The Journal of Experimental Biology 202, 987–995 (1999)
Printed in Great Britain The Company of Biologists Limited 1999JEB1918
RESPONSIVENESS OF GILL Na+/K+-ATPase TO CORTISOL IS RELATED TO GILL CORTICOSTEROID RECEPTOR CONCENTRATION IN JUVENILE RAINBOW TROUT
J. MARK SHRIMPTON* AND STEPHEN D. MCCORMICK
Conte Anadromous Fish Research Center, Biological Resources Division, USGS, Turners Falls, MA 01376, USA and
Department of Biology, University of Massachusetts, Amherst, MA 01002, USA
*Present address: Biology Program, College of Science and Management, University of Northern British Columbia, 3333 University Way,
Prince George, British Columbia, Canada V2N 4Z9 (e-mail: [email protected])
Accepted 3 February; published on WWW 22 March 1999A positive relationship between receptor concentration significant reduction in CR numbers. The decrease in CR and tissue responsiveness is an often-assumed and rarely Bmax corresponded to a reduction in gill responsiveness to tested principle in endocrinology. In salmonids, seasonal cortisol. Triiodothyronine, but not growth hormone, changes in levels of plasma cortisol and gill corticosteroid treatment was found to increase CR Bmax significantly. The receptors (CRs) during the spring indicate a potential role increase in CR numbers was correlated with a marked for this hormone in the parr–smolt transformation. It is not increase in gill responsiveness to cortisol. A significant known whether these seasonal changes result in alterations positive linear relationship exists between the in vitro gill in gill responsiveness to cortisol. The relationship between Na+/K+-ATPase activity response to cortisol and CR Bmax CR concentration and tissue responsiveness was, therefore, (r2=0.614, P<0.001). We have demonstrated that binding examined in the gills of juvenile rainbow trout sites for cortisol in the gills of rainbow trout have high (Oncorhynchus mykiss). Gill CR concentration (Bmax) and affinity, high specificity and saturable binding and that the affinity (Kd) were assessed using a radioligand binding number of binding sites is correlated with the tissue assay with the synthetic glucocorticoid triamcinolone response to cortisol. acetonide. Gill responsiveness to cortisol was quantified by measuring in vitro Na+/K+-ATPase activity. Gill CR
Key words: cortisol, corticosteroid receptor, growth hormone,
concentration was manipulated by stress or hormonal
triiodothyronine, gill, Na+/K+-ATPase activity, rainbow trout,
treatments. Repeated handling stresses resulted in a Introduction
Many salmonids undergo a developmental process, the
in the gills of salmonids (Chakraborti et al., 1987; Maule and
parr–smolt transformation, that is controlled by seasonal
Schreck, 1990). A direct relationship, however, between tissue
changes in both photoperiod and temperature. These
sensitivity to cortisol and corticosteroid receptor concentration
environmental cues stimulate a series of physiological changes
or affinity has not been reported in the literature for teleosts.
in juvenile salmon that culminate in the transformation of
To understand the significance of changes in receptor
stream-dwelling parr into migratory smolts capable of
concentration, it is important to establish a relationship
surviving in the marine environment (Hoar, 1988). A variety
between the tissue response and the receptor concentration.
of physiological and morphological changes have been shown
Plasma cortisol and gill CR concentration have been observed
to be associated with the parr–smolt transformation. Notable
to change seasonally in conjunction with smolting in coho
among these is an increase in gill Na+/K+-ATPase activity,
salmon Oncorhynchus kisutch (Shrimpton et al., 1994),
which is correlated with the development of seawater tolerance
steelhead trout Oncorhynchus mykiss (McLeese et al., 1994)
(McCormick and Saunders, 1987). This enzyme has been
and Atlantic salmon Salmo salar (Shrimpton and McCormick,
shown to be regulated by cortisol in vitro (McCormick and
1998a). McCormick et al. (1991a) showed that seasonal
Bern, 1989) and in vivo (for a review, see McCormick, 1995).
changes in gill responsiveness to cortisol occurs in coho and
The action of cortisol in the gills is probably mediated by
Atlantic salmon. The seasonal changes in responsiveness to
intracellular corticosteroid receptors (CRs). Hormone
cortisol and receptor concentration associated with smolting
receptors are characterized by high affinity, high specificity
suggest a functional relationship between these two variables;
and saturable binding, and by stimulating a response when
however, this relationship has not been established in a single
bound to the appropriate hormone (Clark and Peck, 1977).
Protein molecules that fit the first three criteria have been found
CR concentration has been shown to be altered by hormone
treatment. Shrimpton et al. (1995) found that growth hormone
(100 mg l−1; neutralized and buffered with sodium bicarbonate,
(GH) treatment increased the number of gill CRs in coho salmon.
pH 7.0). Length and mass were measured, and the fish were
Shrimpton and McCormick (1998b) found that triiodothyronine
injected with one of the following: vegetable oil as vehicle,
(T3) augmented the effect of growth hormone on increasing the
5.0 µg g−1 ovine growth hormone (GH; National Institute of
number of CRs in Atlantic salmon. Cortisol treatment and stress
Health, Bethesda, MD, USA), 1.6 µg g−1 T3 (Sigma, St Louis,
led to a decrease in the number of gill CRs (Maule and Schreck,
MO, USA) or 5.0 µg g−1 GH plus 1.6 µg g−1 T3. Groups were
1991; Shrimpton and Randall, 1994) and liver CRs (Pottinger et
identified by coloured acrylic paint injected between the fin rays
al., 1994). Methods exist, therefore, to manipulate the number of
of the anal fin. After recovery, the fish were placed in a circular
CRs in the gills of salmonids. Gill organ culture has been used
tank 1 m in diameter. Four days later, fish were reinjected with
to assess the responsiveness of the gill to hormones by measuring
the same treatment. On day 8, the fish were sampled as described
the increase in Na+/K+-ATPase activity in vitro (McCormick and
below. Fish were not fed throughout the experiment.
Bern, 1989; McCormick et al., 1991a; Madsen and Bern, 1993). To assess the effect of differences in CR B
responsiveness, CR Bmax was manipulated by stress and
Fish were rapidly removed from their tanks and placed in
hormonal treatment, and responsiveness was measured from
200 mg l−1 tricaine methane sulphonate (buffered with sodium
changes in Na+/K+-ATPase activity in organ culture.
bicarbonate, pH 7.0). Length and mass were measured within5 min of first disturbing the fish to ensure that a stress-associated rise in cortisol level did not occur. The right first
Materials and methods
gill arch was removed and placed in minimal essential medium
(MEM with Hanks’ salts, Gibco) on ice. Approximately 6–8
primary gill filaments were removed and placed in 100 µl of
Juvenile rainbow trout [Oncorhynchus mykiss (Walbaum)]
were transported from Sunderland State Trout Hatchery,
50 mmol l−1 imidazole, pH 7.3) on ice for later determination
Sunderland, MA, USA, to the Conte Anadromous Fish
of Na+/K+-ATPase activity. Samples were frozen at −80 °C
Research Center in Turner Falls, MA, USA, on 8 August 1995.
within 30 min. The rest of the gill tissue was removed, placed
Fish were reared in dechlorinated city water (19.4–19.8 °C)
in 2 ml of TEMS (10 mmol l−1 Tris-HCl, 1 mmol l−1 Na2EDTA,
under natural photoperiod and fed to satiation twice daily. On
12 mmol l−1 monothioglycerol, 20 mmol l−1 sodium molybdate,
16 August 1995, juvenile rainbow trout (16.8±0.2 cm, 55.7±
10 % v/v glycerol, pH 7.4) and frozen immediately at −80 °C
2.3 g; means ± S.E.M., N=6) were placed into two tanks 1 m in
for later analysis of CR concentration and affinity.
diameter. Fish in the first tank were left undisturbed for 10days. Fish in the second tank were acutely stressed twice daily
for 10 days. One of three stressors was used to prevent
The protocol of McCormick and Bern (1989) was followed
accommodation of the fish to a specific stressor: confinement,
for gill organ culture. Primary gill filaments were severed just
water removal or chasing. For the confinement stress, fish were
above the septum and separated from one another. Filaments
caught in a dip net and held for 15 min at a density such that
were moved using a positive displacement pipette to minimize
all fish were in physical contact with one another. For the water
tissue damage. Five to six primary gill filaments were placed
removal stress, the water was drained from the tank until the
in 0.5 ml of MEM with 25 mmol l−1 Hepes buffer, 4 mg ml−1
fish were partially stranded and on their sides. After 60 s, the
bovine serum albumin (Sigma radioimmunoassay grade),
water was replaced. The third stress involved chasing the fish
with a dip net for 15 min. At the end of the 10 day treatment,
fish were left undisturbed for 3 days and then sampled as
(adjusted to pH 7.8 with NaOH) in sterile 24-well culture
described below. This protocol has been shown to result in a
plates. Gill filaments were preincubated in this medium on ice
significant reduction in CR concentration (Shrimpton and
for up to 5 h. A stock solution of cortisol was prepared by
Randall, 1994). Fish were not fed throughout the experiment.
dissolving 5 mg ml−1 in ethanol. Serial dilutions of cortisol inethanol were made, and 60 µl was added to 100 ml of MEM to
achieve final cortisol concentrations of 0.1, 1 and 10 µg ml−1.
Juvenile rainbow trout were transported from McLaughlin
The same volume of ethanol was added to the control MEM,
State Trout Hatchery, Belchertown, MA, USA, on 16 September
but without cortisol. Ethanol at this concentration (0.06 %)
1996. We found no difference in CR Bmax and responsivenes of
does not affect Na+/K+-ATPase activity during in vitro
gill Na+/K+-ATPase to cortisol between fish from the
exposure (McCormick and Bern, 1989). The preincubation
McLaughlin and Sunderland Hatcheries (J. M. Shrimpton and S.
medium was removed and replaced with 0.75 ml of MEM
D. McCormick, unpublished results). Fish were held in filtered
containing 50 units ml−1 penicillin and 50 µg ml−1 streptomycin
water drawn from the Connecticut River (13.5–14.1 °C) under
and cortisol or vehicle that had been equilibrated with a 99 %
natural photoperiod and fed to satiation twice daily. On 6
oxygen and 1 % carbon dioxide gas mixture. Gill filaments
October 1996, rainbow trout (13.5±0.2 cm, 26.9±1.0 g; N=6)
were incubated at 15 °C for 48 h in a humidified chamber with
were anaesthetized with tricaine methane sulphonate
99 %:1 % O2:CO2 with gentle shaking. After culture, gill
Responsiveness of gill Na+/K+-ATPase to cortisol
filaments were removed with forceps and placed in 80 µl of
ligand was separated from bound ligand by centrifugation at
SEI buffer on ice and then frozen at −80 °C until analysis of
3000 g for 15 min in a Beckman GPKR refrigerated centrifuge.
The supernatant (0.5 ml) was added to 3 ml of aqueouscounting scintillant (Scintisafe Econo 2 Fisher Scientific).
Samples were counted on a Beckman LS 6000IC liquid
Gill Na+/K+-ATPase activity was measured according to the
scintillation counter. Specific binding was determined by
microassay protocol of McCormick (1993). Gill filaments were
subtracting the non-specific bound ligand from the total bound
homogenized in SEI buffer containing 0.1 % sodium
deoxycholate. Following centrifugation (at 3000 g for 2 min) to
Although the origin of CRs in the gills may be cytosolic or
remove insoluble particles, ouabain-sensitive ATPase activity
nuclear, they are referred to as cytosolic as they are found in
was determined kinetically by following the hydrolysis of ATP
the cytosol fraction following tissue processing. The CR
linked to the oxidation of nicotinamide adenine dinucleotide
concentration measured consists of the unbound receptor
(NADH), measured at 340 nm for 10 min at 25 °C in the
population. The equilibrium dissociation constant (Kd) and the
presence and absence of 0.5 mmol l−1 ouabain. Protein content
concentration of corticosteroid receptor sites (Bmax) were
in the gill homogenate was measured using a bicinchoninic acid
calculated according to Scatchard (1949). Bmax was divided by
(BCA) protein assay (Pierce, Rockford, IL, USA). Specific
the homogenate protein concentration, and CR concentration
activities were expressed as µmol ADP mg−1 protein h−1.
was expressed as fmol mg−1 protein. To estimate cooperativitybetween CR and ligand, the Hill coefficient was calculated
The method of Maule and Schreck (1990) as modified by
Shrimpton and Randall (1994) was used for analysis of
corticosteroid receptors. All procedures were carried out with
For experiment 1, a t-test was conducted to determine
samples on ice. Thawed gill tissue was scraped away from the
differences in initial Na+/K+-ATPase activity, Bmax and Kd
cartilage and then homogenized in 2.0 ml of TEMS using a
between the control and stressed fish. A two-way analysis of
Tekmar TP 18/10S1 homogenizer for two 10 s bursts.
variance (ANOVA) was used to determine the effect of
Homogenates were centrifuged in a Beckman GPKR knee-well
hormone treatment and cortisol concentration on in vitro
centrifuge at 3000 g for 15 min. The supernatant was removed
responsiveness of gill Na+/K+-ATPase activity. For experiment
and placed on ice. The pellet was resuspended with 0.5 ml of
2, a two-way ANOVA was used to determine the effects of GH
TEMS containing 50 µg ml−1 bacitracin, 20 µg ml−1 benzidine,
and T3 on in vivo Na+/K+-ATPase activity, Bmax and Kd. A
0.5 µg ml−1 aprotinin and 10 µg ml−1 o-phenanthroline, to wash
three-way ANOVA was used to determine the effects of GH
more CRs from the pellet, and then recentrifuged at 3000 g for
treatment, T3 treatment and cortisol concentration on in vitro
15 min. The supernatants were combined and centrifuged at
responsiveness of gill Na+/K+- ATPase activity, followed by a
48 000 g for 2 h in a Beckman J2-21M centrifuge with a JA-21
Tukey’s test to find significant differences among the means.
rotor. After this centrifugation, the supernatant was removed,
Data from all the experiments were combined for regression
mixed with 1.0 ml of TEMS containing 10 % (w/v) activated
analysis. Responsiveness of gill Na+/K+-ATPase activity to
charcoal and 1.0 % (w/v) dextran and incubated for 10 min to
cortisol was regressed on Bmax, Kd and Bmax/Kd. Statistical
remove endogenous steroids. To separate the charcoal from the
significance was taken at a level of P=0.05. All values are
liquid, the samples were centrifuged at 3000 g for 15 min. The
final supernatant was used to quantify cortisol binding. Proteincontent was assayed with Bradford reagent (Bradford, 1976)using bovine serum albumin as a standard.
Cortisol binding receptor studies were conducted with
Representative binding curves, Scatchard plots and Hill
[3H]triamcinolone acetonide (TA; 1,4-pregnadien-9α-fluoro-
plots are shown in Fig. 1 for fish sampled in August 1995. The
11β,16α,-17β,21-tetrol-3,20-dione-16,17 acetonide) with a
analysis indicated saturable binding. A single class of receptor
specific activity of 1620 GBq mmol−1 (Dupont-NEN). In
was indicated by the linear Scatchard analysis. There was no
binding and competition studies on duplicate gill homogenates,
indication of cooperative binding because the Hill plot was
TA and cortisol bound to the same number of receptors, but
linear and the Hill coefficient was equivalent to 1.
TA had a higher affinity. To determine the number of high-
Specificity for the receptor is shown in Fig. 2. The
affinity cortisol receptors, 100 µl of the final supernatant was
competition hierarchy of steroid competitors for gill CRs can
incubated in duplicate with 100 µl of buffer containing [3H]TA
be summarized as TA>cortisol=dexamethosone>11-deoxy-
with or without a 500-fold excess of cold TA. The final
concentration of [3H]TA ranged from 0.1 to 6 nmol l−1. The
tubes were vortexed and incubated for 2 h on ice. After theincubation period, 0.5 ml of TEMS containing 2.5 % (w/v)
activated charcoal and 0.25 % (w/v) dextran was added and
Following stress treatment, Na+/K+-ATPase activity was
vortexed. After 10 min, the charcoal containing unbound
1.22±0.09 and 1.40±0.14 µmol ADP mg−1 protein h−1 for the
protein h−1). GH+T3 treatment induced the highest Na+/K+-
ATPase activity of 2.94±0.14 µmol ADP mg−1 protein h−1.
Two-way ANOVA indicated that there was a significant effect
of GH on in vivo gill Na+/K+-ATPase activity (P<0.01), but noeffect of T3 (P=0.45) and no interaction effect (P=0.41).
There was a twofold increase in CR Bmax with T3 and
GH+T3 treatment compared with the vehicle-injected controls
(Fig. 5). Two-way ANOVA indicated a significant effect of T3
treatment on CR Bmax (P<0.01), but no effect of GH (P=0.76)
and no interaction effect (P=0.84). Kd was not altered by any
of the hormone treatments (Fig. 5). Two-way ANOVA results
for GH, T3 and their interaction were P=0.32, P=0.35 andP=0.54, respectively. In vitro gill Na+/K+-ATPase activity was significantly
affected by cortisol concentration in the incubation medium
(P<0.001), T3 treatment of the fish (P=0.02), but not GH
treatment of the fish (P=0.87) (Fig. 6). There were no
interaction effects between any of the factors. T3- and GH+T3-
treated fish showed the greatest responsiveness to cortisol. The
effect of GH treatment alone, however, was not significantly
different from that for vehicle-injected fish. At 10 µg ml−1 invitro cortisol, the percentage increase in gill Na+/K+-ATPase
activity of the GH+T3- and T3-treated groups was twofoldgreater than for the vehicle- and GH-treated groups (Fig. 6;P<0.05).
Fig. 1. Representative binding plot (A), Scatchard plot (B) and Hill
plot (C) for gill corticosteroid receptors (CRs) in juvenile rainbow
trout. , total binding; ᭡, non-specific binding; ᭹ specific (total
minus non-specific) binding. Values are means ± 1 S.E.M. of six
fish. Units are fmol mg−1 protein for bound CRs and nmol l−1 for
, where B=bound ligand at different
control and stress groups, respectively. These values did not
differ significantly (P=0.33). Kd, also, did not differsignificantly between the two groups (Fig. 3, P=0.12). Therewas, however, a significant 33 % reduction in B
stressed group compared with the control (P<0.01).
The response of the gill to cortisol was significantly affected
by stress treatment (P<0.001) and the cortisol concentration in
the medium (P=0.011), but there was no interaction effect
(P=0.77) (Fig. 4). At cortisol concentrations of 0.1, 1 and
10 µg ml−1, gill tissue from stressed fish consistently exhibiteda lower response than that from control fish.
Fig. 2. Specificity of corticosteroid binding in rainbow trout gillhomogenates. Pooled gill homogenates from four individuals were
incubated in duplicate with 4 nmol l−1 [3H]TA with or without 10, 40,100, 400, 1000, 4000 and 10 000 nmol l−1 of unlabelled competitor.
Hormone treatment had a significant effect on gill Na+/K+-
Specific binding was calculated as the difference between total
ATPase activity. Enzyme activity in the vehicle-injected fish
binding and binding in the presence of 10 000 nmol l−1 unlabelled
was 1.94±0.22 µmol ADP mg−1 protein h−1. GH treatment
TA. Values are means of duplicate homogenates. P, progesterone;
increased this activity to 2.56±0.29 µmol ADP mg−1 protein h−1.
17-HP, 17-hydroxyprogesterone; 11-D, 11-deoxycortisol; F, cortisol;
The gill Na+/K+-ATPase activity of T3-treated fish was similar
S, cortisone; TA, triamcinolone acetonide; Dex, dexamethosone; C,
to that of the vehicle-injected group (1.93±0.20 µmol ADP mg−1
Responsiveness of gill Na+/K+-ATPase to cortisol
Fig. 3. Concentration (Bmax, fmol mg−1 protein) and dissociation
Fig. 5. Concentration (Bmax, fmol mg−1 protein) and dissociation
constant (Kd, nmol l−1) of gill corticosteroid receptors from juvenile
constant (Kd, nmol l−1) of gill corticosteroid receptors from juvenile
rainbow trout sampled 3 days after cessation of daily handling stress
rainbow trout treated with vehicle (V, vegetable oil), 5.0 µg g−1 ovine
(N=6). The fish were stressed twice daily over a period of 10 days.
growth hormone (GH), 1.6 µg g−1 triiodothyronine (T3) or 5.0 µg g−1
An asterisk indicates that the value for the stressed group was
GH + 1.6 µg g−1 T3. An asterisk indicates that values for the T3-
significantly different from that for the control group (P<0.01).
treated groups are significantly different from those for groups not
treated with T3 (P<0.01). Values are means + 1 S.E.M. , N=6.
There was a significant relationship between responsiveness
concentration (Fig. 7). Binding sites in the gills of rainbow
of Na+/K+-ATPase activity and cortisol treatment at 10 µg ml−1
trout showed affinity and Bmax values comparable with values
when regressed on Bmax (P<0.001; r2=0.614; Fig. 7). Kd was
reported elsewhere for CRs in the gills of several species of
not significantly correlated with the responsiveness of Na+/K+-
salmonids (Sandor et al., 1984; Chakraborti et al., 1987; Maule
ATPase activity (P=0.612; r2=0.004). The correlation between
and Schreck, 1990; Shrimpton and Randall, 1994). The
CR Bmax and in vitro gill Na+/K+-ATPase activity, however,
specificity of branchial CRs is consistent with published results
was slightly increased when Bmax was divided by Kd (Bmax/Kd)
for several different salmonid tissues: gills (Chakraborti et al.,
1987; Maule and Schreck, 1990), liver (Pottinger et al., 1994)and brain (Lee et al., 1992; Knoebl et al., 1996). The syntheticglucocorticoid TA competed most effectively. Of the natural
steroids, cortisol was the most effective competitor and was
In this study, we have demonstrated that the cortisol-binding
similar in effect to dexamethasone, another synthetic
sites in the gills of rainbow trout meet the four requirementsof a receptor. The receptor has high affinity (Fig. 1), high
specificity (Fig. 2) and shows saturable binding (Fig. 1), and acorrelation exists between tissue response and receptor
Fig. 6. In vitro gill Na+/K+-ATPase activity in response to cortisol
for fish treated with vehicle (vegetable oil), 5.0 µg g−1 ovine growth
hormone (GH), 1.6 µg g−1 triiodothyronine (T3) or 5.0 µg g−1 GH +1.6 µg g−1 T3. Gill filaments were incubated with 0, 0.1, 1 and
Fig. 4. In vitro gill Na+/K+-ATPase activity in response to cortisol
10 µg ml−1 cortisol for 2 days. Values are in vitro gill Na+/K+-
for stressed and control fish. Gill filaments were incubated with 0,
ATPase as a percentage of in vitro gill Na+/K+-ATPase activity in
0.1, 1 and 10 µg ml−1 cortisol for 2 days. Values are in vitro gill
control filaments not treated with cortisol. An asterisk indicates that
Na+/K+-ATPase as a percentage of in vitro gill Na+/K+-ATPase
values for the T3-treated groups are significantly different from those
activity in control filaments not treated with cortisol. Values are
for groups not treated with T3 (P<0.05). Values are means ± 1 S.E.M.,
1996). Using the protocol outlined above, gill CRs are found
in the cytosolic fraction, possibly as a result of redistribution
from the nucleus during tissue processing (Welshons and
Jordan, 1987). We have been unable to quantify CRs in the
nuclear fraction, as has been reported by Pottinger et al. (1994)
and Knoebl et al. (1996). The CR concentration measured,
therefore, consists of the unbound ‘cytosolic’ receptorpopulation. Throughout this study, fish were anaesthetized and
sampled in under 5 min to prevent an increase in plasma
cortisol levels. By sampling rapidly and minimizing anypotential increase in plasma cortisol levels, the number of CRs
bound to cortisol remains low and the majority of CRs exist in
the unbound ‘cytosolic’ receptor pool. The majority of the
binding sites in liver and brain of rainbow trout have been
Fig. 7. In vitro Na+/K+-ATPase activity for gill filaments incubated
reported to be cytosolic (Lee et al., 1992). Since the total
with 10 µg ml−1 cortisol plotted against gill corticosteroid receptor
population of CRs could not be measured, however, we do not
concentration (Bmax). Values are in vitro gill Na+/K+-ATPase as a
know the extent to which the nuclear receptor population may
percentage of in vitro gill Na+/K+-ATPase activity in control
have changed and affected the relationship between CR Bmax
filaments not treated with cortisol. Values are for fish from
and in vitro responsiveness to cortisol.
experiment 2 injected with vehicle (vegetable oil), 5.0 µg g−1 ovine
Responsiveness of gill Na+/K+-ATPase activity to cortisol
growth hormone (GH), 1.6 µg g−1 triiodothyronine (T3) or 5.0 µg g−1
in vitro changes seasonally and during development in
GH + 1.6 µg g−1 T3 and for fish from experiment 1 subjected to daily
salmonids. In coho salmon, the gills were unresponsive to
handling stress. Each point corresponds to one individual. Theequation for the regression line is y=0.311x+103.6 (r2=0.614,
cortisol in November, showed the highest responsiveness in
January, and the response then declined until gill tissue wasunresponsive in April, when in vivo gill Na+/K+-ATPaseactivity peaked (McCormick et al., 1991a). In separate studies
glucocorticoid. 11-Deoxycortisol, the precursor to cortisol,
on coho salmon, gill CR levels were low in November, highest
was less competitive than cortisol. The other precursors to
in the early spring (Shrimpton, 1996) and then declined
cortisol (progesterone and 17-hydroxyprogesterone), the
coincident with the peak in Na+/K+-ATPase activity
breakdown product of cortisol (cortisone) and corticosterone
(Shrimpton et al., 1994). In Atlantic salmon, McCormick et al.
were less effective than cortisol in competing for CRs.
(1991a) reported that presmolts responded to cortisol in vitro,
Most of the work examining the relationship between CR
whereas smolts were unresponsive. Shrimpton and
concentration and tissue responsiveness to cortisol has been
McCormick (1998a) found that CR Bmax in Atlantic salmon
conducted on mammalian cell lines, many of which are cancer
was significantly greater in presmolts than in smolts, further
cell lines. CR concentration has been correlated with a
supporting a relationship between CR Bmax and response to
physiological response in mouse thymoma-derived cells
cortisol. Another line of evidence within the literature that
(Danielsen and Stallcup, 1984), but not in human leukocytes
supports a relationship between gill CR concentration and gill
from leukaemia patients (Homo et al., 1980). Although
responsiveness to cortisol was provided by Shrimpton et al.
conflicting results do exist in the literature, most studies
(1994). They found that wild coho salmon exhibited a higher
indicate that CR concentration is closely correlated with the
gill CR Bmax and a greater increase in plasma cortisol level,
magnitude of the response (Bamberger et al., 1996). Although
which corresponded to a significantly greater increase in gill
cortisol receptors have been found in most tissues of fish, little
Na+/K+-ATPase activity and the development of seawater
is known of the relationship between cortisol receptor numbers
tolerance, compared with their hatchery-reared counterparts.
and tissue responsiveness. In leukocytes from the anterior
Corticosteroid receptor Bmax was three times greater in
kidney of coho salmon, Maule et al. (1993) found a correlation
experiment 1 (August, Fig. 3) that in experiment 2 (October,
between the in vitro immune response to cortisol and CR
Fig. 5). Seasonal changes in Bmax of this magnitude have been
number. They did not find a similar relationship for splenic
observed in the gills of Atlantic salmon (Shrimpton and
leukocytes, and speculated that other factors must be involved.
McCormick, 1998a) and coho salmon (Shrimpton et al., 1994;
In the present study, we have demonstrated a direct
Shrimpton, 1996). Seasonal changes of smaller magnitude
relationship between gill cytosolic CR Bmax and in vitro
have also been found in the gills of hybrid rainbow/steelhead
responsiveness of the gill to cortisol.
trout, but no significant seasonal differences were seen in
The subcellular distribution of CRs in fish has not been
steelhead trout (McLeese et al., 1994). It is not known whether
investigated. In mammals, however, considerable controversy
changes of this magnitude are characteristic of this time of the
exists over the subcellular location of CRs. Recent studies have
year because most of the work examining seasonal changes in
found the unoccupied CR to be located in the nucleus (Brink
CRs has focused on the parr–smolt transformation during the
et al., 1992; Pekki et al., 1992) or the cytoplasm (Sackey et al.,
spring. Shrimpton and McCormick (1998a) sampled Atlantic
Responsiveness of gill Na+/K+-ATPase to cortisol
salmon in early October and found little change in Bmax until
has been shown to bind to membrane receptors in the liver,
December. Kd changes seasonally in association with changes
gill and kidney (Fryer and Bern, 1979; Sakamoto and Hirano,
in Bmax in most species examined (Shrimpton and McCormick,
1991). The increase in hypo-osmoregulatory ability due to
1998a; Shrimpton, 1996). If Bmax differences were associated
GH may also be associated with the production of insulin-
with seasonal patterns of development, Kd might also be
like growth factor-I (IGF-I). McCormick et al. (1991b)
expected to differ. Values for Kd in the two experiments are
showed levels IGF-I to increase hypo-osmoregulatory ability
very similar (0.484±0.015 and 0.476±0.015 nmol l−1 for
significantly in rainbow trout. Whether the interaction
experiment 1 and experiment 2, respectively), suggesting that
between GH and cortisol may be mediated by IGF-I is not
seasonal differences may be small and that some other factor
known, but McCormick (1996) showed that IGF-I and
may account for the differences observed in Bmax.
cortisol are additive in increasing gill Na+/K+-ATPase
Temperature may have directly influenced CR Bmax because
it was approximately 19.6 °C for experiment 1 and 13.8 °C for
The upregulation of gill CR Bmax by T3 has not been
experiment 2. The temperature of the water during experiment
demonstrated previously. Shrimpton and McCormick (1998b)
1 was above the optimum temperature for growth in rainbow
found that T3 had an effect on Bmax in Atlantic salmon, but that
trout, but well below the thermal maximum for this species
the increase was significant only when given in combination
(Bidgood and Berst 1969), and the fish exhibited good feeding
with GH. Endocrine factors modulating CR Bmax thus appear
performance and growth. Since CR Bmax was higher in fish
to differ between species of salmonids. We do not know the
from experiment 1 than from experiment 2, it is unlikely that
mechanism by which T3 increases gill CR Bmax. In rat pituitary
the warmer temperature was stressful for the fish because CR
cells, T3 treatment caused a significant increase in levels of CR
Bmax is downregulated by stress (Shrimpton and Randall, 1994;
Pottinger et al., 1994; this study). In Atlantic salmon, we found
The increase in CR Bmax following T3 treatment suggests
that the seasonal decline in CR Bmax occurred earlier in fish
that an interaction between T3 and cortisol exists. Evidence for
where the spring increase in water temperature was advanced.
an interaction between thyroid hormones and cortisol in fish is
This response was independent of photoperiod (J. M.
limited. Thyroxine (T4) has been shown to enhance the effect
Shrimpton and S. D. McCormick, unpublished data). Further
of cortisol on stimulating gill Na+/K+-ATPase activity in
investigations are required to determine how temperature
tilapia Oreochromis mossambicus (Dangé, 1986). In rainbow
affects the seasonal cycles of CR Bmax in rainbow trout.
trout, Madsen (1990c) found that treatment with T4 and cortisol
Endocrine factors are known to alter the abundance of CRs.
for 1 week resulted in a significant increase in gill Na+/K+-
Cortisol and stress have been shown to decrease CR numbers
ATPase activity compared with controls, whereas either
in coho salmon gills (Maule and Schreck, 1991; Shrimpton and
hormone alone did not stimulate gill Na+/K+-ATPase activity
Randall, 1994) and rainbow trout liver (Pottinger et al., 1994).
significantly. T4 treatment, however, was without effect after
In the present study, we confirmed this finding for gill cytosolic
14 and 28 days and did not alter the stimulatory effect of
CRs in rainbow trout. The reduction in CR number may be due
cortisol. The results of the present study indicate that
to a decrease in the rate of production of CRs, because CR
upregulation of cortisol receptors by thyroid hormones is one
mRNA levels decline in response to dexamethasone treatment
mechanism for the interaction between these hormones.
(Kalinyak et al., 1987). It is also possible that the rate of
The effectiveness of hormone receptors in the regulation of
breakdown of CRs may change with stress or cortisol treatment
gene transcription is also correlated with hormone binding
because the degradation rate of CRs has been shown to
affinity (Bamberger et al., 1996). Mutations of the
increase following treatment with the steroid agonist TA
glucocorticoid receptor are associated with decreased hormone
(McIntyre and Samuels, 1985). Both these mechanisms may
binding affinity of the receptor and are associated with clinical
contribute to the downregulation of CRs in the gills of rainbow
syndromes of glucocorticoid hyposensitivity (Hurley et al.,
1991). Although changes in affinity due to mutations of the CR
Increases in gill CR numbers have been found following
are much greater than differences in Kd that occur seasonally,
GH treatment in coho salmon (Shrimpton et al., 1995) and
they indicate that Kd changes can have an effect on tissue
Atlantic salmon (Shrimpton and McCormick, 1998b). The
sensitivity. McCormick and Bern (1989) found a hierarchy of
increase in CR Bmax induced by GH has been proposed as a
in vitro responsiveness of coho salmon gill filaments to
mechanism for the interaction between GH and cortisol that
dexamethasone, cortisol, 11-deoxycortisol and cortisone. The
has been found in sea trout Salmo trutta (Madsen, 1990b),
affinity of gill CRs for these four steroids was identical to that
Atlantic salmon (McCormick, 1996) and rainbow trout
found for brook trout Salvelinus fontinalis (Chakraborti et al.,
(Madsen, 1990a). GH treatment in the present study on
1987) and American eel Anguilla rostrata (Sandor et al.,
rainbow trout, however, was without effect on gill CR Bmax
1984). The relative affinity of the steroid for gill CRs,
and affinity. The absence of an effect of GH on CR in
therefore, appears to play an important role in tissue sensitivity.
rainbow trout brings into question the proposed mechanism
The differences in Kd in the present study were not significant,
for interaction between GH and cortisol for this species. GH-
and the potential effects on cortisol responsiveness are small
induced increases in gill Na+/K+-ATPase activity may work
in comparison with the changes in Bmax observed. Regression
directly through interaction with the GH receptor because GH
of Bmax on cortisol responsiveness accounted for 61 % of the
variance in the data. Division of Bmax by Kd, however,
Bidgood, B. F. and Berst, A. F. (1969). Lethal temperatures for Great
strengthened the relationship slightly to account for 66 % of the
Lakes rainbow trout. J. Fish. Res. Bd Can. 26, 456–459.
variance in the data. CR affinity changes have been observed
Bradford, M. M. (1976). A rapid and sensitive method for the
seasonally in gills of coho salmon (Shrimpton et al., 1994;
quantitation of microgram quantities of protein using the principle
Shrimpton, 1996) and Atlantic salmon (Shrimpton and
of protein dye binding. Analyt. Biochem. 72, 248–252. Brink, M., Humbel, B. M., De kloet, E. R. and Van Driel, R.
McCormick, 1998a) and in coho salmon leukocytes (Maule et
(1992). The unliganded glucocorticoid receptor is localized in the
al., 1993), but not in steelhead trout gills (McLeese et al.,
nucleus, not in the cytoplasm. Endocrinology 130, 3575–3581.
1994). Cortisol has also been shown to decrease CR affinity in
Chakraborti, P. K., Weisbart, M. and Chakraborti, A. (1987). The
coho salmon gills (Maule and Schreck, 1991; Shrimpton and
presence of corticosteroid receptor activity in the gills of brook
Randall, 1994) and rainbow trout liver (Pottinger et al., 1994).
trout, Salvelinus fontinalis. Gen. Comp. Endocr. 66, 323–332.
GH also caused a decrease in CR affinity in gills of Atlantic
Clark, J. H. and Peck, E. J. (1977). Steroid hormone receptors:
salmon parr (Shrimpton and McCormick, 1998b).
Basic principles and measurement. In Receptors and Hormone
Other regulatory factors may also affect the responsiveness
Action (ed. B. W. O’Malley and L. Birnbaumer), pp. 383–410. New
of the gills to cortisol. Intracellular metabolism of cortisol by
11β-hydroxysteroid dehydrogenase (11β-HSD) (Bamberger et
Danielsen, M. and Stallcup, M. R. (1984). Down-regulation of
al., 1996) and removal of cortisol from the cell by specific
glucocorticoid receptors in mouse lymphoma cell variants. Mol. Cell. Biol. 4, 449–453.
membrane transporters (Thompson, 1995) are both
Dangé, A. D. (1986). Branchial Na/K-ATPase in freshwater or
mechanisms that prevent the interaction between
saltwater acclimated tilapia Oreochromis mossambicus: effects of
glucocorticoids and CRs and alter the sensitivity of the tissue.
cortisol and thyroxin. Gen. Comp. Endocr. 62, 341–343.
Variation around the regression line exists for all treatment
Fryer, J. N. and Bern, H. A. (1979). Growth hormone binding to
groups (Fig. 7). The stress-treated group, however, showed in
tissues of normal and stunted juvenile coho salmon, Oncorhynchusvitro Na+/K+-ATPase activity in response to cortisol that was
kisutch. J. Fish Biol. 15, 527–533.
lower than that for other fish with a similar CR Bmax because
Hoar, W. S. (1988). The physiology of smolting salmonids. In Fish
they consistently fell below the regression line (Fig. 7). One of
Physiology, vol. 11B (ed. W. S. Hoar and D. J. Randall), pp.
these other regulatory factors may account for the observed
275–344. New York: Academic Press.
difference. It is known that the half-life of cortisol in the
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plasma decreases with stress in juvenile coho salmon (Redding
(1980). Heterogeneity of the in vitro
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et al., 1984). We do not know whether the intracellular
Hurley, D. M., Accili, D., Stratakis, C. A., Karl, M.,
metabolism of cortisol in the gills was increased in the stress-
Vamvakopoulos, N., Rorer, E., Constantine, K., Taylor, S. I.
treated group, but this could account for the difference seen in
and Chrousos, G. P. (1991). Point mutation causing a single amino
acid substitution in the hormone binding domain of the
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Clin. Invest. 87, 680–686.
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increases, CR abundance, resulting in significant differences in
(1987). Tissue-specific regulation of glucocorticoid receptor
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abundance that have been observed during the parr–smolt
Knoebl, I., Fitzpatrick, M. S. and Schreck, C. B. (1996).
transformation in several species of salmonids alter the
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We thank the staff at the Sunderland and McLauglin State
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Hatcheries, Massachusetts Department of Fish and Wildlife,
Madsen, S. S. (1990a). Enhanced hypoosmoregulatroy response to
for providing the fish used in this study. We also thank Judy
growth hormone after cortisol treatment in immature rainbow trout,
Carey, Jill Leonard, Juan-Miguel Mancera, Michael O’Dea
Salmo gairdneri. Fish Physiol. Biochem. 8, 271–279.
and Joe Zydlewski for assistance in sampling and Dr Graham
Madsen, S. S. (1990b). The role of cortisol and growth hormone in
Young, Phil Veillette and two anonymous referees for their
seawater adaptation and development of hypoosmoregulatorymechanisms in sea trout (Salmo trutta trutta). Gen. Comp. Endocr.
critical review of the manuscript. NIH generously provided
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Editor’s note: opinion released October 17, 2000 SUPREME COURT OF LOUISIANA No. 00-C-0492 BERT J. WAINWRIGHT, ET AL. ROMONA FONTENOT, ET AL. ON WRIT OF CERTIORARI TO THE COURT OF APPEAL, THIRDAt issue in this delictual action is whether a factfinder errs as a matter of law when it declines toaward general damages after finding defendant at fault for plaintiff’s injuries and award
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