11
Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Sparus aurata L.) Rosa Do ´ pido, Covadonga Rodrı ´guez, Toma ´s Go ´ mez, Nieves G. Acosta, Mario Dı ´az * Laboratorio de Fisiologı ´a Animal, Departamento de Biologı ´a Animal, Facultad de Biologı ´a, 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 Mg 2+ -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 response to changes in environmental salinity (Lahlou, 1983; Colin et al., 1985; Lorenzo and Bolan ˜os, 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., 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. Dı ´az). Comparative Biochemistry and Physiology, Part A 139 (2004) 21– 31 www.elsevier.com/locate/cbpa

Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Sparus aurata L.)

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www.elsevier.com/locate/cbpa

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

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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.

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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-

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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.

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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.

Page 6: Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Sparus aurata L.)

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.

Page 7: Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Sparus aurata L.)

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

Page 8: Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Sparus aurata L.)

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

Page 9: Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Sparus aurata L.)

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

Page 10: Isolation and characterization of enterocytes along the intestinal tract of the gilthead seabream (Sparus aurata L.)

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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