Am J Physiol Regul Integr Comp Physiol 288: R1598 –R1605, 2005.
First published February 3, 2005; doi:10.1152/ajpregu.00741.2004.
Tissue culture of sockeye salmon intestine: functional
response of Na⫹-K⫹-ATPase to cortisol
Philip A. Veillette1 and Graham Young1,2
1
Department of Zoology, University of Otago, Dunedin, New Zealand; and 2School
of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington
Submitted 2 November 2004; accepted in final form 26 January 2005
A PRIMARY MECHANISM OF ION transport across intestinal epithelium is the basolateral Na⫹-K⫹-ATPase. This ATP-requiring
enzyme provides an electrochemical gradient that drives solute
and water transport (24, 48). The vertebrate intestine is a major
osmoregulatory organ, and, in fish, intestinal salt and water
uptake is essential for maintaining internal water balance,
especially in seawater where osmotically lost water must constantly be replaced (44).
Electrolyte transport across the vertebrate intestine is modulated by steroids of the adrenal cortex, the glucocorticoids,
and mineralocorticoids (5, 6, 22, 40). In teleost fishes, cortisol
is the primary corticoid secreted by the diffuse interrenal
tissue (adrenal homologue), and, because aldosterone is
generally not found in plasma, cortisol exhibits a wide spectrum of action, including both ionoregulatory and metabolic
functions (32).
In vivo, intestinal ion and water transport is regulated by
cortisol in teleosts. In several species of the genera Anguilla
(eels) and in Carassius auratus auratus (goldfish) and Fundu-
lus heteroclitus (cyprinodont), adrenocorticotropic hormone or
cortisol stimulates intestinal water absorption and Na⫹-K⫹ATPase activity in intact fish and restores these mechanisms
that are otherwise decreased with interrenalectomy or hypophysectomy (12–14, 16, 19, 20, 34). The augmentation of water
absorption is specific for cortisol, since other C21 steroids are
without effect (20, 36).
In juvenile migratory salmonids, intestinal Na⫹-K⫹-ATPase
activity and fluid uptake become elevated during the seasonal
event termed parr-smolt transformation that takes place in fresh
water and is preparatory for seawater survival (4, 33, 50, 53).
These changes in the intestine are part of the complex of
physiological mechanisms that lead to increased salinity tolerance (21, 31). Cortisol is thought to mediate osmoregulatory
adaptations of the salmonid intestine (46). The cortisol-synthesizing capacity of the coho salmon’s interrenal tissue and
circulating concentrations of cortisol in sockeye salmon are
elevated during this period of development (15, 54), and
cortisol stimulates intestinal Na⫹-K⫹-ATPase activity and
fluid uptake in anadromous rainbow trout and brown trout (25,
39), Atlantic salmon (8), and chinook salmon (51). Finally,
intestinal fluid absorption in Atlantic salmon (Salmo salar) is
inhibited by the glucocorticoid receptor antagonist RU-486,
indicating a cortisol-specific regulation (49).
Despite this body of evidence, the regulation of osmoregulatory mechanisms by cortisol in fish intestine have been
limited to in vivo studies. A culture system would be advantageous to discriminate between direct and indirect actions of
regulatory signals, particularly in salmon, since a host of
endocrine factors are elevated during parr-smolt transformation
that likely interact with cortisol and contribute to increased
hypoosmoregulatory ability (28). These include growth hormone, insulin-like growth factor-I (IGF-I), and thyroid hormones (1, 37, 55).
In the present paper, a short-term culture system for intestinal explants from freshwater-adapted sockeye salmon (Oncorhynchus nerka) was developed. Two regions of the intestine
were examined due to their prominent absorptive functions: the
numerous pyloric ceca, which are attached and open to the
anterior intestine, and the posterior region of the intestine.
Although ceca are the primary site for nutrient uptake (3), both
ceca and posterior intestine are major sites of salt and water
balance (4, 50, 51). Cytological and physiological changes
during culture were assessed. Na⫹-K⫹-ATPase activity was
the endpoint for analyses of functional responses to cortisol.
Part of these data have appeared in abstract form (52).
Address for reprint requests and other correspondence: P. A. Veillette,
Graduate School of Oceanography, Box 14, Univ. of Rhode Island, 218 South
Ferry Rd., Narragansett, RI 02882-1197 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
teleost; Oncorhynchus nerka; osmoregulation; pyloric ceca
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Veillette, Philip A., and Graham Young. Tissue culture of sockeye
salmon intestine: functional response of Na⫹-K⫹-ATPase to cortisol.
Am J Physiol Regul Integr Comp Physiol 288: R1598 –R1605, 2005. First
published February 3, 2005; doi:10.1152/ajpregu.00741.2004.—A
method to culture tissue explants of the intestine from freshwateradapted sockeye salmon (Oncorhynchus nerka) was developed to
assess possible direct effects of cortisol on Na⫹-K⫹-ATPase activity.
As judged by several criteria, explants from pyloric ceca and the
posterior region of the intestine remained viable during short-term
(6-day) culture, although Na⫹-K⫹-ATPase activity declined and basolateral components of the enterocytes were observed to be partially
degraded. Addition of cortisol to the culture medium maintained
Na⫹-K⫹-ATPase activity (over 2–12 days) above that of control
explants and, in some cases, was similar to levels before culture. The
response to cortisol was dose dependent (0.001–10 ␮g/ml). Within the
physiological range, the response was specific for cortisol and showed
the following hierarchy: dexamethasone ⱖ cortisol ⬎ 11-deoxycortisol ⬎ cortisone. Insulin maintained Na⫹-K⫹-ATPase activity over
controls in explants of ceca but not posterior intestine. To compare in
vivo and in vitro responses, slow-release implants of cortisol (50
␮g/g) were administered to salmon for 7 days. This treatment elevated
plasma cortisol levels and stimulated Na⫹-K⫹-ATPase activity in
both intestinal regions. The results demonstrate that the teleost intestine is a direct target of cortisol, this corticosteroid protects in vitro
functionality of Na⫹-K⫹-ATPase, and explants retain cortisol responsiveness during short-term culture.
IN VITRO RESPONSE OF SALMON INTESTINE TO CORTISOL
MATERIALS AND METHODS
of protein (BCA assay), yielded ouabain-sensitive Na⫹-K⫹-ATPase
activity.
Statistics. For all experiments, each gut region was analyzed
separately. In experiments with a control and a single treatment,
significant differences (P ⬍ 0.05) were assessed by one-way
ANOVA. In all experiments involving results for multiple days, there
was a significant interaction between treatment and day (two-way
ANOVA). Therefore, for these results, each day was analyzed separately to determine differences between treatments. Two-way
ANOVA was used to assess the simultaneous effects of 1) fetal bovine
serum and cortisol and 2) insulin and cortisol. When appropriate,
Tukey’s honestly significant difference procedure was applied for post
hoc comparisons between means. We used one-way ANOVA to
analyze dose-response curves, followed by Dunnett’s post hoc test to
determine significant differences from controls (0 ␮g/ml). Analyses of
half-maximal responses (effective concentrations ⫽ EC50) to cortisol
and dexamethasone were performed with the use of four-parameter
curve fitting on mean Na⫹-K⫹-ATPase activity for each dose.
RESULTS
Cytological changes in culture. Light microscopy analyses
showed that, after 3 or 6 days in culture, shrinkage of the tissue
occurred in general, including a reduction in size of the
mucosal folds and cells of the epithelium (Fig. 1). A few parts
of the tissue exhibited signs of necrosis, and there was some
edema of the tissue underlying the epithelium, the lamina
propria. One of the more pronounced changes during culture
was partial degradation at the base of the single layer of
epithelial cells, resulting in a separation of the epithelium from
the underlying tissue in some sections. However, at the ultrastructural level, the cells exhibited functionality on the basis of
intact nuclei and other organelles, intact microvilli of the brush
border, and intact cell-to-cell borders and junctions (Fig. 2).
Cortisol-treated explants were not examined cytologically.
Tissue protein and sodium concentrations in culture. Total
protein and sodium content of intestinal explants remained
relatively unchanged in both ceca and posterior intestine after
6 days in culture, although sodium content of ceca rose moderately (Table 1).
Effect of cortisol on tissue protein in culture. Potential
effects of cortisol on tissue protein during culture were examined because measurements of Na⫹-K⫹-ATPase activity were
normalized to total protein and changes in protein content
could affect estimations of enzyme activity. Table 2 shows the
effects of cortisol exposure (1 ␮g/ml) on protein content of
intestinal explants in culture for 6 days. The amount of protein
in explants of pyloric ceca was unaffected by cortisol, but this
was significantly lowered in the posterior intestine compared
with controls.
Time course of cortisol action on Na⫹-K⫹-ATPase in culture. Explants were cultured from 2 to 6 days in the presence
and absence of 1 ␮g/ml cortisol and/or 10% fetal bovine
serum. This concentration of cortisol was chosen because it has
been shown to stimulate Na⫹-K⫹-ATPase activity in the cultured gill of several salmonids (29, 30). Although BSA was a
constituent of the culture medium, addition of fetal bovine
serum was explored to determine whether it affected Na⫹-K⫹ATPase activity or could potentiate a response to cortisol,
perhaps through growth factors (or other factors) present in the
serum.
In the present and subsequent experiments, Na⫹-K⫹ATPase activity generally declined after several days in culture
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Fish husbandry. Juvenile sockeye salmon, between 1 and 2 yr of
age, were maintained in recirculating, freshwater aquaria at 12°C
under a natural photoperiod. Before dissections, salmon were lethally
anesthetized in 3-aminobenzoic acid ethyl ester (MS-222, 200 mg/l)
buffered with sodium bicarbonate (400 mg/l). All maintenance and
experimental manipulations of salmon and tissues were approved by
the University of Otago Committee on Ethics in the Care and Use of
Laboratory Animals and adhered to the APS’s Guiding Principles in
the Care and Use of Animals.
Implants. Salmon were anesthetized in 0.2% 2-phenoxyethanol and
given intraperitoneal injections of cortisol (50 ␮g/g body wt) suspended in a lightly heated mixture (1:1) of vegetable oil and vegetable
shortening as described in detail elsewhere (45). Controls were injected with vehicle.
Tissue culture. The technique used was adapted from a previously
published method for culturing gill filaments from salmon (29).
Several pyloric ceca and the posterior region of the intestine were cut
from the gastrointestinal tract, gently sliced open along the long axis,
and laid flat (serosal side down) on an ice-cold glass sheet. Each
intestinal region was then cut into pieces of ⬃1 ⫻ 1.5 mm and
carefully placed in 24-well culture plates (in duplicate) containing
preincubation medium (MEM with Hanks’ salts, 5 mg/ml BSA, 250
U/ml penicillin G, and 250 ␮g/ml streptomycin sulfate, adjusted to pH
7.8 at 14°C). After several hours, the medium was replaced with
MEM containing Earle’s salts (pH 7.8), 4 mg/ml BSA, 292 ␮g/ml
L-glutamine, 50 U/ml penicillin G, and 50 ␮g/ml streptomycin sulfate.
Explants were incubated at 14°C in an air-tight humidified chamber
and gassed daily with 95% O2-5% CO2 (29). Every third day, the
culture medium was replaced with freshly prepared MEM. Steroids
were dissolved in ethanol and bovine insulin in 0.01 N HCl, before
addition to culture media, so that the final solvent concentration in all
plate wells was 1 ␮l/ml (including wells with control tissue). Although explants were occasionally found to adhere to the bottom of
the plate well, they typically remained unattached during culture.
Microscopy. For analyses of tissues by light microscopy, intestinal
explants were fixed in aqueous Bouin’s fixative overnight at room
temperature, dehydrated through an ethanol series, and infiltrated and
embedded in paraffin. Sections were cut at 6 ␮m and stained with
hematoxylin and eosin, all according to standard histological procedures. Transmission electron microscopy was carried out on tissues
that were prepared as follows: explants were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer at 4°C overnight. Postfixation was in
1% osmium tetroxide-1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 1 h. Tissue was then dehydrated and embedded in Agar
100 resin. Ultrathin sections were cut at 80 nm, stained with uranyl
acetate and lead citrate, and viewed on a Philips CM100 transmission
electron microscope.
Measurement of tissue protein and sodium. Before protein and
sodium analyses, explants were dried overnight at 65°C, weighed (for
normalizing protein and sodium concentrations), and then dissolved in
0.1 N HNO3 overnight. Methods for protein determinations followed
the procedures of McCormick and Bern (29). Tissue was homogenized in 0.1% sodium deoxycholate and assayed for protein using
bicinchoninic acid (BCA; Sigma) as chromogen. Sodium concentrations were measured by inductively coupled plasma spectroscopy at
Chemsearch Laboratories (Department of Chemistry, University of
Otago).
Measurement of plasma cortisol and Na⫹-K⫹-ATPase. Concentrations of cortisol were measured by radioimmunoassay in unextracted
plasma (54). The assay for determining Na⫹-K⫹-ATPase activity has
been reported by Veillette and Young (53). Briefly, crude homogenates of intestine were prepared, and the hydrolysis of ATP was
enzymatically coupled to the conversion of NADH to NAD⫹. The
utilization of NADH was measured over 8 min in the presence and
absence of ouabain. The difference in activity, normalized to amount
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IN VITRO RESPONSE OF SALMON INTESTINE TO CORTISOL
in the absence of cortisol. Treatment with cortisol significantly
maintained Na⫹-K⫹-ATPase activity over that shown in control tissue in pyloric ceca within 2 days of culture, and this
response continued through 6 days of culture (Fig. 3). In the
posterior intestine, cortisol exposure maintained Na⫹-K⫹ATPase activity over controls after 4 and 6 days of culture. At
no time did activity exceed initial, preculture levels in any of
the treatment groups. Addition of 10% fetal bovine serum to
the culture medium did not enhance the maintenance of in vitro
Na⫹-K⫹-ATPase activity or change the response to cortisol
(Fig. 3). Fetal bovine serum contained ⬍2 ng/ml cortisol, as
assessed by radioimmunoassay.
DISCUSSION
The most important finding of this study is that the intestine
of sockeye salmon is responsive to cortisol in short-term
culture, as evidenced by the maintenance of Na⫹-K⫹-ATPase
activity. This is the first demonstration of a direct action of
cortisol on the teleost intestine. The response to cortisol was
dose dependent and specific for cortisol in a physiological
range. The present results implicate the intestinal epithelium as
a target for the osmoregulatory actions of cortisol in salmonids.
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Fig. 1. Hematoxylin- and eosin-stained light micrographs of the intestine from
freshwater-adapted sockeye salmon before (A) and after (B–E) culture. A:
several mucosal folds of the epithelium of a pyloric cecum: epithelium (e),
lamina propria (lp), and stratum compactum (sc). Posterior intestine is similar
(not shown). B and C: pyloric cecal epithelium after 4 (B) or 6 (C) days in
culture. D and E: posterior intestinal epithelium after 4 (D) or 6 (E) days in
culture. Enterocytes of sections of the cultured epithelium retain structural
integrity after 4 days in culture (arrows in B and D), but epithelial nuclei are
less organized by day 6 (C and E). Scale bar (100 ␮m) applies to all panels.
Addition of cortisol (1 ␮g/ml) to the culture medium 3 days
after the start of explant culture resulted in significant elevations of Na⫹-K⫹-ATPase activity over controls in both intestinal regions within 2 days. This response continued for 4 days
after addition of cortisol to the medium (Fig. 4).
Effect of insulin and cortisol on Na⫹-K⫹-ATPase in culture.
The in vitro effect of insulin (10 ␮g/ml), with or without
cortisol (10 ␮g/ml), was explored. After 6 days of culture,
cortisol significantly maintained Na⫹-K⫹-ATPase activity
over controls in pyloric ceca and posterior intestine (Fig. 5).
Insulin did not enhance the action of cortisol on either gut
region, although the presence of insulin changed Na⫹-K⫹ATPase activity, resulting in significantly elevated Na⫹-K⫹ATPase activity in pyloric ceca and a small but significant
decrease (main effect of insulin) in the posterior intestine.
Cortisol maintained Na⫹-K⫹-ATPase activity over that of
controls through 12 days of culture in the pyloric ceca and 9
days in the posterior intestine (Fig. 5), indicating a continued
response of explants to hormone beyond 6 days of culture.
Dose-response relationship between several corticosteroids
and Na⫹-K⫹-ATPase in culture. The in vitro response of
Na⫹-K⫹-ATPase activity to cortisol and dexamethasone was
dose dependent in both regions of the intestine (Fig. 6).
Significant responses (compared with controls, 0 ␮g/ml) to
both cortisol and dexamethasone were seen at 0.1 ␮g/ml for
pyloric ceca and at 1 ␮g/ml for posterior intestine. To compare
the relative potency of these two steroids, four-parameter curve
fitting was used to calculate the dose required for a halfmaximal response (EC50). The EC50 for dexamethasone was
lower than that for cortisol in both regions of the intestine
(Table 3).
To further evaluate whether the response of Na⫹-K⫹ATPase activity was specific to cortisol, the effects of cortisol,
11-deoxycortisol, and cortisone were compared at doses of
0 –10 ␮g/ml (Fig. 7). Cortisol was more potent than the other
natural corticosteroids. The only corticoid to induce a response
near a physiological concentration (0.1 ␮g/ml) was cortisol,
although responses were elicited by 11-deoxycortisol and cortisone at higher doses (1–10 ␮g/ml). EC50 results were not
calculated for these steroids because a maximal response was
not achieved with 11-deoxycortisol or cortisone. These results,
together with those of Fig. 6, suggest the following hierarchy
of potency for these steroids: dexamethasone ⱖ cortisol ⬎
11-deoxycortisol ⱖ cortisone.
In vivo effect of cortisol implants. Peritoneal implants of
cortisol for 7 days elevated plasma cortisol concentrations over
controls and significantly, although moderately, stimulated
Na⫹-K⫹-ATPase activity in both pyloric ceca and posterior
intestine (Table 4).
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IN VITRO RESPONSE OF SALMON INTESTINE TO CORTISOL
The specificity of the cortisol response reported here for
salmon intestine is supported by corticoid-binding studies of
the eel intestinal mucosa and responses of intestinal osmoregulatory functions to cortisol in vivo. DiBattista et al. (9, 10)
demonstrated high-affinity and low-capacity cortisol binding
sites in eel (Anguilla rostrata) intestinal mucosa, with relative
binding affinities for various corticoids that matched our functional responses in culture. In addition, the in vitro potency of
these corticosteroids in salmon intestine is corroborated by the
relative ability of these steroids to stimulate in vivo intestinal
sodium and water absorption in eel and goldfish (20, 36).
More recently, two glucocorticoid-like receptors have been
cloned from rainbow trout (designated rtGR1 and rtGR2);
these are highly expressed (mRNA) in intestine and share
cortisol as the major ligand (2, 11). Interestingly, high concentrations of 11-deoxycortisol and corticosterone induce transcriptional activity in COS-7 cells transfected with rtGR2 (but
not rtGR1), similar to the response of Na⫹-K⫹-ATPase activity
in cultured salmon intestine. In a cichlid fish, four subtypes of
corticoid receptors are present that all show high ligand selectivity for cortisol, although the one mineralocorticoid-like
receptor has a higher affinity for cortisol than the glucocorticoid-like receptors (18). In contrast, only a single class of
receptors is suggested by radioreceptor assay for gills of coho
and Atlantic salmon (41, 43). Finally, a rainbow trout mineralocorticoid-like receptor exhibits higher affinity for cortisol
than for dexamethasone, based on characterization of the
steroid-binding domain or transactivation properties (7, 47),
whereas, in the present study, the potency of dexamethasone
was equal to or greater than the potency of cortisol. From this
evidence and those from Colombe et al. (7) and Sturm et al.
(47), changes in Na⫹-K⫹-ATPase activity in the cultured
salmon intestine in response to cortisol are mediated by what
would be operationally defined as a glucocorticoid-like receptor(s). At this time, specific functions are not known for each
of the corticoid receptors identified in teleost fish.
The concentration of cortisol necessary to induce a response
by the intestine in vitro compares favorably with those determined for other osmoregulatory epithelia in culture. Cortisol
either maintains or stimulates Na⫹-K⫹-ATPase activity in the
cultured gill of coho salmon at 10 ␮g/ml and opercular membrane of tilapia at 0.1 ␮g/ml (27, 29). In addition, the intestine
and coho salmon gill share a similar specificity for cortisol and,
along with the tilapia opercular membrane, a similar timeframe of action on Na⫹-K⫹-ATPase activity. For example, the
response to cortisol is seen within 2 days in both gill and
Table 1. Total protein and sodium content of intestinal
explants before and after 6 days in culture
Table 2. Effect of cortisol on protein content of intestinal
explants cultured for 6 days
Total Protein, ␮g protein/mg dry wt
Total Sodium, ␮mol Na⫹/mg dry wt
Protein Content, ␮g protein/mg dry wt
Day
Pyloric ceca
Posterior intestine
Pyloric ceca
Posterior intestine
Group
n
Pyloric ceca
Posterior intestine
0
6
306⫾27 (6)
336⫾24 (6)
363⫾32 (6)
344⫾15 (6)
0.35⫾0.02 (5)
0.42⫾0.02 (6)*
0.54⫾0.04 (5)
0.57⫾0.06 (6)
Control
Cortisol (1 ␮g/ml)
6
6
378⫾27
362⫾21
419⫾13
335⫾18*
Values are means ⫾ SE of tissues from 5 or 6 fish (n in parentheses).
*Significantly different from day 0 (ANOVA, P ⬍ 0.05).
Values are means ⫾ SE of tissues from 6 fish. *Significantly different from
control (ANOVA, P ⬍ 0.05).
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Fig. 2. Transmission electron micrographs of enterocytes from freshwater-adapted sockeye salmon before
(A) and after 6 days in culture (B–D). A: apical portion
of several enterocytes of a pyloric cecum showing
microvilli (mv). Posterior intestine is structurally similar (not shown). B: apical portion of enterocytes from
cultured pyloric cecum retain cellular ultrastructure and
microvilli. C: nuclei (n) of enterocytes in culture. D:
anchoring (white arrow) and occluding (black arrow)
cell-cell junctions from cultured posterior intestine.
Scale bars ⫽ 2 ␮m.
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IN VITRO RESPONSE OF SALMON INTESTINE TO CORTISOL
intestine. These data indicate that the intestine shares a similar
sensitivity to cortisol with other osmoregulatory tissues.
Cortisol treatment enabled explants to retain Na⫹-K⫹ATPase activity but did not increase activity over levels found
Fig. 5. Effect of insulin (10 ␮g/ml) and/or cortisol (10 ␮g/ml) on Na⫹-K⫹ATPase activity of cultured intestinal explants for 6 –12 days. Before culture,
activity was 6.09 ⫾ 0.15 and 3.11 ⫾ 0.09 ␮mol ADP 䡠 mg protein⫺1 䡠 h⫺1 for
pyloric ceca and posterior intestine, respectively. Data are means ⫾ SE of
tissues from 11 or 12 fish. Ceca and intestine were analyzed separately by
2-way ANOVA (insulin ⫻ cortisol) at each day. Significant (P ⬍ 0.05) main
effects of cortisol are indicated by horizontal lines with letters: bars grouped
under the same line are different from the other grouped bars (a vs. b). There
was a significant interaction between insulin and cortisol for pyloric ceca on
day 6: values not sharing the same letter are different (Tukey’s test, P ⬍ 0.05).
Fig. 4. Effect of cortisol (1 ␮g /ml) addition to culture media on intestinal
Na⫹-K⫹-ATPase activity 3 days after culture of explants was started. Data are
means ⫾ SE Na⫹-K⫹-ATPase activity of tissues from 6 fish. *Significant
difference from control tissue on that day (ANOVA, P ⬍ 0.05).
in tissue before the start of culture. This is often the case for
salmon gill in culture (30). In the present study, it is not clear
whether the action of cortisol was due to direct effects on
Na⫹-K⫹-ATPase or due to the potential for cortisol to retain
explant integrity. Both are likely however. With regard to
architecture of explants, glucocorticoids have direct actions on
the morphology of mammalian intestine in organ culture,
including the preservation of mucosal structure in rabbit ileum
and increased mucosal maturation in fetal mouse (17, 38).
A possible explanation for the in vitro decrease in Na⫹-K⫹ATPase activity is the partial degradation of basal components
of the intestinal epithelium. Because Na⫹-K⫹-ATPase is localized in the basolateral membrane of the epithelium (39), any
degradation in this region would presumably result in decreased activity. The absence of factors that are important for
basement-membrane attachment, or the possibility of dedifferentiation occurring, might also account for some of the de-
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Fig. 3. Response of intestinal Na⫹-K⫹-ATPase activity to tissue culture
incubation for 2– 6 days in MEM, with (⫹) or without (⫺) 10% fetal bovine
serum, and in the presence (Cort) or absence (Con) of 1 ␮g cortisol/ml. Data
are means ⫾ SE Na⫹-K⫹-ATPase activity of tissues from 6 fish. *Significant
difference from control tissue on that day (2-way ANOVA, P ⬍ 0.05).
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IN VITRO RESPONSE OF SALMON INTESTINE TO CORTISOL
creased enzyme activity in culture. Decreasing Na⫹-K⫹ATPase activity is common during culture of the salmon gill
(26, 30), although in the case of the gill Na⫹-K⫹-ATPase is
concentrated in chloride cells.
The possibility that factors absent in culture might potentiate
a cortisol response or interact positively with cortisol warrants
further attention. Shrimpton and McCormick (42) have shown
that growth hormone and triiodothyronine increase the abundance of corticosteroid receptors in Atlantic salmon gill and
therefore may increase cortisol responsiveness. Also, IGF-I
protects against a decline in Na⫹-K⫹-ATPase activity during
culture of gill from coho salmon (26).
Table 3. Summary of half-maximal response (EC50) of
Na⫹-K⫹-ATPase activity to cortisol or dexamethasone
after 6 days of exposure in culture
Fig. 7. Comparison of the effect of 3 corticoids on intestinal Na⫹-K⫹-ATPase
activity after 6 days in culture. Data points are means ⫾ SE of tissues from 6
fish. Before culture, Na⫹-K⫹-ATPase activity from ceca and intestine was
4.81 ⫾ 0.20 and 1.97 ⫾ 0.16 ␮mol ADP 䡠 mg protein⫺1 䡠 h⫺1. *Significant
difference (P ⬍ 0.05) from control (0 ␮g/ml) (ANOVA, followed by Dunnett’s
test).
Protein content of the cultured posterior intestine declined
by ⬃20% over 6 days of cortisol exposure. An increase in
catabolism may explain the decrease of protein concentrations,
although it is unclear why no change occurred in ceca.
In pyloric ceca, Na⫹-K⫹-ATPase activity was higher in the
presence of insulin, whereas insulin slightly decreased Na⫹K⫹-ATPase activity in the posterior intestine. Although the
reason for this is not clear, it may be that sensitivity of ceca to
insulin is associated with a high capacity for nutrient uptake in
Table 4. Plasma cortisol and intestinal Na⫹-K⫹-ATPase
activity of sockeye salmon 7 days after being implanted with
or without (control) 50 ␮g cortisol/g body wt
Na⫹, K⫹-ATPase Activity,
␮mol ADP 䡠 mg protein1 䡠 h⫺1
EC50, ng/ml
Cortisol
Dexamethasone
Pyloric ceca
Posterior intestine
30.4
15.6
13.0
2.2
EC50 results were calculated from the curve of best fit (4-parameter) through
the means (n ⫽ 6 fish) from Fig. 6.
Group
n
Body Weight, g Plasma Cortisol, ng/ml Pyloric ceca Posterior intestine
Control 14
Cortisol 13
103⫾6
105⫾5
9⫾1
202⫾9*
8.3⫾0.3
9.8⫾0.2*
2.9⫾0.2
3.4⫾0.1*
Values are means ⫾ SE. *Significantly different from control (ANOVA,
P ⬍ 0.05).
AJP-Regul Integr Comp Physiol • VOL
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Fig. 6. Effect of cortisol (Cort) or dexamethasone (Dex) on intestinal Na⫹K⫹-ATPase activity after 6 days in culture. Data points are means ⫾ SE
Na⫹-K⫹-ATPase activity of tissues from 6 fish. Before culture, Na⫹-K⫹ATPase activity from ceca and intestine was 4.87 ⫾ 0.38 and 1.63 ⫾ 0.10
␮mol ADP 䡠 mg protein⫺1 䡠 h⫺1. *Significant difference (P ⬍ 0.05) from
control (0 ␮g/ml) (ANOVA, followed by Dunnett’s test).
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IN VITRO RESPONSE OF SALMON INTESTINE TO CORTISOL
ACKNOWLEDGMENTS
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
The expert histological teachings of, and assistance from, the late Gerald
Stokes are gratefully acknowledged. We thank the staff at Chemsearch Laboratories (Otago) for ion analyses and Matthew Downes for invaluable contributions in preparing tissues for electron microscopy. Discussions with Dr.
Jennifer Specker (University of Rhode Island) were particularly helpful while
preparing the manuscript.
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