Bicarbonate Transport

Question 1

describe the proximal tubule cell model for bicarbonate transport

Answer 1

1. HCO₃^- + H⁺ > H₂O + CO₂ (reaction enhanced by carbonic anhydrase IV in the membrane)
2. CO₂ crosses the A membrane (probably facilitated by AQP1)
3. CO₂ + H₂O > H⁺ + HCO₃^- (facilitated by carbonic anhydrase II)
4. HCO₃^- is transported across the BL membrane by NBC in a 1 Na:3 HCO3 stochiometry

Question 2

1. name 4 factors that stimulate HCO3 transport
2. what are stimulating factors linked to?
3. name 3 inhibiting factors that downregulate HCO3 transport
4. what are downregulating factors linked to?

Answer 2

1. angiotensin II, endothelin 1, NA and adenosine
2. increase in Ca and PKC
3. ANP, parathyroid hormone and DA
4. cAMP and PKA

Question 3

1. what are out of equilibrium solutions and what are they used for?
2. what type of epithelia were used in this study? what type of solutions was the apical side perfused with
3. what happened when the BL side was perfused with pure CO2? (in terms of HCO3 reabsorption)
4. what happened when the BL side was perfused with pure HCO3? (in terms of HCO3 reabsorption)
5. how was [Ca]i examined?
6. what was the effect of the following solutions on [Ca]i?
   a) pure CO2
   b) pure HCO3
7. what are the overall conclusions of this study?

Answer 3

1. they are used to manipulate CO2 and HCO3 levels independently of pH

a pure CO2 solution will contain a mix of 10% CO2 at pH 5.4 with 0 CO2/HCO3 at pH 7.5

a pure HCO3 solution will contain a mix of 0 CO2/HCO3 at pH 6.99 with 44mM HCO3 at pH 9.4

2. isolated rabbit proximal tubules; apical side was continuously perfused with equilibrated CO2/HCO3 solution
3. increased bicarb reabsorption (and decrease pHi)
4. reduced CO2 reabsorption (and increased pHi)
5. using fura-2
   6a) increase
   2b) small increase (not significant)
6. the presence of basolateral CO2 causes an increase in HCO3 reabsorption, by acting on a CO2 sensor which causes an increase in [Ca]i

CO2 removal from the BL side (i.e. pure HCO3 solutions) cause a decrease in HCO3 reabsorption by reducing the stimulation of the CO2 receptor.

Question 4

1. Name 2 reasons why bicarbonate transport is important
2. describe the split drop micropuncture technique
3. how has this technique shown that bicarbonate transport is crucial for water transport?

Answer 4

1. maintaining plasma pH; water reabsorption
2. drops of oil are introduced into the proximal tubule. a fluid drop is introduced in between the oil drops. The volume of oil decreases as water is reabsorbed from the tubule
3. the rate of fluid reabsorption was faster in the presence of HCO3, and slower in the presence of DIDs which inhibits bicarbonate transport.

Question 5

1. what type of ion transport does apical NHE3 mediate?
2. what is the phenotype of NHE3 KO?
3. What did in vivo microperfusion techniques show about KO mice?
4. Describe 3 inibitors that were used to see if any other channels are upregulated in NHE3 KO mice.
5. What were the overall conclusions of these studies?

Answer 5

1. H⁺ Secretion
2. decreased arterial blood pressure, decreased plasma pH and decreased plasma bicarbonate
3. HCO3 flux was reduced by 60%, and water reabsorption was reduced by 70% in KOs
4. EIPA was used to inhibit other NHE isoforms - had no effect on KOs

Baflomycin was used to inhibit H+ ATPase - had no effect on KOs

sch28080 was used to inhibit K+/H+ - had no effect on KOs

5. No other channels are upregulated in NHE3 KOs, meaning that NHE3 is critically linked to H2O transport.

Question 6

1. why does the cellular model for bicarbonate secretion in the pancreatic duct require a BL HCO3/CO2 transporter?
2. Which transporter performs this role? What is its stochiometry in the pancreatic duct?
3. why might the Cl/HCO3 exchanger be electrogenic?

Answer 6

1. because diffusion of CO2 across the membrane does not provide sufficient bicarbonate for secretion
2. NBC. 1 Na: 2 HCO3
3. to amplify the amount of secretion that can be achieved.

Question 7

describe the interraction between SLC26A6 and CFTR

Answer 7

at rest, SLC26A6 inhibits CFTR

the R domain of CFTR blocks the CFTR pore

PKA stimulation leads to the phosphorylation of the R domain. When phosphorylated, the R domain binds to the STAS domain of SLC26A6

this relieves the inhibition of CFTR and activates SLC26A6

Question 8

1. Describe the methodolgy used to investigate whether CFTR is involved in bicarbonate secretion
2. What were the results of this study?
3. What CF mutant is pancreatic sufficent? What does this mean?
4. What CF mutant is pancreatic insufficient? What does this mean?

Answer 8

1. the pancreatic duct was perfused to set up conditions for HCO3 secretion

IC pH was measured (secretion causes acidification)

DIDs was used to inhibit all other HCO3 transporters

[K⁺] was manipulated to alter the driving force for secretion

experiment was also performed on the ΔF508 mutant

2. lowering the [K⁺] lead to acidification (indicating bicarbonate secretion)

changing [K+] had no effect on IC pH of ΔF508 expressing cells

3. R117H - normal HCO3 transport is seen, but Cl transport is disrupted
4. I148T - normal Cl transport is seen, but bicarbonate transport is disrupted

Question 9

1. describe the structural difference in the kidney and pancreatic isoforms of NBC
2. Describe how these differences in structure were investigated as the cause of the difference in stochiometry between the 2 isoforms
3. what was found in this experiment and what does this imply?
4. How were the effects of phosphorylation on stochiometry investigated?
5. what was found?
6. how were the effects of IC Ca on NBC stochimetry investigated?
7. what were the findings?
8. How do these findings link to the regulation of bicarbonate transport?

Answer 9

1. the initial 41aa of kNBC are replaced with 85 aas in pNBC
2. proximal tubule cells or CCD cells (model of pancreatic duct cell) were transfected with either kNBC or pNBC.

cells were mounted using ussing chambers and the voltage clamped. the Vrev measured gives an indication of the stochiometry of NBC

3. when pNBC was transfected in PT cells, the stochiometry was 1:3, but 1:2 in CCD cells. The same was found when kNBC was transfected
4. PKA was used to induce phosphorylation. Also looked at the stochiometry of S1026A (phosphoinhibiting mutations) and S1026D (phosphomimicking mutations)
5. before stimulation, the stochiometry was 1:3; following stimulation, the stochiometry was 1:2

the phosphoinhibiting mutant had a stochiometry of 1:3 that was unaffected by PKA phosphorylation

the phosphomimicking mutant had a stochiometry of 1:2 even prior to PKA phosphorylation

6. IC Ca was changed; Vrev was measured in xenopus oocytes using macropatching
7. when IC Ca was low, stochiometry was 1:2

when IC Ca was high, stochiometry was 1:3

8. stimulating factors are linked to an increase in Ca - change in stochiometry to 1:3

downregulating factors are linked to PKA - change in stochiometry to 1:2