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?