Carbonate precipitates and bicarbonate secretion in the intestine of sea bass, Dicentrarchus labrax

ORIGINAL PAPER

Carbonate precipitates and bicarbonate secretion in the intestineof sea bass, Dicentrarchus labrax

Caterina Faggio • Agata Torre • Gabriele Lando •

Giuseppe Sabatino • Francesca Trischitta

Received: 21 June 2010 / Revised: 8 November 2010 / Accepted: 10 November 2010 / Published online: 9 December 2010

� Springer-Verlag 2010

Abstract The aim of this paper was to study the chemical

composition of the precipitates found in the intestine of

Dicentrarchus labrax and the source of HCO3- secreted

into the intestinal lumen. The chemical analysis was per-

formed by employing the potentiometric double titration

method and by means of an electron microscope coupled

with a spectrometer and X-ray powder diffraction. The

results obtained suggest the presence of very insoluble

intestinal precipitates, presumably formed by a mixture of

CaCO3 and MgCO3, with a higher quantity of the former

with respect to the latter. HCO3- secretion rate was

investigated with the aid of the pH stat method in isolated

tissues mounted in Ussing chamber, where the transepi-

thelial electrical parameters were also measured. When the

serosal surface of the intestinal mucosa was bathed in

HCO3--Ringer bubbled with 1% CO2 in O2 while the

serosal surface was bathed in HCO3- free Ringer solution

bubbled with pure O2, bicarbonate secretion proceeded at

an almost stable rate of 0.9 ± 0.05 leq cm-2 h-1 for

about 3 h while Isc maintained a constant value of

38 ± 1.5 lA cm-2. The carbonic anhydrase inhibitor

ethoxyzolamide elicited a progressive reduction of HCO3-

secretion that was about 75% of the initial value after

80 min. When serosal HCO3-–CO2 saline was substituted

with Hepes–O2 saline base secretion progressively declined

reaching a value of about 20% of the initial value. It was

also strongly inhibited when Na? was substituted with the

impermeant cation choline and when either DIDS or oua-

bain were added to the basolateral side. These results

suggest that most of the bicarbonate secreted is of extra-

cellular source and is probably transported across the

basolateral membrane by both Na? independent mecha-

nism and Na? dependent transporter, presumably a

NaHCO3 cotransport.

Keywords Fish intestine � Carbonate precipitates �Bicarbonate secretion � pH stat method

Introduction

The blood of marine teleosts has an osmotic pressure of

300–370 mOsm kg-1 despite the hyperosmoticity of sea

water (1,000 mOsm kg-1) and the high osmotic perme-

ability of the large area of the respiratory surface (the

branchial lamellar epithelium) exposed to the surrounding

medium, leading to a continuous diffusive water loss

(Evans et al. 2005; Marshall and Grossell 2005). To

withstand this osmoregulatory challenge, these fish drink

large amounts of seawater that is desalinated in the water-

impermeable oesophagus that selectively absorbs NaCl

(Hirano and Mayer–Gostan 1976; Parmelee and Renfro

1983). The lowering of osmotic pressure of intestinal fluid

is necessary to allow an active transport of Na? and Cl- at

intestinal level that drives water absorption (see Evans and

Claiborne 2006).

Communicated by G. Heldmaier.

C. Faggio � A. Torre � F. Trischitta (&)

Facolta di Scienze MM.FF.NN.,

Dipartimento di Scienze della vita ‘‘M. Malpighi’’,

Messina, Italy

e-mail: ftrischi@unime.it

G. Lando

Facolta di Scienze MM.FF.NN., Dipartimento di Chimica

Inorganica, Chimica Analitica e Chimica Fisica, Messina, Italy

G. Sabatino

Facolta di Scienze MM.FF.NN., Dipartimento di Scienze della

Terra, Messina, Italy

123

J Comp Physiol B (2011) 181:517–525

DOI 10.1007/s00360-010-0538-y

Since sea water is rich in both Ca2? and Mg2?, marine

teleosts introduce the divalent cations at high rate with

ingested water. In order to reduce the osmotic pressure of

intestinal fluid, the intestine actively secretes HCO3- to

form CaCO3 and MgCO3 rich precipitates (Whittamore

et al. 2010; Wilson et al. 2002) that are excreted into the

environment as discrete pellets or incorporated with feces

(Walsh et al. 1991; Wilson et al. 2002) Ca2? precipitation

would also have the role to reduce Ca2? absorption and

hence avoid renal stone formation (Wilson and Grosell

2003). Both Ca2? movements and bicarbonate secretion

can be regulated by two calcitropic hormones such as

stanniocalcin1 and parathyroid hormone-related protein

(Fuentes et al. 2006, 2010).

A recent study suggests that the carbonates excreted by

fish could contribute 3–15% of total carbonate budget in

the ocean (Wilson et al. 2009), conventionally attributed to

the carbonated skeleton released by coccolithophores and

foraminifera upon death (Feely et al. 2004; Schiebel 2002).

The estimate of teleost contribution is based on the

assumption that all teleosts produce large amount of car-

bonates as part of their osmoregulatory strategy as already

demonstrated in a small number of teleosts adapted to

seawater (Genz et al. 2008; Grosell and Genz 2006; Grosell

et al. 2001; Walsh et al. 1991; Wilson and Grosell 2003;

Wilson et al. 1996, 2002; Kurita et al. 2008) and on cal-

culation of global marine biomass (Wilson et al. 2009).

Consequently, it is interesting to know whether the ability

to produce carbonate is an ubiquitous feature of the intes-

tine of marine teleosts.

This study was performed with the aim to add a small

block to this intriguing research field, by evaluating the

presence of carbonate and the source of HCO3- secreted in

the isolated intestine of the sea bass, Dicentrarchus labrax.

In order to determine the chemical composition of the

intestine precipitates, potentiometric titrations, carried out

following the method reported in Wilson et al. (2002) and

analysis by means of an electron microscope coupled with

a spectrometer and X-ray powder diffraction were per-

formed. HCO3- secretion was studied in the isolated

intestine of sea bass, mounted in Ussing chamber; the pH

stat method was employed.

Materials and methods

Sea bass (D. labrax, 60–100 g) were obtained from a local

offshore fish farm, transported to the Centro di Ittiopato-

logia Sperimentale della Sicilia (C.I.S.S.) and kept in 800-l

tanks with flowing artificial seawater with the same com-

position of the natural one (37.5 PSU). The water tem-

perature was 18�C. Fish were acclimated for at least

3 weeks prior to experimentation. They were fed with dry

pellets of seabass (Aller Aqua, DK-6070 Christiansfeld;

46% crude protein; 20% crude fat; 10% ash; 1.5% fiber)

but were not fed for 96 h prior to the experiments. Fish

were killed by overdose of tricaine methanesulfonate (MS

222; 0.5 g l-1) and the entire intestine was obtained by

dissection.

Analysis of intestinal precipitates

All the samples of intestinal precipitates collected for this

study were analyzed with the double titration method and

by scanning electron microscope (SEM), in order to gain as

much information as possible. For the potentiometric

double titration method, NaOH and HNO3 were prepared

by diluting concentrated ampoules (Fluka). NaOH(aq)

solutions were preserved from atmospheric CO2 by means

of soda lime traps. NaNO3 aqueous solutions were prepared

by weighing the pure salt previously dried in an oven at

t = 110�C. Ultrapure water (R C 18 MX) and grade A

glassware were used for all the measurements. Some

potentiometric titrations (at t = 25.0 ± 0.1�C, controlled

by means of a waterbath) were performed in order to

determine the amount of carbonate in the intestinal samples

of the analyzed fishes. The samples were weighed by

means of an analytical balance. We added HNO3 standard

solution to the samples, measuring the electromotive force

values up to pH *3.8–4.0. We continued acid addition

until the pH remained stable for a minimum of 15 min

under continuous gassing of N2 (CO2 free). At this point all

CaCO3 should be dissolved and a back titration can be

initiated by the addition of NaOH to the solution. The

difference in the number of moles of HNO3 and NaOH

required to return to the starting pH is then equal to the

number of HCO3- ? CO3

2- equivalents in the original

sample. This procedure is reported in Wilson et al. (2002)

and described in Hills (1973). The free hydrogen ion

concentration was measured with a Metrohm model 713

potentiometer (resolution 0.1 mV, reproducibility

0.15 mV) connected to a model 8101 Ross type Orion

electrode, coupled with a standard calomel electrode.

NaNO3 was added in the samples in order to reach an ionic

strength value of 0.1 mol l-1 (this precaution allowed the

stabilization of the electromotive force readings). NaOH

0.1 mol l-1 and HNO3 0.2 mol l-1 were used as titrant

solutions. During the titrations, N2 (CO2 free) was gently

bubbled into the solution.

The intestinal precipitates were also analyzed by SEM.

This analysis was necessary in order to know the elemental

composition of the precipitates before and after the disso-

lution in pure water for the double titration method.

Analysis were performed using a FEI INSPECT-S electron

microscope coupled with an Oxford EDX spectrometer and

Si(Li) detector with an ultra-thin window and a resolution

518 J Comp Physiol B (2011) 181:517–525

123

of 136 eV at Mn Ka. The spectral data were acquired at

1,500–2,000 counts s-1 with dead time below 25%, using

the ZAF correction. Analyses were carried out at a working

distance of 19 mm, at acceleration voltage of 20 kV and

550 pA (PROBE).

Data were collected using a BRUKER D8ADVANCE

Diffractometer with Cu Ka radiation on a Bragg–Brentano

theta–theta goniometer, equipped with a SiLi solid-state

detector, Sol-X. Acquisition conditions were 40 kV and

40 mA. Scans were obtained typically from 2� to 80� 2h,

with step size of 0.02� 2h, with a count time of 1 s. Raw

diffraction scans are stripped of ka2 component, back-

ground corrected with a digital filter (or fourier filter).

Observed peak positions are matched against the ICDD

JCPDS database.

Ussing chamber experiments

The middle intestine (3.0 cm caudal the insertion of the

pyloric caeca) of seawater-acclimated sea bass was

removed, stripped of muscle layers using two pairs of fine

forceps and fixed in a modified Ussing chamber (CHM6,

World Precision Instruments, Berlin, Germany; membrane

area: 0.13 cm2), where it was perfused on both sides by

isotonic salines (Ringer solutions), which compositions are

indicated in Table 1. The temperature of the perfusing

solution was kept constant at 18�C.

Tissues were connected to an automatic short circuit

current device (DVC-1000, World Precision Instruments)

by four Ag/AgCl electrodes (two voltage electrodes and

two current electrodes) that made contact with the bathing

solutions via agar-Ringer filled cartridges. The short circuit

current (Isc) was measured by passage of sufficient current

through Ag/AgCl electrodes to reduce the spontaneous Vt

to zero (resistance of the chamber fluid was subtracted

automatically). The preparations were kept open circuited

throughout the experiments, except for a few seconds every

5 min for recording Isc. Vt was measured with respect to the

mucosal side (grounded). The Isc is referred to as negative

when current flows across the tissue from the apical side to

the serosal side. Transepithelial resistance (Rt) was mea-

sured by pulsed current injection (33 lA cm-2, 500 ms)

through the tissue. This injected current produces a voltage

deflection from which Rt was calculated.

The secretory HCO3- fluxes were determined by a pH

stat titration apparatus (Abu-80; Combined electrode, GK

2421C; Titrator TTT1, Radiometer, Copenhagen). The

serosal side of the epithelium was perfused with standard

Ringer’s solution bubbled with 99% O2, 1% CO2 (Table 1,

sol. 1) while the mucosal side was kept unbuffered and

nominally HCO3- free (Table 1, sol. 5). Mixing of the

solution was achieved by gassing with pure oxygen, pass-

ing through a 3 M KOH trap, through airlifts. The alka-

linization arising from HCO3- transport was automatically

titrated by clamping pH at 7.74 with the addition of HCl

5 mM, which was delivered by a polyethylene tube near

the pH electrode.

In the calculation of the fluxes we subtracted the minute

quantities (about 0.0004 ml) of titrant delivered by the

burette during the measures of Isc due to the interference of

current pulse with pH.

All the drugs were obtained from Sigma–Aldrich (St.

Louis, MO, USA). The drugs were prepared as concen-

trated stocks and dissolved in the experimental solution to

Table 1 Ionic composition

of the solutions (concentration

in mM)

Solutions 1, 2 and 3 were

bubbled with a mixture of 1%

CO2 and 99% O2 to yield a pH

of 7.74 ± 0.1, solutions 4 and 5

were bubbled with 100% O2,

1 N NaOH was added to

solution 4 to obtain pH 7.74.

In Cl--free solution Ca2?

concentration was raised to

compensate for binding by

gluconate.

p = 370 ± 3 mOsm kg-1

Ringer

(1)

Ringer

Na? free

(2)

Ringer Cl-

free

(3)

Hepes-Ringer

(4)

Unbuffered

Ringer

(5)

NaCl 167 – – 167 167

KCl 3.2 3.2 – 3.2 4.0

MgCl2 1.0 1.4 – 1.4 1.0

CaCl2 2.5 2.5 – 2.5 2.5

NaHCO3 10 10 10 – –

KH2PO4 0.8 0.8 0.8 – –

Glucose 15 15 15 15 15

Hepes – – – 3.5 –

Na? gluconate – – – 10 10

Cl- choline – 167 – – –

HCO3- choline – 10 – – –

Na? gluconate – – 167 – –

K? gluconate – – 3.2 – –

Ca2? gluconate – – 12.5 – –

MgSO4 – – 1 – –

J Comp Physiol B (2011) 181:517–525 519

123

obtain the final concentration. Bumetanide, DIDS and

ouabain were dissolved in DMSO, its final percentage in

the chamber (0.01%) did not alter neither transephitelial

parameters nor basal bicarbonate secretion.

The data are given as mean ± SE. Statistical analyses

were performed using the Student’s t test.

Results

Carbonate precipitates

Accumulations of precipitates were clearly visible inside

the intestine of all fish observed, kept unfed for 96 h

(Fig. 1). We performed two different kinds of chemical

analysis on the intestinal precipitates, the double titration

method and the microscopical investigation; both analyses

were performed to determine the chemical composition of

the precipitates. The potentiometric analysis were per-

formed as described above, but the amounts of acid and

base, used in the double endpoint titration method, resulted

equal, meaning that no dissolution of the intestinal pre-

cipitate, or only very slight, occurred in the sample. This

fact suggests the Mg presence in the precipitate sample. In

fact, it is well known that Mg-rich calcium carbonates are

less soluble with respect to the simple CaCO3 (Krauskopf

and Bird 1995). Hence, we performed SEM and X-ray

analysis on the precipitates without sample preparation and

on the precipitate kept in pure water (for 48 h) and then

filtered over 0.45 lm filters. The various spectra collected

were homogenous and the results (for the pre and after

preparation) of the intestinal precipitates are reported in

Table 2, and were comparable among them in any case,

demonstrating that the precipitates did not dissolve in pure

water. Most of the precipitates contained carbon, oxygen,

magnesium and calcium. These results confirm the insol-

ubility of intestinal precipitates, presumably formed by a

mixture of MgCO3 and CaCO3 (Krauskopf and Bird 1995).

This high insolubility is consistent with the fact that the

potentiometric measurements did not show any result.

Although a mixture of MgCO3 and CaCO3 can be inter-

preted as dolomite, the semiquantitative nature of the

SEM-EDX measurements cannot allow any theory about

the presence of this mineral. Higher quantity of calcium

with respect to magnesium (Fig. 2) is also consistent with

the physiology of the fishes that clear large amount of

MgCl2 by renal excretion (Marshall and Grosell 2005) even

if the majority of ingested Mg2? is eliminated with rectal

fluid (Genz et al. 2008). In order to confirm the presence of

a carbonate, a powder diffraction was also performed as

described above. This technique allows to determine the

presence of crystalline structure in the samples. In our case

we found the main peak of the calcite (Fig. 3), demon-

strating the presence of some crystals of this mineral. As an

example, we report in Fig. 4 a spectra of the sample

(without pre-treatment in this case) enlarged by the SEM

technique. The sample appears homogeneous, because the

color associated to the various zones of the sample is equal

in any case. This fact suggests that the elementar analysis

in some selected points can be representative of all the

sample.Fig. 1 Precipitates formed in the intestine of Dicentrarchus labrax

Table 2 Elementar composition of the intestinal precipitates

analyzed

1 2

CO2 69.91 74.59

Na2O 1.15 1.15

MgO 9.37 8.96

P2O5 2.51 1.74

SO3 2.42 1.75

Cl 0.64 0.51

K2O 0.21 0.21

CaO 12.5 9.92

CuO 0.56 0.54

Total 99.27 99.37

Results are reported as oxides. Column 1 refers to the sample after the

dissolution in water and the filtration, while column 2 to the sample

without any pre-treatment. Semiquantitative SEM-EDX analysis

Fig. 2 Punctual elemental analysis (EDS) of a sample (values in

column 1 of Table 2). The point of the sample referring to this EDS

analysis is shown in Fig. 4 and is denoted by a square

520 J Comp Physiol B (2011) 181:517–525

123

Transepithelial parameters in the control conditions

In order to assess the viability of the tissue in vitro, pre-

liminary experiments were performed. The tissue were

isolated, mounted in Ussing chamber and perfused with

identical salines (Table 1, sol. 1) from both sides, mucosal

and serosal, generated a serosa negative potential differ-

ence, Vt, of -2.3 ± 0.2 mV (the minus sign refers to

the serosal side), and a short circuit current (Isc) of

-30.7 ± 2.2 lA cm-2; the transepithelial resistance was

75 ± 4.8 X cm2 (n = 4). All the electrical parameters

remained constant for almost 3 h.

Both Vt and Isc were nullified when Cl- was substituted

with equimolar amount of gluconate (Table 1, sol. 3) in

both the mucosal and the serosal side (Table 3). Vt and Isc

were significantly inhibited by bumetanide (10-5 M) and

ouabain (10-3 M) added to the mucosal and serosal bath,

respectively. In all these experimental conditions Rt was

not significantly modified.

pH stat experiments

When the serosal surface of the intestinal mucosa was

bathed in HCO3--Ringer bubbled with 1% CO2 in O2

(Table 1, sol. 1) while the mucosal surface was bathed in

unbuffered HCO3- free Ringer solution bubbled with

pure O2 (Table 1, sol. 5), bicarbonate secretion proceeded

at an almost stable rate of 0.9 ± 0.05 leq cm-2 h-1 for

about 3 h while Isc maintained a constant value of

-38 ± 1.5 lA cm-2 (n = 4).

To assess whether the HCO3- secreted is derived from

intracellular hydratation of CO2 or is of extracellular

source, i.e. transported across the basolateral membrane,

we performed two series of experiments. In the first series,

after the luminal alkalinization reached stable values, the

lipophilic carbonic anhydrase inhibitor, ethoxyzolamide

(10-3 M), was added to the serosal side of the epithelium.

We avoided the luminal addition because preliminary

experiments showed that the drug produced a slight acid-

ification of the unbuffered solution that could cause an

underestimation of base secretion.

As Fig. 5 shows, ethoxyzolamide elicited a progressive

reduction of HCO3- secretion that became significant after

20 min. After 80 min it was about 75% of the value before

addition of the inhibitor. Isc was not altered in a significant

way.

In order to verify whether the HCO3- secretory fluxes

were dependent on serosal HCO3-, after the secretion had

reached stable values, serosal HCO3-–CO2 saline was

substituted with Hepes–O2 saline (Table 1, sol. 4).

Fig. 3 Diffraction spectra of a sample of intestinal precipitate

Fig. 4 Electron microscope image of a sample (column 1 in Table 2)

Table 3 Effect on the transepithelial electrical parameters of Cl-

removal and of various inhibitors

Experimental

conditions

Vt

(mV)

Isc

(lA cm-2)

Rt

(Xcm2)

n

Control -3.0 ± 0.7 -38.8 ± 1.5 76 ± 9.5 4

Cl- free (M, S) 0.0 ± 0.0* 0.0 ± 0.0* 78 ± 9.2

Control -2.3 ± 0.2 -30.7 ± 2.2 75 ± 4.8 4

Bumetanide

(10-5 M), M

-0.7 ± 0.1* -7.6 ± 0.8* 75 ± 4.2

Control -2.4 ± 0.6 -28.6 ± 8.7 85 ± 5.6 4

Ouabain

(10-3 M), S

-0.4 ± 2.1* -5.3 ± 2.5* 85 ± 5.1

Vt = transepithelial voltage; Isc = short circuit current; Rt = trans-

epithelial resistance; n = number of tissues; M = mucosal side;

S = serosal side

Values are mean ± SE. The minus sign of Vt indicates that the serosal

side is negative when referred to the mucosal (grounded) side. The

minus sign of Isc indicates current flowing from the mucosal to the

serosal side. * p \ 0.001

J Comp Physiol B (2011) 181:517–525 521

123

As Fig. 6 shows, in this experimental condition base

secretion progressively declined reaching a value of about

20% of the initial value, while Isc tended to reach values

significantly more negative than the values in the presence

of serosal HCO3-.

Figure 7 shows that bicarbonate flux was strongly

inhibited by serosal 5�10-4 M DIDS (4,40-Diisothiocyano-

2,20-stilbenedisulfonic acid), that produced also a small but

significant increase of serosa negative Isc.

In order to assess the Na? dependence of serosal HCO3-

uptake, the luminal alkalinization was measured in tissues

perfused with serosal solution in which Na? was substi-

tuted with the impermeant cation choline (Table 1, sol. 2).

As shown in Fig. 8, in this experimental condition the

secretory flux rapidly declined. Na? gradient from mucosa

to serosa, obviously, produced large Na? diffusional fluxes

altering Vt; in addition Rt increased (not shown).

In Fig. 9 the effects of serosal ouabain (10-3 M) on

Isc and on bicarbonate fluxes are reported. The Na?–K?-

ATPase inhibitor elicited a gradual reduction of HCO3-

secretion and almost nullified Isc.

Discussion

This work confirms the presence of carbonate in the pre-

cipitates found inside the intestine of Dicentrarchus labrax.

The precipitates were detected in all fish observed. As they

were kept unfed until 96 h prior to experimentation, it is

evident that they are formed via a process that is

independent of digestion. Indeed a highly alkaline intesti-

nal microenviroment, necessary to facilitate precipitation

was demonstrated even in unfed fish (Wilson et al. 2002).

Carbonate precipitates have been already detected in other

teleosts adapted to sea water such as Oncorhynchus mykiss

(Shehadeh and Gordon 1969) Opsanus beta (Walsh et al.

Ethoxtyzolamide, S

0

0,4

0,8

1,2

0 10 20 30 40 50 60 70 80 90 100

-45

-36

-27

-18

-9

0

time (min)

I sc(

µAcm

-2)

J (H

CO

3- ) (µe

q cm

-2 h

-1)

*

Fig. 5 Effect of ethoxyzolamide (10-3 M) on HCO3- secretion and

Isc, S = serosal side. The drug was added for the period indicated by

the horizontal bar. The serosal bath contained 10 mM HCO3- and

was bubbled with 1% CO2 and 99% O2, while the unbuffered mucosal

solution was HCO3- free and bubbled with 100% O2. The values are

mean ± SE (n = 4). The JHCO3- reduction was significant from the

time labelled with asterisk

HCO3- -free, S

0

0,4

0,8

1,2

0 10 20 30 40 50 60 70 80 90 100 110

-60

-40

-20

0

time (min)

I sc(μA

cm-2

)

J (H

CO

3- ) (µe

q cm

-2 h

-1)

*

**

Fig. 6 Effect on HCO3- secretion and Isc of serosal substitution of

HCO3- buffer with Hepes buffer bubbled with 100% O2, for the

period indicated by the horizontal bar. The unbuffered mucosal

solution was HCO3- free and bubbled with 100% O2. S = serosal

side. The values are mean ± SE (n = 4). The difference from control

values was significant from the time labelled with asterisk for

JHCO3- and with double asterisk for Isc

DIDS, S

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 20 40 60 80 100 120 140

-45

-36

-27

-18

-9

0

time (min)

I sc(

µAcm

-2)

J (H

CO

3- ) (µe

q cm

-2 h

-1)

*

**

Fig. 7 Effect on HCO3- secretion and Isc of serosal DIDS

(5 9 10-4 M), S = serosal side. The values are mean ± SE

(n = 4). The drug was added for the period indicated by the

horizontal bar. The serosal bath contained 10 mM HCO3- and was

bubbled with 1% CO2 and 99% O2, while the unbuffered mucosal

solution was HCO3- free and bubbled with 100% O2. The difference

from control values was significant from the time labelled with

asterisk for JHCO3- and with double asterisk for Isc

522 J Comp Physiol B (2011) 181:517–525

123

1991), Takifugu obscurus (Kurita et al. 2008), Platichtys

flesus (Wilson et al. 2009).

The chemical analysis of intestinal precipitates of

D. labrax performed in our study showed that most of the

precipitate were homogeneous and formed by both CaCO3

and MgCO3. Being the mixture of this salt much more

insoluble than the simple calcite, the precipitates did not

dissolve in water, and the double titration method did not

evidence any result. By diffraction analysis we evidenced

the presence of some calcite crystals, meaning that effec-

tively Ca2? and Mg2? are extruded by complexation with

carbonate. Time of rest considerably long can be also

supposed, because the transformation from amorphous

to crystalline calcite requires quite long time of deposition

(2 or 3 days in the intestine).

Since, to our knowledge, this is the first Ussing chamber

study performed in the isolated intestine of the sea bass, we

performed some preliminary experiments to test the suit-

ability of our preparation for ‘‘in vitro’’ studies. For this

aim we measured the time course of the transepithelial

parameters and the effect of drugs known to inhibit the

main transport mechanisms operating on the mucosal and

the serosal membranes of the enterocyte of sea water

adapted teleosts, i.e. the luminal Na?–K?–2Cl- cotrans-

port and the serosal Na?–K?-ATPase (Musch et al. 1982;

Halm et al. 1985; Aguenaou et al. 1989; Trischitta et al.

1992a, 1992b, 2004). The above membrane transporters are

known to be inhibited by bumetanide and ouabain

respectively. We found that the transepithelial parameters

remained stable for at least 3 h; luminal bumetanide and

serosal ouabain strongly inhibited Isc and Vt without

altering tissue resistance (Table 3), as already observed in

other intestinal preparation of marine teleosts (Trischitta

et al. 1992a, 1992b, 2004).

Having established the viability of our preparation in

Ussing chamber we started the pH stat experiments. In the

intestine of sea bass the rate of base secretion in the

presence of serosal HCO3-/CO2 was 0.9 ± 0.05 leq

cm-2 h-1, a value slightly higher than the values measured

in other teleost fish ranging from 0.3 to 0.7 lmol l-1

(Dixon and Loretz 1986; Fuentes et al. 2010; Grosell and

Genz 2006; Grosell et al. 2005; Wilson and Grosell 2003)

but it was far below the value reported by Ando and

Subramanyam (1990) in the Japanese eel that was

3.0 leq cm-2 h-1. These contrasting findings could sug-

gest the strong dependence of HCO3- secretion from the

serosal HCO3- concentrations since the work in the Japa-

nese eel was performed employing serosal saline with high

level of HCO3- (25 mM). In the other studies, including

ours, salines with HCO3- concentrations near to the values

generally measured in extracellular fluid of teleost fish

(4–10 mM) were used (Marshall and Grossell 2005; Wil-

son 1999). Indeed a dependence of HCO3- secretion rate

from serosal HCO3- concentration was suggested by the

study of Taylor et al. (2010) in which the rate of HCO3-

secretion in the toadfish intestine was measured as a

function of serosal HCO3- concentration ranging from 0 to

20 mmol l-1.

However, the contribution of extracellular HCO3- to the

overall HCO3- secretion is different in the intestine of

Na+ -free, S

time (min)

J (H

CO

3- ) (µe

q cm

-2 h

-1)

*

0

0,2

0,4

0,6

0,8

1

1,2

0 10 20 30 40 50 60 70

Fig. 8 Effect on HCO3- secretion of Na? removal from serosal

solution, S = serosal side. The drug was added for the period

indicated by the horizontal bar. The serosal bath contained 10 mM

HCO3- and was bubbled with 1% CO2 and 99% O2, while the

unbuffered mucosal solution was HCO3- free and bubbled with 100%

O2. The values are mean ± SE (n = 3). The JHCO3- reduction was

significant from the time labelled with asterisk

Ouabain, S

0

0,2

0,4

0,6

0,8

1

0 10 20 30 40 50 60 70 80 90

-45

-30

-15

0

time (min)

J (H

CO

3- ) (µe

q cm

-2 h

-1)

I sc(

µAcm

-2)

*

**

Fig. 9 Ouabain (10-3 M) almost nullified Isc and produced a

decrease of HCO3- secretion significant from the time labelled with

asterisk for JHCO3- and with double asterisk for Isc. S = serosal side.

The drug was added for the period indicated by the horizontal bar.

The serosal bath contained 10 mM HCO3- and was bubbled with 1%

CO2 and 99% O2, while the unbuffered mucosal solution was HCO3-

free and bubbled with 100% O2. The values are mean ± SE (n = 3)

J Comp Physiol B (2011) 181:517–525 523

123

various teleost fish. The extracellular HCO3- transported

across the intestinal epithelium is the main source of the

bicarbonate secreted into the intestinal lumen of the Japa-

nese eel (Ando and Subramanyam 1990), while the HCO3-

derived from the intracellular hydration of the metabolic

CO2 provides a significant source of the HCO3- secreted in

the intestine of Platichthys flesus (Grosell et al. 2005), of

O. beta (Grosell and Genz 2006), of Gillichthys mirabilis

(Dixon and Loretz 1986) and of O. mykiss (Grosell et al.

2009). In the Sparus auratus intestine roughly 50% of

bicarbonate secretion seems driven via transcellular path-

way while the remaining 50% is produced by cytosolic

hydration of CO2 (Fuentes et al. 2010).

In the D. labrax intestine we showed that HCO3-

secretion proceeded although with a 20% reduction when

carbonic anydrase was inhibited by ethoxyzolamide

(Fig. 5), as already observed by Grosell et al. (2005) in

Platichthys flesus and by Grosell and Genz (2006) in

O. beta. These results suggest that the endogenously

formed HCO3- provides a source for luminal bicarbonate

secretion but that it is not the main source. It is likely that

extracellular bicarbonate transported across the epithelium

gives an higher contribution since the serosal removal of

HCO3- elicited a pronounced reduction of luminal alka-

linization (Fig. 6). The transport mechanism responsible of

HCO3- uptake by serosal membrane is DIDS sensitive,

since the stilbene derivative produced a sustained reduction

of HCO3- secretory fluxes (Fig. 7). In this respect, the sea

bass intestine behaves differently from the European

flounder intestine in which a lack of sensitivity of intestinal

bicarbonate secretion to serosal DIDS was observed

(Grosell and Jensen 1999).

It is known that different transport mechanisms are

DIDS sensitive: Cl-/HCO3- exchanger, Na?–HCO3

- co-

transporter and the Na?-driven Cl-–HCO3- exchanger

(Grichtchenko et al. 2001; Romero and Boron 1999).

Two observations led us to suppose that the serosal

mechanism of HCO3- uptake is a Na?-dependent

transporter:

1. HCO3- secretion was strongly reduced when Na? in

the saline bathing basolateral side was substituted with

the impermeant cation, choline (Fig. 8).

2. Blocking of the Na?–K?-ATPase produced a large

inhibition of HCO3- flux (Fig. 9), suggesting that the

electrochemical Na? gradient created by the pump is

necessary to drive the bicarbonate movement as

already observed by Grosell and Genz (2006) in the

O. beta.

Na?–HCO3- cotransporter is a possible target of DIDS

since a basolateral Na?–HCO3- cotransport involved in

formation carbonate precipitates has been recently descri-

bed in the intestine of T. obscurus and of O. beta (Kurita

et al. 2008; Taylor et al. 2010). However, we cannot

exclude that the Na? dependence of HCO3- secretion can

be partly due to a reduced Na?-dependent extrusion across

the basolateral membrane of H? derived from the cytosolic

hydration of CO2; this H? extrusion is necessary to

maintain intracellular pH while HCO3- is excreted across

the luminal membrane as suggested from Grosell and Genz

(2006). Other studies will be necessary to have information

about the mechanism(s) involved.

Since the percentage inhibition of HCO3- secretion in

the absence of serosal Na? (55%) is less than the per-

centage inhibition in the absence of serosal HCO3- (85%)

it is conceivable that a Na? independent bicarbonate

transport is also operating on the serosal membrane as

already suggested (Dixon and Loretz 1986). It is interesting

to note that the serosa negative Isc increased when bicar-

bonate secretion was strongly inhibited, i.e. both in the

absence of HCO3- in the serosal saline (Fig. 6) and in the

presence of serosal DIDS (Fig. 7), suggesting that the

secretory flux of bicarbonate contributes to the overall

short circuit current. These parameters are algebraic sum of

all ionic currents across the epithelium. In the sea bass

intestine the serosa negative Isc in the control condition

would be due to a net Cl- absorptive flux that mainly

depends on the operation of the luminal Na?–K?–2Cl-

cotransport, as suggested by the effect of bumetanide on

Isc (Table 3). So the serosa negative Isc increases when the

secretory HCO3- flux is inhibited. The observation that in

the presence of serosal ouabain Isc is almost nullified

(Fig. 9; Table 3) suggests that the Na? gradient maintained

by the Na?–K?ATPase drives not only the HCO3- secre-

tory flux but also Cl- absorption. Since Isc is nullified by

the removal of Cl- from both the mucosal and the serosal

salines (Table 3), it is evident that all ionic fluxes, included

HCO3- secretory fluxes, are Cl- dependent.

In conclusion, the intestine of sea bass is capable of

actively secreting HCO3-, the majority of which are of

extracellular origin and is transported across the basolateral

membrane by a Na?-dependent mechanism. This secretion

is responsible for the large amount of Ca2? and Mg2?

carbonates found inside the intestinal lumen of the fish.

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