 10

Whats the action of S18 on ASL in HBE?

Answer 10

S18 – height increases – stays at a higher level as ENaC is inhibited

Question 11

What is the action of S18 on ENaC function in HBE?

Answer 11

Transepithelial potenial reduced - almost completely inhibited
S18 – reduced amiloride sensitive short circuit currents – inhibits proteolytic cleavage, open probability stays low, less Na absorption, currents low

Question 12

What is the action of S18 on ENaC function in alveolar cells?

Answer 12

• Role of alveolar – reabsorb Na and Cl
• Anything that blocks ENaC – increased ASL in alveolar cells
• S18 – height increased of ASL

Question 13

what are the systemic renal effects of S18?

Answer 13

• ENaC – important role for Na reabsorption in principal cells in collecting duct
• S18 – fewer systemic effects (effects in kidney)
No impact on urine flow rate, no inhibition of ENaC in the kidney

Question 14

what are the systemic renal effects of amiloride?

Answer 14

impacts urine flow rate - blocks ENaC - blocks water reabsorption
increased loss of Na
massive decrease in loss of K in urine – Na absorption drives K secretion – so if you inhibit ENaC and Na reabsorption, you inhibit K secretion into the urine – rise in plasma K
– rise in plasma K+ - cardiac arrhythmias and sudden cardiac death

Question 15

Effect of S18 on CF sheep model?

Answer 15

• Exposed sheep to CFTR inhibitor –
o Blocks CFTR channels
o TMV drops
maintained recovery