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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: [email protected]
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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