Oubain-sensitive HCO 3 - secretion & acid absorption by the marine teleost fish play a role in...
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Oubain-sensitive HCO3- secretion & acid absorption by
the marine teleost fish play a role in osmoregulation
M. Grosell & J. Genz
9.31.7K+
9.61.8Ca2+
2697SO42-
50159Mg2+
2.263∑CO2
(HCO3-)
1049405Total
51352Cl-
43930Na+
SW (mM)
Intestinal fluid* (mM)
Extracellular fluid
[Na+] = 150mM, [Cl-] = 145 mM
Osmoregulation in marine teleost fish
Drinking
Active extrusion of Na+ and Cl-
Na+, Cl- and water absorption
Diffusive water loss
Diffusive salt gain
* Gulf Toadfish in 35‰
Study Goals
• Determine the contribution of endogenous metabolic CO2 vs extracellular HCO3
- as sources of secreted HCO3-
• Determine role of carbonic anhydrase (CA) in hydrating endogenous CO2
• Determine fate of liberated H+ from CA-mediated hydration reaction
Endogenous CO2 is hydrated in a reaction regulated by carbonic anhydrase (CA). The resulting HCO3
- is actively transported across the apical membrane via Cl-/HCO3
- exchange (AE). This is driven by the + potential inside the cell set up by NKA.
Fig. 11 Cellular mechanisms in intestinal epithelial transport of HCO3
-
Results• Resting tissue
– Viable and stable for >5 h
– secretion rates are between 0.3-0.5 umol/cm2/h
– TEP = -20 mV
– Conductance = 10 mS/cm2
• HCO3- secretion decreases when deprived of O2
• Secretion is strongly temperature dependant
Extracellular HCO3- vs. Endogenous CO2
• HEPES vs. 5 mM HCO3- saline
• When exposed to HCO3-/CO2,
base secretion increased twofold
Figure 4
• Hydration of endogenous CO2 accounts for ~50% of total HCO3
- secretion
• Percentage contributed by hydrated endogenous CO2 is species-dependant– 30-60% CO2 in European flounder (Grosell et al 2001)
and goby (Dixon & Loretz 1986)
– 100% extracellular HCO3- in Japanese eel (Ando &
Subramanyam 1990)
% of endogenous CO2
Role of CA
• Pharmacological test
- Etoxzolamide
Figure 5
What happens to H+?
• Must be removed to– Maintain constant intracellular pH– Prevent reversal of CA-catalyzed hydration
Possible pathways for removal of H+ across the basolateral membrane is either Na+/H+ exchange (NHE) or an H+ pump.
Na+ dependence
(Fig. 8) suggests NHE.
Altering serosal pH
pH % Reduction
7.4 33
7.0 44
6.6 47
Directly measure H+ transport
Pharmacology• Not inhibited by EIPA or Amiloride
– Does not discount NHE • Significant reduction when exposed to oubain
(NKA inhibitor)
Net acid/base flux
Salinity (ppt)
9 33 50
Aci
d-b
ase
flu
xes
(eq
v kg
-1 h
-1)
-600
-400
-200
0
200
400total HCO3
-
Precipitate HCO3-
Total H+
NH4+
Ca:Mg ratio in precipitate
Salinity (ppt) 9 33 50
Ca2+ (mmol/g) -0.20 ± 0.14 0.44 ± 0.14 0.65 ± 0.04
Mg2+ (mmol/g) -0.02 ± 0.02 4.13 ± 1.96 0.47 ± 0.02
Ca:Mg ratio 6.54 ± 1.95 0.16 ± 0.03 1.36 ± 0.08
Intestinal
Rectal
Salinity (ppt) 9 33 50
Ca2+ (mmol/g) 0.15 ± 0.05 0.08 ± 0.03 0.14 ± 0.07
Mg2+ (mmol/g) 0.03 ± 0.01 0.13 ± 0.03 0.19 ± 0.03
Ca:Mg ratio 4.92 ± 1.23 -2.36 ± 3.15 0.45 ± 0.29
Further Discussion
• Possibility of global impact of precipitates• Whole-animal acid/base balance• This process is similar to mammalian pancreatic
secretion– Could intestinal HCO3
- secretion be common to all vertebrates?