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Comparative Biochemistry and Physio
Isolation and characterization of enterocytes along the intestinal tract
of the gilthead seabream (Sparus aurata L.)
Rosa Dopido, Covadonga Rodrıguez, Tomas Gomez, Nieves G. Acosta, Mario Dıaz*
Laboratorio de Fisiologıa Animal, Departamento de Biologıa Animal, Facultad de Biologıa, Universidad de La Laguna, 38206 Tenerife, Spain
Received 5 March 2004; received in revised form 14 June 2004; accepted 18 June 2004
Abstract
Epithelial cells were successfully isolated along the intestine of the gilthead seabream using a dissociation method based on intracellular-
like solutions. Biochemical and physiological tests revealed highly viable cells from all intestinal segments. Image analysis was used to
identify cell types in the epithelial preparations which were highly enriched in enterocytes (N95%) over mucous cells. Several digestive
hydrolases were determined in the isolated cells. Maltase (M), sucrase (S), leucine aminopeptidase (LA), 5Vnucleotidase (5VN), but not g-glutamyl transferase (g-GT) or alkaline phosphatase (AP) activities were found to be enriched in the epithelial preparations versus the
corresponding intestinal homogenates. Comparison of digestive hydrolases revealed the existence of a clear heterogeneity in their expression
pattern in the enterocytes, along the intestine. Na+–K+-ATPase, Na+-ATPase and Cl�-ATPase activities were also determined in the
membrane fraction of isolated cells. Analyses of enzymatic profiles revealed a clear asymmetry in the distribution of all Mg2+-dependent
ATPases; that is, maximal Na+–K+- and Na+-ATPase activities were observed in the enterocytes from pyloric caeca, while Cl�-ATPase
activity was about twice as high in the enterocytes from anterior and posterior intestines compared with pyloric caeca. This is the first report
demonstrating the existence of heterogeneous metabolic and enzymatic profiles in different enterocyte populations from euryhaline teleosts.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Cell isolation; Enterocyte heterogeneity; Enzymatic profiles; Enterocyte ATPases; Digestive hydrolases; Enterocyte metabolism; Enterocyte
morphometry; Intestinal epithelia; Fish intestine
1. Introduction
The epithelial lining of the gastrointestinal tract plays a
major role in the maintenance of body homeostasis. Enter-
ocytes are the first cells to contact ingested food and
develop digestive functions to render nutrients which are
transported across the enterocyte membranes, towards the
underlying mucosal blood circuits. Evidence accumulated
over the last century has shown that the digestive tract is
also actively involved in fish osmoregulation, especially in
euryhaline fish, and several studies have shown that many
fish are able to adapt their intestinal transport processes in
1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpb.2004.06.013
* Corresponding author. Tel.: +34 922 318343; fax: +34 922 318311.
E-mail address: [email protected] (M. Dıaz).
response to changes in environmental salinity (Lahlou,
1983; Colin et al., 1985; Lorenzo and Bolanos, 1989).
It is well known that fish intestine comprises several
structurally distinct regions in the proximal–distal axis,
usually beginning with a variable number of blind out-
growths known as pyloric caeca, whose function in fish
largely differs from the fermentation chamber role in
mammals and birds (Buddington and Diamond, 1986,
1987). Such anatomical differences are correlated with
functional differences between intestinal regions. Indeed,
several reports have shown that membrane-bound digestive
hydrolase activities are distributed along the digestive tract
according to different feeding habits (Ugolev and Kuz’mina,
1994; Harpaz and Uni, 1999). Likewise, the existence of
regional differences has been demonstrated for metabolic
enzymes (Mommsen et al., 2003), active glucose and amino
acid transport (Ferraris and Ahearn, 1983; Lorenzo et al.,
logy, Part A 139 (2004) 21–31
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–3122
1989), electrical resistance and transmural potential differ-
ence (Lorenzo et al., 1989) and potassium transport (Loretz
and Fourtner, 1991), between the proximal and distal
portions in several species of teleosts. Moreover, differences
in the activity of the Na+–K+-ATPase along the different
regions of the gilthead seabream intestinal tract have been
demonstrated, which have been attributed to different
isoenzyme and lipid microenvironments surrounding the
enzyme (Almansa et al., 2001, 2003).
However, most of the studies about these processes have
been developed using complete intestinal preparations.
Others have studied specific transport processes on isolated
brush-border membranes (Di Costanzo et al., 1983; Storelli
et al., 1989; Cahu et al., 2000; Boge et al., 2002), but the
physiological role that the enterocyte, as a whole digestive
and absorptive cell, directly plays in the intestinal functions
of marine fish has not been studied in depth. In this sense,
suspensions of isolated fish enterocytes would help the
understanding of the mechanisms of intestinal transport and
digestive functions because they provide both easy accesses
to the apical and basolateral membranes and to the
manipulation of cell environment.
The aim of the present work was to develop an isolation
procedure to obtain fish enterocytes from the different
regions in the proximal–distal axis of the intestine of the
gilthead seabream, Sparus aurata. We have also accom-
plished a rigorous characterization of enterocyte preparations
from the biochemical, physiological and morphological
points of view to look into the cellular traits accounting for
the different roles of enterocytes within the pyloric caeca and
the anterior and posterior intestines.
2. Materials and methods
2.1. Animals
Specimens of gilthead seabream (S. aurata L.) weighing
400–600 g were obtained from Centro Oceanografico de
Canarias (Instituto Espanol de Oceanografıa, Tenerife,
Spain). Fish were reared in 500-l tanks in seawater (35x)
at 20 8C and fed commercial fish pellets containing (in dry
weight basis) 44–45% protein, 10.5–14.5% carbohydrate
and 21–25% lipid (of which ~2.0% were n-3 HUFA). Once
in the laboratory, fish were maintained in small aquariums
for ~3 days without feeding until they were sacrificed by
decapitation. The whole intestine was removed and placed
in aerated physiological saline solution (PS) maintained at 4
8C. All procedures were performed in accordance with the
regulations by the Committee for the Care and Use of
Laboratory Animals at Universidad de La Laguna.
2.2. Cell isolation
Epithelial cells were obtained using the hyperosmolar
intracellular-like method originally described by Del Cas-
tillo (1987) with modifications. The different intestinal
sections, i.e., pyloric caeca, anterior intestine and posterior
intestine, were dissected and their contents removed by
gently washing with cold PS. As a rule, a 1-cm segment
between anterior and posterior intestines was routinely
rejected to avoid possible transitions between both intestinal
sections. Each intestinal segment was then filled and
incubated for 10 min at room temperature with a hyper-
osmolar (intracellular-like) solution (Solution I) containing
7.0 mM K2SO4, 44.0 mM K2HPO4, 9.0 mM NaHCO3, 10.0
mM HEPES, 180.0 mM glucose (pH 7.4) and was clamped
both ends with forceps. Afterwards, the intestinal solution
was discarded (to remove remaining food particles and
mucus) and intestinal sections were filled and incubated
again for additional 3 min with Solution I supplemented
with EDTA and dithiothreitol (DTT) as disjunctive agents
[Solution II: solution I+0.5 mM DTT, 0.2 mM EDTA].
During this time, intestinal segments were gently palpated
with the fingers. Then, the luminal solution containing cells
loosened from the epithelia was filtered through a 100-Amnylon mesh. Intestinal sections were then filled again with
Solution II for 3 min and the resultant cell suspension was
pooled together and centrifuged at 1500�g for 10 min in a
refrigerated centrifuge. The resulting pellets obtained from
the three intestinal sections were separately resuspended in a
Ringer-type solution (Solution III) containing, 116.0 mM
NaCl, 6.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 10.0
mM NaHCO3, 1.0 mM NaH2PO4, 10.0 mM K2SO4, 9.0
mM NaHCO3, 10.0 mM HEPES (pH 7.4) with collagenase
(0.1 mg/ml) and incubated under shaking for 15 min at 25
8C. Then, cell suspensions were filtered through a 60-Amnylon mesh and centrifuged at 1500�g for 10 min. Pellets
were resuspended in DMEM (Dulbecco’s modified Eagle
medium).
2.3. Homogenates
Isolated cells were homogenized at a concentration of
60�106 cells/ml using a Potter–Elvehjem homogenizer for 2
min, in the appropriate buffer depending on the type of
subsequent assay. The homogenization procedure was
performed using cold solutions kept on ice. Homogenates
were divided into 1–1.5 ml aliquots and stored at �80 8Cuntil assayed. For comparative purposes, the tissues
remaining after epithelial cell isolation (mainly underlying
smooth muscle), as well as untreated whole intestinal
segments, were also subjected to homogenization in
appropriate buffers at 200 mg wet mass/ml homogenization
buffer.
2.4. Viability tests
Cell integrity was tested by the colorant exclusion
method using Trypan blue (0.4% in PS). Cell suspensions
were placed on a Neubauer chamber and counted under
microscope at room temperature immediately after isolation.
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–31 23
Lactate dehydrogenase (LDH) activities and ATP con-
tents from isolated enterocytes were measured spectrophoto-
metrically by enzymatic reactions coupled to NADH
oxidation using Sigma Kits DG1340-K and 366-A, respec-
tively. Intracellular LDH activity and ATP content assays
were performed on, respectively, 100 and 500 Al samples,
homogenized in a sucrose buffer (SB) containing, 50 mM
sucrose, 20 mM Tris, 1 mM Na2EDTA and 1 mM PMSF
(pH=7.4 with 1 M HCl). Extracellular LDH activities were
also determined as a measure of cell integrity, in 100-Alsamples taken from the supernatant of centrifuged samples.
Oxygen consumption was measured on freshly isolated
cells in an O2-saturated closed system using a Clark
electrode connected to a computerized data acquisition
system and processed with RESPI software (Jeulin SAV,
France). Approximately 120�106 cells in 3 ml of DMEM
were added to the incubation chamber and maintained at 20
8C. After a 5-min stabilization period, O2 concentrations
were linear with time and were plotted on-line for 10–15
min. Experimental zero O2 concentration was obtained by
adding sodium dithionite. The rate of O2 consumption
(ROxygen) was computed as the slope of the linear relation-
ship in the plots [O2] versus time, and expressed in nmol O2/
mg protein/h. Protein contents in homogenates were
determined using the Coomassie blue method (Bradford
1976).
2.5. Cellular staining
Freshly isolated cells (300 Al cell suspension) were
seeded on polylysine-coated slides and were allowed to
sediment for 15 min. After washing with PS, 600 Al of a 1:3dilution of 1% Alcian blue were added to the slides and left
for 90 s. Then, slides were washed twice with PS and 600 Alof 1% erythrosin were added for 30 min. Afterwards, slides
were washed three times with PS, drained, and immediately
a few drops of Glycerol:PS mixture (1:1) were added onto
the preparation and covered with a coverslip. Preparations
were observed under light microscopy and images were
digitally acquired using Leica Q500MC morphometry
analysis software. Some preparations were also submitted
to haematoxylin–eosin staining (H–E). Briefly, cells seeded
on slides as stated before, were first exposed to 600-Al eosinfor 5 min and washed twice in PS (containing 0.1%
ethanol). Then, preparations were exposed to 600 Al Harris’haematoxylin for 1 min, washed three times in PS and
mounted as indicated above.
2.6. Digestive enzyme activities
The activities of six digestive enzymes were assayed in
homogenates from both isolated enterocytes and whole
intestine, i.e., sucrase (S), maltase (M), alkaline phosphatase
(AP), 5Vnucleotidase (5VN), leucine aminopeptidase (LA)
and g-glutamyl transferase (g-GT). g-GT was also assayed
in smooth muscle homogenates. The composition of
homogenization buffers varied depending on the specific
requirements of each enzyme activity under study. Thus, for
disaccharidases S and M, the homogenization buffer
contained, 50.0 mM sodium citrate, 1.35 mM EDTA, 20.0
mM Tris, 50.0 mM mannitol and 1.0 mM PMSF (pH=7);
while for AP, 5VN, LA and g-GT, the buffer contained, 50.0
mM sucrose, 20.0 mM Tris, 1 mM Na2EDTA, 1 mM PMSF
(PMSF was omitted for LA and g-GT; pH=7.5).
Sucrase and maltase activities were determined by
measuring the rate of hydrolysis of sucrose and maltose,
respectively, according to Dahlquist (1964) and Harpaz and
Uni (1999) with some modifications. Briefly, 50 Al enter-ocyte or whole intestine homogenates were mixed with 50
Al citrate buffer (CB) [50 mM citric acid, pH 4.8] and 50 Al150 mM of appropriate enzyme substrate (sucrose or
maltose in CB) and were incubated at 37 8C for 30 min.
Reactions were stopped by incubation in ice for 4 min.
Then, 50 Al was taken to measure the amount of glucose
produced by the hydrolysis of corresponding substrates.
Glucose was measured using standard methods (Sigma kits
510-Da and 17–25). Corrections were made for unspecific
glucose production from substrates in the absence of
homogenates.
Alkaline phosphatase activity was determined spectro-
photometrically by the rate of p-nitrophenyl phosphate
hydrolysis (Sigma kit 104-LS) on homogenates from whole
intestine or enterocytes and from fractionated enterocytes
(see later for fractionation procedure).
5VNucleotidase activity was determined according to the
method described in detail by Aronson and Touster (1974),
using Na-AMP (50 mM) as substrate in a medium containing
0.5 M glycine and 0.1 M MgCl2. Whole intestine homoge-
nate (50 Al) or enterocyte homogenate (25 Al) were added to
450 and 475 Al medium, respectively, and incubated for 30
min at 37 8C. The reaction was stopped by adding 2.5 ml of
8% trichloroacetic acid (TCA) and the inorganic phosphate
released during the reaction was measured spectrophoto-
metrically by the method of Forbush (1983).
Leucine aminopeptidase activity in whole intestine and
enterocyte homogenates (10 Al) was determined spectro-
photometrically by the rate of l-leucil-h-naphthylamide
hydrolysis using Sigma kit 251-AW. g-GT activity was
measured using Sigma kit (ref. 419-20) to determine the
hydrolysis of l-g-glutamyl-3-carboxy-4-nitroanilide by
whole intestine, and enterocyte homogenates (50 Al).Hydrolase activities were measured in duplicates and
expressed as Amol product/mg protein/h, except for the
case of sucrase which was expressed in nmol product/mg
protein/h.
2.7. ATPase activities
Three different ATPases activities, i.e., Na+–K+-, Na+-
and Cl�-ATPases were measured in enterocytes isolated
from the three intestinal sections. Cells were homogenized
in SB and divided into 1 ml aliquots in 1.5-ml micro-
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–3124
centrifuge tubes. Samples were first centrifuged for 5 min at
120�g and the resulting supernatant collected and centri-
fuged at 12,000�g for 30 min. The new pellet (P; mainly
formed by cell membranes) was resuspended in 450 Al ofSB. Both the pellet (P) and the final supernatant (S) were
submitted to ATPase activity assays.
ATPases activities were measured by adding 50 Almembrane suspension (P) or supernatant (S) to 1 ml of
appropriate reaction media [Na+–K+-ATPase: 200 mM
NaCl, 10 mM KCl, 5.0 mM MgCl2 and 25 mM HEPES (pH
7.4; Dıaz et al., 1998); Na+-ATPase: 5.0 mM MgCl2, 25.0
HEPES (pH 7.0) and 1.0 mM ouabain (Moretti et al., 1991);
Cl�-ATPase: 10.0 mM KCl, 6.0 mM Mg(CH3COO)2, 1.0
mM EDTA, 2.0 mM NaN3, 1.0 mM ouabain, 100.0 mM
Tris/MES (pH 6.0; Shiroya et al., 1989)]. The reaction was
started by the addition of vanadate-free ATP (5.0 mM for
the Na+–K+-ATPase and 6.0 mM for the Cl�-ATPase) or
Tris-ATP (2.0 mM for the Na+-ATPase) and incubated for
10 min at 25 8C. The inorganic phosphate released from
ATP (Pi) was determined by the method of Forbush (1983)
using Na2HPO4 as Pi standard. Specific Na+–K+-ATPase
activity was determined as the difference in the Pi
production from ATP in the presence or absence of 1.0
mM ouabain. Na+-ATPase activity was determined as the
difference in Pi production from ATP in the presence or
absence of sodium (40 mM NaCl). Specific Cl�-ATPase
activity was determined as the difference in Pi production in
the presence or absence of 0.3 mM ethacrynic acid.
Corrections for unspecific phosphate hydrolysis were made
by measuring Pi liberated in the absence of protein
suspension. ATPases activities were measured in triplicate
and expressed as Amol Pi/mg protein/h.
2.8. Statistical analysis
Mean values of the three different intestinal regions were
compared using either a one-way analysis of variance
(ANOVA) followed by Tukey’s or Duncan’s multiple
comparison test, or by Kruskall–Wallis analysis followed
Table 1
Viability tests performed on isolated epithelial cells from the different intestinal r
Pyloric caeca
LDH 98.91F24.61
(mU/mg protein) (3)
Protein 10.25F1.70
(lg/106 cells) (5)
ATP 15.89F4.74
(nmol/mg protein) (4)
Oxygen consumption 264F29
(nmol/mg protein/h) (5) a
Dye exclusion 92.63F1.71
(% Trypan blue exclusion) (6)
RLDH 10.65F3.09
(intracellular/extracellular) (3)
Results are expressed as meanFS.E.M. Values in the same row bearing different le
sample sizes. LDH: Lactate dehydrogenase. RLDH: intracellular to extracellular L
by a Mann–Whitney U-test to compare between groups.
Student’s t test was used to check the statistical difference
of mean values between enterocyte and whole intestine
data.
2.9. Materials
Vanadate-free Na2ATP was purchased from Boehringer
Mannheim (Germany). HEPES, EDTA, DMSO and Tris
were obtained from Merck (Germany). All other reagents
were purchased from Sigma (Biosigma, Spain). Ouabain
and ethacrynic acid were dissolved in DMSO and stored in
50 and 15 mM stock solutions, respectively. Carbonyl
cyanide 3-chlorophenylhydrazone (CCCP) was freshly
prepared in methanol (0.5 mM stock solutions) before each
experiment.
3. Results
3.1. Isolation of epithelial cells and validation of the
preparation
The method described here using a hyperosmolar intra-
cellular-like solution provided highly enriched preparations
mostly constituted by viable epithelial cells. Cell viability
was judged by different criteria including trypan blue
exclusion, oxygen consumption, intracellular ATP content
and lactate dehydrogenase activity. Results showed in Table
1 demonstrate that at the end of the isolation procedure,
more than 92% of the cells in the three intestinal segments
excluded trypan blue. Isolated cells exhibited oxygen
consumption values ranging from 67 to 264 nmol/mg
protein/h which were significantly different between intes-
tinal segments (Table 1). Thus, maximal O2 consumption
values (ROxygen) were observed in cells isolated from pyloric
caeca, while posterior intestine exhibited the minimal
ROxygen, revealing the existence of a proximal–distal
gradient regarding respiratory activity. Addition of carbonyl
egions of gilthead seabream (Sparus aurata)
Anterior intestine Posterior intestine
131.39F15.10 154.17F35.56
(4) (3)
8.97F0.63 9.16F1.60
(5) (5)
7.10F1.54 18.07F1.83
(4) (4)
150F43 67F13
(5) ab (5) b
94.42F0.44 94.37F0.37
(6) (6)
15.69F1.67 9.34F1.31
(4) (3)
tters are significantly different with pb0.05. Numbers in parentheses indicate
DH activity ratio.
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–31 25
cyanide 3-chlorophenylhydrazone (CCCP, 0.5 AM), a
mitochondrial uncoupler, to the incubation chamber,
increased ROxygen by 127%, 73% and 41% in pyloric caeca,
anterior intestine and posterior intestine preparations,
respectively. Measurement of intracellular ATP contents
showed values around 13 nmol/mg protein which are in the
range reported for other isolated intestinal cells and are
indicative that mitochondrial respiration was intact. Meas-
urements of intra- and extracellular LDH activity, revealed
high levels of intracellular lactate dehydrogenase activity
and that less than 10% of LDH activity was lost during the
isolation procedure (as inferred from the intracellular/
extracellular ratio), which is in good agreement with the
viability value aforementioned.
3.2. Morphological characterisation
The staining procedures set-up and used in this study
allowed the identification of two main cell types in the
epithelial preparation from the three intestinal segments, i.e.,
Fig. 1. Representative light micrographs of isolated epithelial cells from anterior in
obtained after 2 h of isolation showing different cell types in the epithelial pre
magnification micrograph of a mucous cell (H–E). Panels C and D: Detailed ima
different cellular types in the epithelial preparation stained with erythrosin–Alcian b
panel E, showing a mucous cell and several enterocytes. Panel G: Detailed image
AB staining. The apical membrane brush-border appears stained blue (arrows), a
enterocytes and mucous (Goblet) cells (Fig. 1). Upon
isolation, cells adopted rounded shapes, which may be a
consequence of disruption of intercellular junctions. In all
cases, enterocytes accounted for more than 95% of the overall
cell population (Fig. 1A, E). Haematoxylin–eosin staining of
epithelial preparations showed enterocytes exhibiting well-
defined excentric nuclei (Fig. 1C, D) and often additional
basophilic granules. The relative abundance of eosinophilic
cytoplasm compared to enterocytes allowed the identification
of mucous cells (Fig. 1G). The higher affinity of Alcian blue
dye for mucopolysaccharide molecules allowed the identi-
fication of mucous cells as larger cells mainly stained blue
due to their higher mucopolysaccharide content (Fig. 1E).
Enterocytes displayed a typical light pink-stained cytoplasm
and a membrane domain (presumably the brush-border
membrane) stained blue (Fig. 1F), which strongly reveals
the characteristic polarity of enterocytes. Preparations were
subjected to quantitative morphometrical analyses, the results
being summarized in Table 2. After isolation, cells tended to
adopt rounded shapes showing equivalent diameters around 5
testine of gilthead seabream (Sparus aurata). Panel A: Panoramic field view
paration following Haematoxylin–Eosin (H–E) staining. Panel B: Higher
ges of several enterocytes (H–E). Panel E: Panoramic field view showing
lue (E–AB). Panel F: Higher magnification micrograph of the area boxed in
of several enterocytes, showing their characteristic cellular polarity after E–
nd the cytoplasm stained pink.
Table 2
Morphometric analyses of isolated enterocytes (A) and mucous cells (B) from the different intestinal regions of gilthead seabream (Sparus aurata)
Area (Am2) Length (Am) Width (Am) Perimeter (Am) Eq. diameter (Am)
(A) ENTEROCYTES
Pyloric caeca 25.75F0.98 6.34F0.15 5.24F0.12 19.68F0.48 5.66F0.12
(47) a a a a a
Anterior intestine 20.06F1.58 5.50F0.22 4.59F0.20 17.26F0.66 4.97F0.19
(23) b b b b b
Posterior intestine 23.53F0.72 5.97F0.10 5.11F0.13 18.49F0.31 5.46F0.08
(15) ab ab ab ab ab
(B) MUCOUS CELLS
Pyloric caeca 75.49F34.57 9.4F1.72 8.22F1.59 29.28F5.28 8.51F1.62
(10) ab* ab* ab* ab* ab*
Anterior intestine 220.5F157.3 17.85F6.60 14.89F6.59 54.76F21.37 15.75F6.39
(5) a* a* a* a* a*
Posterior intestine 52.99F28.27 8.30F2.06 6.33F1.58 25.13F6.09 7.02F1.74
(7) b b b b b
Data are expressed as meansFS.E.M. Values in the same column bearing different letters are significantly different with pb0.05. Numbers in parentheses
indicate sample sizes.* Statistically different from enterocytes with pb0.01.
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–3126
and 8 Am for enterocytes and mucous cells, respectively.
Mucous cells from anterior intestine and pyloric caeca
exhibited larger diameter, area and perimeter than enterocytes
from the same intestinal regions. Similarly, mucous cells
from anterior intestine displayed slightly larger dimensions
than equivalent cells from pyloric caeca and posterior
intestine, although no differences were observed when
compared to pyloric cells.
3.3. Digestive enzymes
We have measured the activity of six digestive mem-
brane-bound hydrolases in both whole intestine and enter-
Table 3
Digestive hydrolase activities measured in isolated enterocytes (A) and whole tissu
(Sparus aurata)
S M AP
(A) ENTEROCYTES
Pyloric caeca 185.40F70.09 3.85F1.02 40.37F(7) ab (8) ab (8) b
Anterior intestine 153.24F47.06 2.83F0.53 76.38F(5) b (8) b (8) a
Posterior intestine 354.61F76.82 7.18F2.12 43.84F(8) a (8) a (8) ab
(B) WHOLE INTESTINE
Pyloric caeca 59.81F13.51 1.72F0.25 144.31F(4) (4) (4)**
Anterior intestine 67.04F5.73 1.46F0.02 106.71F(4)* (4) (4)
Posterior intestine 74.52F15.78 1.56F0.20 84.04F(4)** (4)** (4)
Activities are indicated as meansFS.E.M. (n). Values in the same column bearin
indicated in parentheses.
S: sucrase, M: maltase, AP: alkaline phosphatase, 5VN: 5Vnucleotidase, LA: leucexpressed as Amol product/mg protein/h except for S, which is expressed in nmo
* Statistically different from enterocytes with pb0.1.** Statistically different from enterocytes with pb0.05.
ocyte preparations from all three intestinal sections.
Determined enzyme activities included sucrase, maltase,
leucine aminopeptidase, 5Vnucleotidase, alkaline phospha-
tase and g-glutamyl transferase (g-GT) and the results are
summarized in Table 3. No significant differences between
intestinal sections were found on enzymatic activities from
preparations of whole intestine. However, enterocyte prep-
arations exhibited a clear heterogeneity in the distribution of
enzyme activities along the digestive tract. Thus, maximal
sucrase, maltase and leucine aminopeptidase activities were
observed in posterior intestine, whereas anterior intestine
displayed the lowest values. On the contrary, alkaline
phosphatase and 5Vnucleotidase activities were lower in
e (B) homogenates from the different intestinal regions of gilthead seabream
5VN LA gGT
8.78 2.85F0.62 97.20F32.45 0.13F0.05
(8) b (8) ab (3)
15.74 10.44F2.81 60.04F15.36 0.15F0.03
(8) a (7) b (3)
8.31 3.31F0.79 162.98F37.10 0.18F0.04
(8) b (8) a (3)
31.74 2.13F0.21 25.17F8.90 0.16F0.02
(4) (4)** (3)
14.69 1.87F0.20 38.33F3.82 0.11F0.01
(4)** (4) (3)
25.61 1.74F0.25 24.50F5.42 0.20F0.05
(4) (4)** (3)
g different letters are significantly different with pb0.05. Sample sizes are
ine aminopeptidase, gGT: g-glutamyl transferase. Hydrolase activities are
l product/mg protein/h.
Fig. 2. Enrichment of hydrolase activities in the isolated enterocytes from
different intestinal regions of gilthead seabream (Sparus aurata). Enrich-
ment factors were calculated as the ratios between enterocyte and whole
tissue activities for each enzyme. S: sucrase, M: maltase, AP: alkaline
phosphatase, 5VN: 5Vnucleotidase, LA: leucine aminopeptidase, gGT: g-
glutamyl transferase.
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–31 27
pyloric caeca and posterior intestine, with maximal activity
values observed in the anterior region. Finally, g-GT
activities were similar in enterocyte preparations from the
three intestinal sections.
We have also calculated the enterocyte-to-whole intestine
activity ratios and plotted the results to estimate the
fractional enrichment factor in the intestinal longitudinal
and transverse axes. Values higher than 1 indicate that
activity is concentrated in the enterocyte. The results shown
in Fig. 2 demonstrate that sucrase, maltase, leucine amino-
peptidase and 5Vnucleotidase activities are predominantly
concentrated in the enterocyte with regards to the whole
intestine, which are in agreement with the digestive nature
Table 4
Alkaline phosphatase activities in enterocyte homogenates (H) and in the fractio
intestinal segments of gilthead seabream (Sparus aurata)
H
Pyloric caeca
Proteins R% 100
Alkaline phosphatase SA 66.62 F 6
RSA 1.00
R% 100
Anterior intestine
Proteins R% 100
Alkaline phosphatase SA 85.98F9.4
RSA 1.00
R% 100
Posterior intestine
Proteins R% 100
Alkaline phosphatase SA 69.20F11.
RSA 1.00
R% 100
SA: specific activity, expressed in Amol product/mg protein/h; RSA: relative specif
the homogenate; R%: percentage of proteins or total activity of the homogenate
Alkaline phosphatase activities and R% values are indicated as meanFS.E.M. of th
significantly different with pb0.05.
of these membrane-bound enzymes. The data also revealed
that sucrase, maltase, leucine aminopeptidases were sur-
prisingly prominent in enterocytes from posterior intestine
compared to the other segments. On the contrary, alkaline
phosphatase and g-glutamyl transferase activities appear to
be homogeneously distributed in both transverse and
longitudinal intestinal axes. Alkaline phosphatase seemed
to be predominantly expressed in tissues other than
epithelia, presumably the underlying tissues, including
submucosal and smooth muscle layers. However, because
alkaline phosphatase is considered to be a good marker of
brush-border membranes, we performed activity assays on
purified enterocyte membranes. The results shown in Table
4 show that after cell fractionation all alkaline phosphatase
activity measured in enterocyte homogenates is completely
recovered in the membrane pellets but none in the
supernatants.
3.4. ATPases activities
Results shown in Table 5 demonstrate that three Mg2+-
dependent ATPases, i.e., Na+–K+-, Na+- and Cl�-ATPase
activities can be detected in homogenates of isolated
enterocytes from the three intestinal segments. ATPase
activities were mainly found in the pellet fractions compared
to supernatants, indicating a major proportion of cell
membranes in the former. The distribution of the different
ATPase activities was not uniform along the intestinal tract.
Thus, Na+–K+-ATPase activity decreased in the proximal to
distal axis; reaching its maximal value in the pyloric caeca
(this trend was also evident in the corresponding supernatant
fractions). Significant differences were detected for the Na+-
ATPase between sections, whose activity seemed to follow a
ned pellet (P) and supernatant (S) from enterocytes isolated from different
S P
37.88F7.18 57.45F2.61
.89 ND 77.10F5.29 ab
0.0 2.01
0.0 115.73F7.94
38.68F5.02 67.23F4.86
8 ND 98.72F4.52 a
0.0 1.77
0.0 114.82F5.26
33.41F7.77 79.81F15.41
74 ND 74.40F8.80 b
0.0 1.34
0.0 107.51F12.72
ic activity, calculated as the ratio between the SA of each fraction and that of
recovered in the fraction.
ree different animals. Values in the same column bearing different letters are
Table 5
Activities of three Mg2+-dependent ATPases measured in the pellet (P) and supernatant (S) fractions of enterocyte homogenates
Na+–K+-ATPase Na+-ATPase Cl�-ATPase
P S P S P S
Pyloric caeca 1.97F0.40 0.52F0.14 4.19F0.92 0.26F0.03 0.19F0.01 0.09F0.01
(4) a (6) (4) a* (6) (3) b (4)
Anterior intestine 1.12F0.23 0.37F0.12 1.45F0.34 0.48F0.16 0.56F0.05 0.14F0.03
(6) ab (6) (5) b (5) (3) a (3)
Posterior intestine 0.75F0.23 0.21F0.08 2.07F0.32 0.46F0.12 0.44F0.07 0.05F0.01
(4) b (5) (5) b** (5) (4) a (3)
ATPase activities are expressed as Amol Pi/mg protein/h and indicated as meansFS.E.M. Values in the same column bearing different letters are significantly
different with pb0.05. Sample sizes are indicated in parentheses.* Statistically different from Na+–K+-ATPase with pb0.1.** Statistically different from Na+–K+-ATPase with pb0.05.
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–3128
U-shaped distribution along the intestine, with largest
activity values found in pyloric caeca. Conversely, Cl�-
ATPase activity was minimal in that same intestinal region
and significantly increased in anterior and posterior intes-
tines. Comparison of Na+-transporting Mg2+-dependent
ATPase activities for each intestinal section indicated that
the Na+-ATPase was always predominant over Na+–K+-
ATPase, irrespective of the enterocyte origin, with pyloric
caeca exhibiting the highest activity values (Table 5).
4. Discussion
The present study describes a successful procedure for
the isolation of epithelial cells from anterior and posterior
intestines as well as from pyloric caeca of the euryhaline
teleost S. aurata. The use of a hyperosmolar, intracellular-
like (containing high K+ and low Na+) solution has allowed
us to obtain enterocyte preparations from the three intestinal
regions exhibiting high purity and viability. Viability tests
performed here showed a very high degree of membrane
integrity, as indicated by the fact that more than 90% of
epithelial cells in the three segments excluded trypan blue
and retained most of the lactate dehydrogenase activity
intracellularly, as judged from the intracellular/extracellular
LDH ratios. Moreover, mitochondrial functional state of
isolated cells appeared to be well preserved based on the
results of oxygen consumption rates, response to CCCP and
intracellular ATP concentrations. Oxygen consumption rates
were within the range of values published for isolated cells
from sea raven and ocean pout (Legate et al., 1998).
Interestingly, respiratory activity decreased in the proximal–
distal axis of the intestine, with maximal values being
observed in the enterocytes from pyloric caeca and minimal
ROxygen in the posterior intestine preparation. Similarly,
although respiratory activity was increased by CCCP in all
enterocyte populations, the response to this mitochondrial
uncoupler was maximal in pyloric caeca and minimal in
posterior intestine. These findings suggest that enterocyte
energetic metabolism is subjected to an aerobic-to-anaerobic
gradient in the proximal–distal axis. This hypothesis was
further reinforced by the observation that LDH activities
followed an opposite trend, with maximal and minimal
activities detected in posterior intestine and pyloric caeca,
respectively. Interestingly, a similar metabolic gradient has
been demonstrated for mitochondrial citrate synthase,
glutamate dehydrogenase and malate dehydrogenase activ-
ities in juvenile trout intestine (Mommsen et al., 2003), yet
this trend was not always followed by homologous enzymes
in other studied teleosts. Finally, it was noticeable that,
whereas oxygen consumption rates were well below the
values reported for intestinal epithelial cells isolated from
endotherms (Brown and Sepulveda, 1985; Ferrer et al.,
1986; Del Castillo, 1987), intracellular ATP concentrations
were found to be markedly similar, reflecting the different
metabolic efficiency between endo- and ectothermic
animals.
Isolated intestinal cells from gilthead seabream consisted
mostly of enterocytes (N95%), and a smaller proportion of
mucous (Goblet) cells, which exhibited a clear distinctive
dye affinity and morphometric biometry when compared
with enterocytes. As judged from light micrographs, as
shown in Fig. 1, most of the enterocytes keep their polarity
showing a brush-border and a basolateral plasma membrane.
Overall, the data discussed up to now demonstrate that the
isolation procedure set-up in this study yields highly pure
and viable enterocyte-enriched epithelial cell preparations,
which preserve much of the morphological and functional
characteristics of the intestinal cells in situ.
Most of the current knowledge on the digestive processes
taking place in the intestinal tract of fish have been
performed on whole intestinal homogenates (Ugolev and
Kuz’mina, 1994; Harpaz and Uni, 1999). Although, in some
cases, these studies have allowed establishment of general
patterns of digestive enzyme distribution for fish feeding
and ecological niches (Chakrabarti et al.,1995; Harpaz and
Uni, 1999), the contaminating inclusion of non-epithelial
tissues (mainly submucosal and muscle, which are not
directly involved in digestive-absorptive functions) in the
enzyme assays, raises important concerns and makes the
results difficult to interpret at the cellular level. It is only in
the epithelial layer of the intestine where the final stage of
digestion, where nutrient absorption takes place and is
carried out by the enterocytes, which acquire differentiated
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–31 29
expression of brush-border digestive enzymes and trans-
porters during their migration to the villus tip (Brown and
Sepulveda, 1985; Ferraris et al., 1992).
We could demonstrate that enterocyte preparations from
S. aurata exhibited a clear heterogeneity in the distribution
of disaccharidase (sucrase and maltase), leucine amino-
peptidase, alkaline phosphatase and 5Vnucleotidase activ-
ities along the digestive tract, which, in turn, appear
concentrated two- to sixfold in the enterocytes versus the
intestinal homogenates. Strikingly, maximal disaccharidase
(sucrase and maltase) and leucine aminopeptidase activities
were observed in posterior intestine, whereas anterior
intestine displayed the lowest values. On the contrary,
alkaline phosphatase and 5Vnucleotidase activities were
lower in pyloric caeca and posterior intestine than in anterior
intestine. The finding that alkaline phosphatase was not
specially enriched in enterocytes versus whole intestine was
astonishing because this enzyme activity has been regarded
as a good marker of brush-border membranes. However,
several pieces of evidence suggest that intestinal alkaline
phosphatase is not exclusive to enterocyte membranes. On
one hand, a soluble form of alkaline phosphatase may be
secreted into the intestinal lumen (Yedlin et al., 1981),
which could contribute to absolute activities found in whole
intestine homogenates. On the other hand, histochemical
mapping of alkaline phosphatase in the digestive tract of
different teleost fishes has shown that mucosal and
submucosal layers display intense alkaline phosphatase
activities (Tyagi et al., 1980), which would explain the
significant specific alkaline phosphatase activity measured
in whole tissue homogenates. Nonetheless, enzyme analyses
performed on membrane preparations from isolated enter-
ocytes from S. aurata have revealed that 100% of overall
alkaline phosphatase activity found in enterocytes is
concentrated in the membrane fraction, with negligible
activities detected in cell homogenate supernatants (Table
4). Therefore, we can conclude that AP activity in gilthead
seabream enterocytes is restricted to membrane domains and
agrees with most literature data on the AP being regarded as
a good marker of enterocyte brush-border membranes.
Finally, g-GT activities were similar in enterocyte
preparations from the three intestinal sections. These
observations suggest that fish intestinal digestion clearly
differ from the general assertion that disaccharidase and
transpeptidase enzymes are mainly expressed in the foregut
epithelium, which have been well documented and estab-
lished for mammal and bird paradigms (Stevens and Hume,
1995). On the other hand, the demonstration of different
digestive activities in caecal enterocytes from gilthead
seabream indicates that the pyloric caeca are capable of
enzymatic hydrolysis. This finding agrees with the emerging
notion that fish caeca increase gut surface area and carry out
enzymatic digestion and nutrient absorption processes,
against the prevalent view that they merely serve as a
fermentation compartment (Buddington and Diamond,
1986, 1987).
It has been established that different fish species exhibit
different patterns of brush-border enzyme activities accord-
ing to their feeding habits (Ugolev and Kuz’mina, 1994;
Harpaz and Uni, 1999) and it has been assumed that
carnivorous fish exhibit lower disaccharidase activity than
the omnivorous and herbivorous counterparts. In this sense,
the gilthead seabream, a carnivorous species, appears to
follow the same trend observed for other freshwater and
marine carnivorous fish, such as striped bass, sea bass, pike
perch, among others (Ugolev and Kuz’mina, 1994; Harpaz
and Uni, 1999). However, the presence of most of the
principal digestive enzymes throughout the digestive tract
observed in most fish, including S. aurata, suggests that the
digestive system of confined aquatic teleosts has no strategy
with regard to the food intake and feeding habits they adopt,
perhaps because of their incomplete segregation of food
niche as well as their evolutionary adaptation. Chakrabarti et
al. (1995) have postulated that, from an evolutionary point
of view, during the early phase of evolution, there was no
specific functional zonation of the digestive system and
most of the zones of the intestinal tract were capable of
producing all the principal digestive enzymes. Only, as
evolution proceeded, with the advent of terrestrial forms,
site-specific enzyme production became restricted.
Our present results also demonstrate the existence of
heterogeneous distribution patterns for different Mg2+-
dependent ATPase activities in the enterocytes of S. aurata.
Thus, Na+–K+-ATPase activity decreased in the proximal–
distal axis, paralleling previous observations from our
laboratory on mucosal homogenates from this same species
(Almansa et al., 2001, 2003). It is noticeable that maximal
Na+–K+- and Na+-ATPase activities were found in the
pyloric caeca, and that the activity of the latter was always
predominant over the Na+–K+-ATPase in all segments.
These observations strongly argue in favour of a crucial role
of the Na+-ATPase supporting the functioning of secondary
active transport processes, namely sodium-dependent
nutrient absorption, and osmo-ionoregulatory mechanisms
in euryhaline and marine fish (Moretti et al., 1991;
Proverbio et al., 1991).
We have also detected significant activity levels of Cl�-
ATPase (anion-sensitive ATPase) in isolated enterocytes
from all segments from S. aurata. Comparison of Cl�-
ATPase activity data showed maximal values in the enter-
ocytes from anterior and posterior intestines, which differs
from the results reported for Salmo gairdneri (Rey et al.,
1991), where similar activities were observed throughout the
intestine, though this study was performed on whole tissue
homogenates. Cl�-ATPase is an enzyme that appears to be
related to active chloride transport in the osmoregulatory
organs (gills, kidneys and intestine) of freshwater teleosts
during adaptation to seawater (Morisawa and Utida, 1976;
Fuentes et al., 1995; Gerencser and Zhang, 2003). Therefore,
it seems reasonable to hypothesize that the high occurrence of
Cl�-ATPase in the intestinal cells of S. aurata could aid the
fulfillment of complex ionoregulatory mechanisms in this
R. Dopido et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 21–3130
marine species. Nevertheless, additional studies will be
required to establish the extent to which changes on
environmental salinity affect the expression pattern of the
different Mg2+-dependent ATPases identified in the isolated
enterocytes of this euryhaline species.
In summary, isolated cells from the intestinal epithelia of
gilthead seabream exhibit segment-specific heterogeneity
regarding respiratory, digestive hydrolase and Mg2+-
dependent ATPase activities along the longitudinal axis of
the intestine. Such asymmetry likely reflects differences in
the functional role of enterocyte populations in each
intestinal segment. The isolated cells are viable and suitable
for further cytological and molecular studies to elucidate the
mechanisms of iono-osmoregulation, and digestive and
nutrient transport processes.
Acknowledgements
This work was supported by grants PI1999/056 and
PI2001/059 from Gobierno Autonomo de Canarias (Spain)
and by grant AGL 2003-06877/ACU from the Ministerio de
Ciencia y Tecnologıa (Spain). R. Dopido is recipient of a
PhD fellowship from the Ministerio de Educacion Cultura y
Deportes (Spain). C. Rodrıguez was supported by a Ramon
y Cajal contract from the Ministerio de Ciencia y Tecnologıa
(Spain). We wish to dedicate this work to the memory of
Juan Manuel Martin Trujillo who, for most of us, was a
magnificent source of scientific stimulus and friendship.
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