Responsiveness of gill na+/k+-atpase to cortisol

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 1999 A 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 cortisol>corticosterone>17-hydroxyprogesterone>cortisone> 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., (P<0.001; r2=0.657).
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 Discussion
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, Oncorhynchus vitro 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 Homo, F., Duval, D., Harousseau, J. L., Marie, J. P. and Zittoun,
plasma decreases with stress in juvenile coho salmon (Redding (1980). Heterogeneity of the in vitro glucocorticoids in acute leukemia. Cancer Res. 40, 2601–2608.
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 The present study demonstrates a strong correlation between glucocorticoid receptor in familial glucocorticoid resistance. J. CR concentration and in vitro responsiveness to cortisol in the Clin. Invest. 87, 680–686.
gills of rainbow trout. Stress decreases, and T3 treatment Kalinyak, J. E., Dorin, R. I., Hoffman, A. R. and Perlman, A. J.
increases, CR abundance, resulting in significant differences in (1987). Tissue-specific regulation of glucocorticoid receptor gill responsiveness to cortisol. The seasonal changes in CR mRNA by dexamethasone. J. Biol. Chem. 262, 10441–10444.
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 Characterization of a glucocorticoid receptor in the brains ofchinook salmon, Oncorhynchus tshawytscha. Gen. Comp. Endocr.
response of the gill to cortisol. This finding supports the role 101, 195–204.
of cortisol as an important endocrine factor in stimulating Lee, P. C., Goodrich, M., Struve, M., Yoon, H. I. and Weber, D.
smolting and seawater tolerance in salmonids.
(1992). Liver and brain glucocorticoid receptor in rainbow trout,Oncorhynchus mykiss: downregulation by dexamethasone. Gen. We thank the staff at the Sunderland and McLauglin State Comp. Endocr. 87, 222–231.
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 79, 1–11.
Madsen, S. S. (1990c). Effect of repetitive cortisol and thyroxine
injections on chloride cell number and Na+/K+-ATPase activity ingills of freshwater acclimated rainbow trout, Salmo gairdneri.
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Nurses’ exposures to antineoplastic drugs in Canada and risk assessment of lifetime cancer incidence Presenter: Avital Jarus-Hakak Authors: Avital Jarus-Hakak, Sharla Drebit, Chun-Yip Hon, Anne-Marie Nicol, George Astrakianakis. Objectives: Oncology as well as non-oncology nurses are at risk for exposure to antineoplastic drugs some of which are known to be carcinogenic. Exposures

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Spring 2007 J e f f e r s o n C a n c e r N e t w o r krecently received a certificate of recognition from the JCN for her News efforts in clinical trial enrollments in 2006. The complete list of recognition awards for 2006 is as follows:For highest overall institutional accrual; Albert Einstein Cancer Message from the Clinical Center, Hematology & Oncology Associates of NEPA, A

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