Fish Physiology and Biochemistry vol. 13 no. 3 pp 183-190 (1994) Kugler Publications, Amsterdam/New York
Cortisol stimulates intestinal fluid uptake in Atlantic salmon (Salmo salar) in the post-smolt stage Sean C. Cornell l, David M. Portesi l, Philip A. Veillette l, Kristina Sundell 12, and Jennifer L. Specker' 1 Department of Zoology, University of Rhode Island, Kingston, Rhode Island 02881, U.S.A.; 2 Deparment of Zoophysiology, University of Goteborg, Medicinargatan 18, S-413 90 Goteborg, Sweden Accepted: March 31, 1994 Keywords: cortisol implants, intestinal transport, Atlantic salmon, Salmo salar, parr-smolt transformation, gill Na+,K+-ATPase activity, seawater adaptation
Abstract The fluid uptake rate of the posterior intestine of salmonids increases during the parr-smolt transformation. Intestinal fluid uptake in post-smolt Atlantic salmon was investigated after treatment with cortisol and growth hormone (GH), alone or together. Two replicate experiments were conducted in August 1991 and August 1992. Cortisol was emulsified in vegetable shortening and vegetable oil (1:1) and implanted into the peritoneal cavity. GH was administered as intraperitoneal injections in a saline vehicle on days 0 and 2. On days 5 and 6, plasma cortisol levels, gill Na + ,K+ -ATPase activity, and in vitro measurements of fluid transport rate (Jv) across the posterior intestine were measured. Implants of cortisol elevated the plasma cortisol levels within a physiological range, and resulted in elevated gill Na + ,K + -ATPase activity, as expected. The fluid uptake rate across the posterior intestine was roughly doubled by cortisol treatment. GH treatment did not affect intestinal fluid transport, gill Na + ,K+ -ATPase activity, or plasma cortisol concentrations. The seawater-adapting increase in the rate of fluid uptake by the posterior intestine of smolting salmon is probably stimulated by elevated plasma cortisol concentrations.
Introduction In teleost fishes, the intestine gills, and kidney are major osmoregulatory organs which maintain internal salt and water balance. Physiological adaptation of migratory and euryhaline fishes to seawater involves functional changes of these organs. Most information on the role of the intestine in seawater adaptation is obtained from studies on eels (Anguilla japonica and Anguilla anguilla; Hirano et al. 1976). The rate of intestinal fluid uptake is higher in seawater-adapted eels than in freshwater-adapted eels. Furthermore, fluid uptake increases before
migration to seawater which indicates a pre-adaptive development of the intestine (Oide and Utida 1967). Juvenile Atlantic salmon (Salmo salar) undergo a complex physiological and morphological preadaptive transformation from parr to smolts in fresh water and become ready for entry into seawater in springtime (Hoar 1988). In salmonids, the most thoroughly studied physiological change is the increase in gill Na+,K+-ATPase activity during the parr-smolt transformation, which has been used as an indicator of increased hypoosmoregulatory ability (see McCormick and Saunders 1987). The functional changes in the intestine of coho salmon
Correspondenceto: Dr. Jennifer Specker, Department of Zoology, University of Rhode Island, Kingston, RI 02881-0816, U.S.A.; FAX (401) 792-4256, Phone (401) 792-2658, e-mail jspecker @ uriacc.uri.edu
184 (Oncorhynchus kisutch) and Atlantic salmon during the parr-smolt transformation include a doubling of the fluid transport rate across the posterior intestine; if salmon are denied access to seawater and retained in fresh water, fluid uptake decreases in the post-smolt stage (Collie and Bern 1982; Veillette et al. 1993b). A complex pattern of endocrine factors mediate the parr-smolt transformation. Cortisol and growth hormone (GH) have been suggested to be of importance for the pre-adaptive increase in hypoosmoregulatory ability (see Bisbal and Specker 1991; McCormick et al. 1991; Sakamoto et al. 1993). Plasma concentrations of GH and cortisol increase in springtime during parr-smolt transformation coincident with increases in hypoosmoregulatory ability (Specker and Schreck 1982; Virtanen and Soivo 1985; Langhorne and Simpson 1986; Bouef et al. 1989; Young et al. 1989) and an elevation of intestinal fluid uptake (Collie and Bern 1982; Veillette et al. 1993b). Furthermore, both GH and cortisol treatment have been shown to stimulate gill Na+,K+-ATPase activity and to improve seawater tolerance in several salmonid species, and cortisol has been shown to stimulate gut Na + ,K+-ATPase activity in one salmonid species (Miwa and Inui 1985; Bjbrnsson et al. 1987; Richman and Zaugg 1987; McCormick and Bern 1989; Madsen 1990a, b, c; Bisbal and Specker 1991; McCormick et al. 1991; Madsen and Bern 1992; see Sakamoto et al. 1993). In the eel, injections of cortisol or ACTH result in increased intestinal fluid uptake (Hirano and Utida 1968, 1971). The increase in fluid uptake in freshwater eels transferred to seawater is accompanied by a transient increase in plasma cortisol levels (Hirano and Utida 1971). The aim of this study was to examine the effects of cortisol and GH treatment, alone or together, on intestinal fluid uptake in Atlantic salmon. Salmon in the post-smolt stage were used because they are known to have fluid uptake rates characteristic of freshwater-adapted salmon (Veillette et al. 1993b). We also examined gill Na+,K+-ATPase activity for concurrent changes since this enzyme is known to be directly stimulated by cortisol (McCormick and Bern 1989).
Materials and methods Animals Juvenile Atlantic salmon were reared at North Attleboro National Fish Hatchery (U.S. Fish and Wildlife Service) under natural photoperiod and constant temperature (10°C). The fish were kept in fresh water until after they underwent parr-smolt transformation and entered the post-smolt stage. The fish were transported to the salmon facility in the Biological Sciences Center, University of Rhode Island, and maintained in a 1.2-m diameter holding tank with flow-through well water (10°C) and simulated natural photoperiod. At the time of the experiments, the fish were age 1 + post-smolts. The average body weight was 128.8 5.1 g (mean SEM) and 181.5 +4.8 g in 1991 and 1992, respectively, and the fork-length was 24.2+0.4 cm in 1991 and 27.2± 0.3 cm in 1992.
Experimental design, injections, handling and sampling Two replicate experiments were conducted in August 1991 and August 1992. The experiment was designed as a two-way factorial, with cortisol and GH being the factors, permitting us to detect a possible interaction between the two factors. There were four treatment groups: control, cortisoltreated, GH-treated and cortisol + GH-treated, each containing twelve fish randomly selected from the holding tank. Salmon were transferred to oval tanks and separated by treatment. Ovine GH (oGH; gift of National Hormone and Pituitary Program, Baltimore, MD) was dissolved in 0.7% saline at a concentration of 300 jig ml-l; salmon GH (sGH; gift of Dr. Penny Swanson, National Marine Fisheries Service, Seattle, WA) was dissolved at a concentration of 7.5 g ml 0.7% saline- 1. Cortisol was emulsified in liquified vegetable shortening: vegetable oil (1:1, by weight) at a concentration of 10 mg ml- . The salmon were anesthetized in 2-phenoxyethanol (0.07%, Sigma, St. Louis, MO) before injections, which were made into the peritoneal cav-
185 ity. The salmon treated with cortisol were injected on day 0 with 0.5 mg cortisol 10 g bw - l. The vegetable shortening: vegetable oil mixture was fluid when injected at 24-34°C and, after injection (0.5 to 1.0 ml, depending on body weight), solidified into an implant in the peritoneal cavity, a methodology described in further detail elsewhere (Specker et al. 1994). The fluid to gel transition occurs at 22-24°C. The salmon receiving GH were injected on days 0 and 2 at 1.5 jig oGH g bw 1, with the exception of day 2 in August 1992 when, because we had used all our oGH, the salmon were injected with sGH at 0.04 gg g bw- . A lower dose of sGH was used because the potency of sGH in salmon has been estimated at 40x that of oGH (Boeuf et al. 1992). Salmon in the control group, best controlling for those fish in the cortisol + GH treatment group, were injected on days 0 and 2 with saline and implanted on day 0 with vegetable shortening: vegetable oil. Due to the length of time required for in vitro determination of fluid uptake, we sampled the fish over a two day period. Half the salmon from each group were sampled on day 5 and the remaining six salmon from each group were sampled on day 6. No significant change occurs between these two days. The salmon were anesthetized, weighed and measured, and blood was collected from caudal vessels into heparanized syringes. Plasma was separated from blood cells by centrifugation and stored at -70°C until analyzed. The spinal cord was severed, the body cavity cut open, and the posterior intestine (the section posterior to the ileorectal valve) carefully removed for measurement of fluid uptake rate (Jv)- Gill filaments were trimmed from the first branchial arches and stored in SEI buffer (in mM: sucrose 300; Na2EDTA 20; imidazole 50; pH 7.3; Zaugg 1982) at -70°C until assayed for Na+ ,K + ATPase activity. Gut mucosa was prepared for analysis of enzyme activity; however, accidental loss of some samples precluded a complete analysis.
Posteriorintestinalfluid transportJv The posterior segment of the intestine was rinsed, filled with salmon Ringer (in mM: NaCl 140; KCI
2.5; NaHCO3 15; CaCl 2 1.5; KH 2PO4 1; MgSO 4 0.8; glucose 10 and HEPES buffer 5; pH 7.8), and tied off at both ends with unwaxed dental floss forming a non-everted sac. This sac was suspended in salmon Ringer aerated with a gas mixture (02: CO2, 95:5%) at 14°C and equilibrated for h. Over the second hour, the gut sac was weighed every ten minutes to the nearest 0.1 mg. Rate of fluid loss from the intestinal sac was determined by regression analysis of the measured sac weights. This rate was normalized by dividing by the surface area of the sac to yield a net rate of fluid movement from mucosal to serosal sides of the intestine, expressed in gl cm - 2h - 1. These methods followed the procedures of Collie and Bern (1982) with modifications by Veillette et al. (1993b).
Na +,K + -A TPase activity Semi-purified enzyme preparations of gill tissue were obtained by using the method of Zaugg (1982), and the determination of Na + ,K+ -ATPase activity was made by modifying the method of Zaugg (1982) as previously described by Bisbal and Specker (1991). Total protein concentrations were determined using the method of Lowry et al. (1951). Absorbance was read at 750 nm using a microplate reader (MR-300, Dynatech Laboratories Inc., Chantilly, VA).
Plasma cortisol levels Cortisol was assayed in unextracted plasma, run in duplicate, using radioimmunoassay (RIA) as described by Young (1986) and validated in our hands (Bisbal and Specker 1991).
Statistical analyses The data were analyzed using two-way analysis of variance, testing for main effects of cortisol and GH and for an interaction (GLM procedure, SAS Inst., Cary, NC). The replicate experiments were analyzed separately. When appropriate, the Tukey
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Fig. 1. Changes in plasma cortisol concentrations (mean SEM, n = 11-12) of Atlantic salmon in the post-smolt stage implanted with cortisol (F) and/or injected with growth hormone (GH) for 5 or 6 days, compared to controls (Con). Upper panel shows experiments conducted in August 1991; lower panel shows experiments conducted in August 1992. In both years, a main effect of cortisol was found (two-way ANOVA); the cortisol-treated salmon had elevated plasma cortisol levels (Tukey HSD, indicated by the horizontal bars).
Fig. 2. Changes in posterior intestinal fluid uptake (J,) (mean + SEM, n = 11-12) of Atlantic salmon in the post-smolt stage implanted with cortisol (F) and/or injected with growth hormone (GH) for 5 or 6 days, compared to controls (Con). Upper panel shows experiments conducted in August 1991; lower panel shows experiments conducted in August 1992. In both years, a main effect of cortisol was found (two-way ANOVA); the cortisol-treated salmon had increased rates of fluid uptake (J,) by the posterior intestine (Tukey HSD, indicated by the horizontal bars).
HSD procedure was used to test for differences among means. Significance was accepted when P < 0.05.
the Tukey analysis indicated that cortisol-treated animals had higher plasma cortisol levels, gill Na+,K+-ATPase activity, and intestinal J, than the animals not receiving cortisol implants (indicated by the common lines above the cortisoltreated groups and the not-cortisol-treated groups, Figs. 1-3).
Results Statistical analyses (two-way ANOVA) of plasma cortisol levels, gill Na + ,K+-ATPase activity, and Jv across the posterior intestine yielded the same results for both experiments: only a main effect of cortisol treatment was found; there was no effect of GH and no interaction (Figs. 1-3). The results of
Discussion Our most significant finding is that exogenous cortisol can stimulate fluid uptake across the posterior
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Fig. 3. Changes in gill Na+,K+-ATPase activity (mean + SEM, n = 11-12) of post-smolt Atlantic salmon implanted with cortisol (F) and/or injected with growth hormone (GH) for 5 or 6 days, compared to controls (Con). Upper panel shows experiments conducted in August 1991; lower panel shows experiments conducted in August 1992. In both years, a main effect of cortisol was found (two-way ANOVA); the cortisol-treated salmon had higher gill Na + ,K + -ATPase activity (Tukey HSD, indicated by the horizontal bars).
intestine of Atlantic salmon. This is the first demonstration of a cortisol-induced change in fluid transport rate in salmonids, adding to the previous findings that cortisol stimulates fluid transport across the eel intestine (Hirano 1967; Hirano and Utida 1968, 1971). Cortisol treatment also induced greater gill Na+,K+-ATPase activity, as demonstrated in a number of earlier studies (see Bisbal and Specker 1991; McCormick et al. 1991). Growth
hormone treatment under these experimental conditions did not affect intestinal fluid transport, gill Na+,K + - ATPase activity, or plasma cortisol concentrations. The cortisol implant used in the present study differs from previously published methods in that cortisol was emulsified in a mixture of partially hydrogenated vegetable shortening (from soybean and palm oil) and vegetable (soybean) oil. The methodology is fully described in Specker et al. (1994). Briefly, it was shown, using serially sampled Atlantic salmon in the post-smolt stage, that plasma cortisol levels at this dose are elevated within 24 h and remain elevated for 28 days. The implant alone causes no elevation in plasma cortisol concentration compared to non-implanted salmon. The vegetable oil: vegetable shortening implant is thus an improvement over those presented in Bisbal and Specker (1991), in which hydrogenated vegetable shortening alone is used, and in Pickering and Duston (1983), in which cocoa butter is used, in that the cortisol implant can be injected at a lower temperature. The plasma cortisol levels achieved by the present method are within a physiological range (cf., Virtanen and Soivo 1985; Barton et al. 1986; Langhorne and Simpson 1986; Bisbal and Specker 1991; Pottinger and Moran 1993). To replace osmotically lost water, teleost fish in seawater are forced to drink and subsequently extract water across the intestine by ion-coupled transport (Skadhauge 1969). In Atlantic salmon, the parr-smolt tranformation is accompanied by an increase in gill Na+,K+-ATPase activity (see McCormick and Saunders 1987) and an increase in intestinal fluid transport across the posterior intestine (Veillette et al. 1993b). These physiological changes are correlated with elevated plasma levels of cortisol and GH (Virtanen and Soivio 1985; Langhorne and Simpson 1986; Bouef et al. 1989). The present study demonstrates that the rate of intestinal fluid absorption in Atlantic salmon is stimulated by cortisol, suggesting that increased plasma cortisol concentrations may be responsible for the pre-adaptive changes of the intestinal mucosa during the parr-smolt transformation of Atlantic salmon. This would parallel the situation in the eel in which cortisol stimulates intestinal sodium and
188 water transport and is probably involved in the developmental adaptation of the intestine when eels migrate to seawater (Hirano 1967; Hirano and Utida 1968; Gaitskell and Chester Jones 1970). Few studies have been conducted on salmonids and the mechanisms behind intestinal fluid transport. However, in coho salmon and Atlantic salmon ouabain is able to reduce fluid uptake by approximately 70°o, indicating that ouabain sensitive Na + ,K + - ATPase activity is responsible for most of the fluid transport (Collie and Bern 1982; Veillette et al. 1993b). In the eel and in killifish, cortisol stimulates Na + ,K+ -ATPase activity (Pickford et al. 1970; Epstein et al. 1971) and the apparent osmotic permeability (Ando 1974) of the intestine. In the rainbow trout, cortisol increases gut Na + ,K + -ATPase activity (Madsen 1990a), whereas, in Atlantic salmon, no effect on Na+,K + ATPase activity was found (Bisbal and Specker 1991). Concurrent electrophysiological experiments performed in our laboratory, using fish from the same tank as used in the present study, showed that a 5-day cortisol implant increased the transepithelial potential (TEP) across the posterior intestine. This increase in TEP was abolished by ouabain (K. Sundell, P.A. Veillette and J.L. Specker, unpublished results). These data, together with the present results, suggest that cortisol stimulates Na+ ,K+ -ATPase activity in the intestinal mucosa of Atlantic salmon, as in trout (Madsen 1990a), and that refinements in the methodology we have used for preparing the gut mucosa are likely to make this apparent. Exogenous GH, either alone or in combination with cortisol, had no effect on plasma cortisol concentration, gill Na+ ,K+ -ATPase activity, or intestinal fluid transport. We expected GH to increase Na + ,K+-ATPase activity in the gills, fluid transport across the intestine, and to act additively with cortisol. Previously, GH had been shown to increase interrenal nuclear diameter and to sensitize the interrenals to ACTH in coho salmon (Higgs et al. 1977; Young 1988). Also, GH in vivo has been shown to increase gill Na + ,K+-ATPase activity in several salmonids (Miwa and Inui 1985; Richman and Zaugg 1987; Bj6rnsson et al. 1987; Boeuf et al. 1992), although GH in vitro does not increase
gill Na+,K+-ATPase activity in coho salmon (McCormick et al. 1991). Furthermore, an additive effect of GH + cortisol treatment in vivo on ionoregulatory ability has been observed for rainbow trout (Oncorhynchus mykiss) and sea trout (Salmo trutta trutta; Madsen 1990b, c). Given the rate of clearance of GH from salmonids (see Sakamoto et al. 1993), our protocol may have been insufficient to observe direct or indirect effects of GH on these osmoregulatory functions, although a similar protocol is sufficient for observing some osmoregulatory effects (see Collie et al. 1989). The sensitivity of tissues to hormones can be seasonal. For example, the sensitivity of gill Na + ,K+-ATPase activity to cortisol, as measured in vitro, and to GH, as measured in vivo, is seasonal (McCormick et al. 1991; Madsen and Bern 1993). Recent evidence from our laboratory shows that the rate of fluid uptake by the posterior intestine is unaffected by cortisol during the peak smolt period (Veilette et al. 1993a). Thus, it is possible that although GH has no effect on the interrenal nor any osmoregulatory action in post-smolt Atlantic salmon in the summer (under these experimental conditions), it may play a role in other phases of development. We conclude that elevated plasma levels of cortisol, achieved by use of cortisol implants, stimulate posterior intestinal fluid uptake and gill Na + ,K+ ATPase activity in post-smolt Atlantic salmon kept in fresh water, and thus that cortisol may be responsible for the pre-adaptive changes in intestinal fluid uptake rate which occur prior to ocean migration. The improved survival in high salinity (37 ppt seawater) and improved plasma Na+ -regulatory ability resulting from cortisol implants in freshwaterphase Atlantic salmon (Bisbal and Specker 1991) may be due to changed intestinal function, in addition to changed branchial function. The duration of these cortisol implants and their longer term effects on growth, survival, and disease resistance are not currently known, but warrant further consideration (see Pickering 1993). We suggest that our non-stressful cortisol implants could be used to facilitate transfer of salmonids to seawater at times of the year, and at developmental stages, outside of the springtime smolt phase.
189 Acknowledgements This publication is the result of research sponsored by NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant #NA89AA-D-SG082 (J.L.S.). The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon. The authors thank Dr. Nathan Collie, Texas Tech University, for his expert advice; Dr. Penny Swanson, National Marine Fisheries Service, for generously supplying salmon GH; Dr. Jerry White, Eastern Washington University, for stimulating discussions; Ms. Rochelle Webster for her expert assistance; and Mr. Kenneth Davignon for graphics.
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tion in the intestine of the eel (Anguilla anguilla) adapted to waters of various salinities. J. Physiol. 204: 135-158. Specker, J.L. and Schreck, C.B. 1982. Changes in plasma corticosteroids during smoltification of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 46: 53-58. Specker, J.L., Portesi, D.M., Cornell, S.C. and Veillette, P.A. 1994. Methodology for implanting cortisol in Atlantic salmon and effects of chronically elevated cortisol on osmoregulatory physiology. Aquaculture 121: 181-193. Veillette, P.A., Sundell, K. and Specker, J.L. 1993a. Developmental changes in the responsiveness of intestinal fluid transport to cortisol and RU 486. Am. Zool. 33: 11A. Veillette, P.A., White, R.J. and Specker, J.L. 1993b. Changes in intestinal fluid transport in Atlantic salmon (Salmo salar L.) during parr-smolt transformation. Fish Physiol. Biochem. 12: 193-202. Virtanen, E. and Soivio, A. 1985. The patterns of T3 , T4, cor+ + tisol, and Na -K -ATPase during smoltification of hatchery-reared Salmo salar and comparison with wild smolts. Aquaculture 45: 97-109. Young, G. 1986. Cortisol secretion in vitro by the interrenal of coho salmon (Oncorhynchus kisutch) during smoltification: relationship with plasma thyroxine and plasma cortisol. Gen. Comp. Endocrinol. 63: 191-200. Young, G. 1988. Enhanced response of the interrenal of coho salmon (Oncorhynchus kisutch) to ACTH after growth hormone treatment in vivo and in vitro. Gen. Comp. Endocrinol. 71: 85-92. Young, G., Bjornsson, B., Th., Prunet, P., Lin, R.J. and Bern, H.A. 1989. Smoltification and seawater adaptation in coho salmon (Oncorhynchus kisutch): plasma prolactin, growth hormone, thyroid hormone, and cortisol. Gen. Comp. Endocrinol. 74: 335-345. Zaugg, W.S. 1982. A simplified preparation for adenosine triphosphatase determination in gill tissue. Can. J. Fish. Aquat. Sci. 39: 215-217.
Cortisol stimulates intestinal fluid uptake in Atlantic salmon (Salmo salar) in the post-smolt stage Sean C. Cornell l, David M. Portesi l, Philip A. Veillette l, Kristina Sundell 12, and Jennifer L. Specker' 1 Department of Zoology, University of Rhode Island, Kingston, Rhode Island 02881, U.S.A.; 2 Deparment of Zoophysiology, University of Goteborg, Medicinargatan 18, S-413 90 Goteborg, Sweden Accepted: March 31, 1994 Keywords: cortisol implants, intestinal transport, Atlantic salmon, Salmo salar, parr-smolt transformation, gill Na+,K+-ATPase activity, seawater adaptation
Abstract The fluid uptake rate of the posterior intestine of salmonids increases during the parr-smolt transformation. Intestinal fluid uptake in post-smolt Atlantic salmon was investigated after treatment with cortisol and growth hormone (GH), alone or together. Two replicate experiments were conducted in August 1991 and August 1992. Cortisol was emulsified in vegetable shortening and vegetable oil (1:1) and implanted into the peritoneal cavity. GH was administered as intraperitoneal injections in a saline vehicle on days 0 and 2. On days 5 and 6, plasma cortisol levels, gill Na + ,K+ -ATPase activity, and in vitro measurements of fluid transport rate (Jv) across the posterior intestine were measured. Implants of cortisol elevated the plasma cortisol levels within a physiological range, and resulted in elevated gill Na + ,K + -ATPase activity, as expected. The fluid uptake rate across the posterior intestine was roughly doubled by cortisol treatment. GH treatment did not affect intestinal fluid transport, gill Na + ,K+ -ATPase activity, or plasma cortisol concentrations. The seawater-adapting increase in the rate of fluid uptake by the posterior intestine of smolting salmon is probably stimulated by elevated plasma cortisol concentrations.
Introduction In teleost fishes, the intestine gills, and kidney are major osmoregulatory organs which maintain internal salt and water balance. Physiological adaptation of migratory and euryhaline fishes to seawater involves functional changes of these organs. Most information on the role of the intestine in seawater adaptation is obtained from studies on eels (Anguilla japonica and Anguilla anguilla; Hirano et al. 1976). The rate of intestinal fluid uptake is higher in seawater-adapted eels than in freshwater-adapted eels. Furthermore, fluid uptake increases before
migration to seawater which indicates a pre-adaptive development of the intestine (Oide and Utida 1967). Juvenile Atlantic salmon (Salmo salar) undergo a complex physiological and morphological preadaptive transformation from parr to smolts in fresh water and become ready for entry into seawater in springtime (Hoar 1988). In salmonids, the most thoroughly studied physiological change is the increase in gill Na+,K+-ATPase activity during the parr-smolt transformation, which has been used as an indicator of increased hypoosmoregulatory ability (see McCormick and Saunders 1987). The functional changes in the intestine of coho salmon
Correspondenceto: Dr. Jennifer Specker, Department of Zoology, University of Rhode Island, Kingston, RI 02881-0816, U.S.A.; FAX (401) 792-4256, Phone (401) 792-2658, e-mail jspecker @ uriacc.uri.edu
184 (Oncorhynchus kisutch) and Atlantic salmon during the parr-smolt transformation include a doubling of the fluid transport rate across the posterior intestine; if salmon are denied access to seawater and retained in fresh water, fluid uptake decreases in the post-smolt stage (Collie and Bern 1982; Veillette et al. 1993b). A complex pattern of endocrine factors mediate the parr-smolt transformation. Cortisol and growth hormone (GH) have been suggested to be of importance for the pre-adaptive increase in hypoosmoregulatory ability (see Bisbal and Specker 1991; McCormick et al. 1991; Sakamoto et al. 1993). Plasma concentrations of GH and cortisol increase in springtime during parr-smolt transformation coincident with increases in hypoosmoregulatory ability (Specker and Schreck 1982; Virtanen and Soivo 1985; Langhorne and Simpson 1986; Bouef et al. 1989; Young et al. 1989) and an elevation of intestinal fluid uptake (Collie and Bern 1982; Veillette et al. 1993b). Furthermore, both GH and cortisol treatment have been shown to stimulate gill Na+,K+-ATPase activity and to improve seawater tolerance in several salmonid species, and cortisol has been shown to stimulate gut Na + ,K+-ATPase activity in one salmonid species (Miwa and Inui 1985; Bjbrnsson et al. 1987; Richman and Zaugg 1987; McCormick and Bern 1989; Madsen 1990a, b, c; Bisbal and Specker 1991; McCormick et al. 1991; Madsen and Bern 1992; see Sakamoto et al. 1993). In the eel, injections of cortisol or ACTH result in increased intestinal fluid uptake (Hirano and Utida 1968, 1971). The increase in fluid uptake in freshwater eels transferred to seawater is accompanied by a transient increase in plasma cortisol levels (Hirano and Utida 1971). The aim of this study was to examine the effects of cortisol and GH treatment, alone or together, on intestinal fluid uptake in Atlantic salmon. Salmon in the post-smolt stage were used because they are known to have fluid uptake rates characteristic of freshwater-adapted salmon (Veillette et al. 1993b). We also examined gill Na+,K+-ATPase activity for concurrent changes since this enzyme is known to be directly stimulated by cortisol (McCormick and Bern 1989).
Materials and methods Animals Juvenile Atlantic salmon were reared at North Attleboro National Fish Hatchery (U.S. Fish and Wildlife Service) under natural photoperiod and constant temperature (10°C). The fish were kept in fresh water until after they underwent parr-smolt transformation and entered the post-smolt stage. The fish were transported to the salmon facility in the Biological Sciences Center, University of Rhode Island, and maintained in a 1.2-m diameter holding tank with flow-through well water (10°C) and simulated natural photoperiod. At the time of the experiments, the fish were age 1 + post-smolts. The average body weight was 128.8 5.1 g (mean SEM) and 181.5 +4.8 g in 1991 and 1992, respectively, and the fork-length was 24.2+0.4 cm in 1991 and 27.2± 0.3 cm in 1992.
Experimental design, injections, handling and sampling Two replicate experiments were conducted in August 1991 and August 1992. The experiment was designed as a two-way factorial, with cortisol and GH being the factors, permitting us to detect a possible interaction between the two factors. There were four treatment groups: control, cortisoltreated, GH-treated and cortisol + GH-treated, each containing twelve fish randomly selected from the holding tank. Salmon were transferred to oval tanks and separated by treatment. Ovine GH (oGH; gift of National Hormone and Pituitary Program, Baltimore, MD) was dissolved in 0.7% saline at a concentration of 300 jig ml-l; salmon GH (sGH; gift of Dr. Penny Swanson, National Marine Fisheries Service, Seattle, WA) was dissolved at a concentration of 7.5 g ml 0.7% saline- 1. Cortisol was emulsified in liquified vegetable shortening: vegetable oil (1:1, by weight) at a concentration of 10 mg ml- . The salmon were anesthetized in 2-phenoxyethanol (0.07%, Sigma, St. Louis, MO) before injections, which were made into the peritoneal cav-
185 ity. The salmon treated with cortisol were injected on day 0 with 0.5 mg cortisol 10 g bw - l. The vegetable shortening: vegetable oil mixture was fluid when injected at 24-34°C and, after injection (0.5 to 1.0 ml, depending on body weight), solidified into an implant in the peritoneal cavity, a methodology described in further detail elsewhere (Specker et al. 1994). The fluid to gel transition occurs at 22-24°C. The salmon receiving GH were injected on days 0 and 2 at 1.5 jig oGH g bw 1, with the exception of day 2 in August 1992 when, because we had used all our oGH, the salmon were injected with sGH at 0.04 gg g bw- . A lower dose of sGH was used because the potency of sGH in salmon has been estimated at 40x that of oGH (Boeuf et al. 1992). Salmon in the control group, best controlling for those fish in the cortisol + GH treatment group, were injected on days 0 and 2 with saline and implanted on day 0 with vegetable shortening: vegetable oil. Due to the length of time required for in vitro determination of fluid uptake, we sampled the fish over a two day period. Half the salmon from each group were sampled on day 5 and the remaining six salmon from each group were sampled on day 6. No significant change occurs between these two days. The salmon were anesthetized, weighed and measured, and blood was collected from caudal vessels into heparanized syringes. Plasma was separated from blood cells by centrifugation and stored at -70°C until analyzed. The spinal cord was severed, the body cavity cut open, and the posterior intestine (the section posterior to the ileorectal valve) carefully removed for measurement of fluid uptake rate (Jv)- Gill filaments were trimmed from the first branchial arches and stored in SEI buffer (in mM: sucrose 300; Na2EDTA 20; imidazole 50; pH 7.3; Zaugg 1982) at -70°C until assayed for Na+ ,K + ATPase activity. Gut mucosa was prepared for analysis of enzyme activity; however, accidental loss of some samples precluded a complete analysis.
Posteriorintestinalfluid transportJv The posterior segment of the intestine was rinsed, filled with salmon Ringer (in mM: NaCl 140; KCI
2.5; NaHCO3 15; CaCl 2 1.5; KH 2PO4 1; MgSO 4 0.8; glucose 10 and HEPES buffer 5; pH 7.8), and tied off at both ends with unwaxed dental floss forming a non-everted sac. This sac was suspended in salmon Ringer aerated with a gas mixture (02: CO2, 95:5%) at 14°C and equilibrated for h. Over the second hour, the gut sac was weighed every ten minutes to the nearest 0.1 mg. Rate of fluid loss from the intestinal sac was determined by regression analysis of the measured sac weights. This rate was normalized by dividing by the surface area of the sac to yield a net rate of fluid movement from mucosal to serosal sides of the intestine, expressed in gl cm - 2h - 1. These methods followed the procedures of Collie and Bern (1982) with modifications by Veillette et al. (1993b).
Na +,K + -A TPase activity Semi-purified enzyme preparations of gill tissue were obtained by using the method of Zaugg (1982), and the determination of Na + ,K+ -ATPase activity was made by modifying the method of Zaugg (1982) as previously described by Bisbal and Specker (1991). Total protein concentrations were determined using the method of Lowry et al. (1951). Absorbance was read at 750 nm using a microplate reader (MR-300, Dynatech Laboratories Inc., Chantilly, VA).
Plasma cortisol levels Cortisol was assayed in unextracted plasma, run in duplicate, using radioimmunoassay (RIA) as described by Young (1986) and validated in our hands (Bisbal and Specker 1991).
Statistical analyses The data were analyzed using two-way analysis of variance, testing for main effects of cortisol and GH and for an interaction (GLM procedure, SAS Inst., Cary, NC). The replicate experiments were analyzed separately. When appropriate, the Tukey
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Fig. 1. Changes in plasma cortisol concentrations (mean SEM, n = 11-12) of Atlantic salmon in the post-smolt stage implanted with cortisol (F) and/or injected with growth hormone (GH) for 5 or 6 days, compared to controls (Con). Upper panel shows experiments conducted in August 1991; lower panel shows experiments conducted in August 1992. In both years, a main effect of cortisol was found (two-way ANOVA); the cortisol-treated salmon had elevated plasma cortisol levels (Tukey HSD, indicated by the horizontal bars).
Fig. 2. Changes in posterior intestinal fluid uptake (J,) (mean + SEM, n = 11-12) of Atlantic salmon in the post-smolt stage implanted with cortisol (F) and/or injected with growth hormone (GH) for 5 or 6 days, compared to controls (Con). Upper panel shows experiments conducted in August 1991; lower panel shows experiments conducted in August 1992. In both years, a main effect of cortisol was found (two-way ANOVA); the cortisol-treated salmon had increased rates of fluid uptake (J,) by the posterior intestine (Tukey HSD, indicated by the horizontal bars).
HSD procedure was used to test for differences among means. Significance was accepted when P < 0.05.
the Tukey analysis indicated that cortisol-treated animals had higher plasma cortisol levels, gill Na+,K+-ATPase activity, and intestinal J, than the animals not receiving cortisol implants (indicated by the common lines above the cortisoltreated groups and the not-cortisol-treated groups, Figs. 1-3).
Results Statistical analyses (two-way ANOVA) of plasma cortisol levels, gill Na + ,K+-ATPase activity, and Jv across the posterior intestine yielded the same results for both experiments: only a main effect of cortisol treatment was found; there was no effect of GH and no interaction (Figs. 1-3). The results of
Discussion Our most significant finding is that exogenous cortisol can stimulate fluid uptake across the posterior
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Fig. 3. Changes in gill Na+,K+-ATPase activity (mean + SEM, n = 11-12) of post-smolt Atlantic salmon implanted with cortisol (F) and/or injected with growth hormone (GH) for 5 or 6 days, compared to controls (Con). Upper panel shows experiments conducted in August 1991; lower panel shows experiments conducted in August 1992. In both years, a main effect of cortisol was found (two-way ANOVA); the cortisol-treated salmon had higher gill Na + ,K + -ATPase activity (Tukey HSD, indicated by the horizontal bars).
intestine of Atlantic salmon. This is the first demonstration of a cortisol-induced change in fluid transport rate in salmonids, adding to the previous findings that cortisol stimulates fluid transport across the eel intestine (Hirano 1967; Hirano and Utida 1968, 1971). Cortisol treatment also induced greater gill Na+,K+-ATPase activity, as demonstrated in a number of earlier studies (see Bisbal and Specker 1991; McCormick et al. 1991). Growth
hormone treatment under these experimental conditions did not affect intestinal fluid transport, gill Na+,K + - ATPase activity, or plasma cortisol concentrations. The cortisol implant used in the present study differs from previously published methods in that cortisol was emulsified in a mixture of partially hydrogenated vegetable shortening (from soybean and palm oil) and vegetable (soybean) oil. The methodology is fully described in Specker et al. (1994). Briefly, it was shown, using serially sampled Atlantic salmon in the post-smolt stage, that plasma cortisol levels at this dose are elevated within 24 h and remain elevated for 28 days. The implant alone causes no elevation in plasma cortisol concentration compared to non-implanted salmon. The vegetable oil: vegetable shortening implant is thus an improvement over those presented in Bisbal and Specker (1991), in which hydrogenated vegetable shortening alone is used, and in Pickering and Duston (1983), in which cocoa butter is used, in that the cortisol implant can be injected at a lower temperature. The plasma cortisol levels achieved by the present method are within a physiological range (cf., Virtanen and Soivo 1985; Barton et al. 1986; Langhorne and Simpson 1986; Bisbal and Specker 1991; Pottinger and Moran 1993). To replace osmotically lost water, teleost fish in seawater are forced to drink and subsequently extract water across the intestine by ion-coupled transport (Skadhauge 1969). In Atlantic salmon, the parr-smolt tranformation is accompanied by an increase in gill Na+,K+-ATPase activity (see McCormick and Saunders 1987) and an increase in intestinal fluid transport across the posterior intestine (Veillette et al. 1993b). These physiological changes are correlated with elevated plasma levels of cortisol and GH (Virtanen and Soivio 1985; Langhorne and Simpson 1986; Bouef et al. 1989). The present study demonstrates that the rate of intestinal fluid absorption in Atlantic salmon is stimulated by cortisol, suggesting that increased plasma cortisol concentrations may be responsible for the pre-adaptive changes of the intestinal mucosa during the parr-smolt transformation of Atlantic salmon. This would parallel the situation in the eel in which cortisol stimulates intestinal sodium and
188 water transport and is probably involved in the developmental adaptation of the intestine when eels migrate to seawater (Hirano 1967; Hirano and Utida 1968; Gaitskell and Chester Jones 1970). Few studies have been conducted on salmonids and the mechanisms behind intestinal fluid transport. However, in coho salmon and Atlantic salmon ouabain is able to reduce fluid uptake by approximately 70°o, indicating that ouabain sensitive Na + ,K + - ATPase activity is responsible for most of the fluid transport (Collie and Bern 1982; Veillette et al. 1993b). In the eel and in killifish, cortisol stimulates Na + ,K+ -ATPase activity (Pickford et al. 1970; Epstein et al. 1971) and the apparent osmotic permeability (Ando 1974) of the intestine. In the rainbow trout, cortisol increases gut Na + ,K + -ATPase activity (Madsen 1990a), whereas, in Atlantic salmon, no effect on Na+,K + ATPase activity was found (Bisbal and Specker 1991). Concurrent electrophysiological experiments performed in our laboratory, using fish from the same tank as used in the present study, showed that a 5-day cortisol implant increased the transepithelial potential (TEP) across the posterior intestine. This increase in TEP was abolished by ouabain (K. Sundell, P.A. Veillette and J.L. Specker, unpublished results). These data, together with the present results, suggest that cortisol stimulates Na+ ,K+ -ATPase activity in the intestinal mucosa of Atlantic salmon, as in trout (Madsen 1990a), and that refinements in the methodology we have used for preparing the gut mucosa are likely to make this apparent. Exogenous GH, either alone or in combination with cortisol, had no effect on plasma cortisol concentration, gill Na+ ,K+ -ATPase activity, or intestinal fluid transport. We expected GH to increase Na + ,K+-ATPase activity in the gills, fluid transport across the intestine, and to act additively with cortisol. Previously, GH had been shown to increase interrenal nuclear diameter and to sensitize the interrenals to ACTH in coho salmon (Higgs et al. 1977; Young 1988). Also, GH in vivo has been shown to increase gill Na + ,K+-ATPase activity in several salmonids (Miwa and Inui 1985; Richman and Zaugg 1987; Bj6rnsson et al. 1987; Boeuf et al. 1992), although GH in vitro does not increase
gill Na+,K+-ATPase activity in coho salmon (McCormick et al. 1991). Furthermore, an additive effect of GH + cortisol treatment in vivo on ionoregulatory ability has been observed for rainbow trout (Oncorhynchus mykiss) and sea trout (Salmo trutta trutta; Madsen 1990b, c). Given the rate of clearance of GH from salmonids (see Sakamoto et al. 1993), our protocol may have been insufficient to observe direct or indirect effects of GH on these osmoregulatory functions, although a similar protocol is sufficient for observing some osmoregulatory effects (see Collie et al. 1989). The sensitivity of tissues to hormones can be seasonal. For example, the sensitivity of gill Na + ,K+-ATPase activity to cortisol, as measured in vitro, and to GH, as measured in vivo, is seasonal (McCormick et al. 1991; Madsen and Bern 1993). Recent evidence from our laboratory shows that the rate of fluid uptake by the posterior intestine is unaffected by cortisol during the peak smolt period (Veilette et al. 1993a). Thus, it is possible that although GH has no effect on the interrenal nor any osmoregulatory action in post-smolt Atlantic salmon in the summer (under these experimental conditions), it may play a role in other phases of development. We conclude that elevated plasma levels of cortisol, achieved by use of cortisol implants, stimulate posterior intestinal fluid uptake and gill Na + ,K+ ATPase activity in post-smolt Atlantic salmon kept in fresh water, and thus that cortisol may be responsible for the pre-adaptive changes in intestinal fluid uptake rate which occur prior to ocean migration. The improved survival in high salinity (37 ppt seawater) and improved plasma Na+ -regulatory ability resulting from cortisol implants in freshwaterphase Atlantic salmon (Bisbal and Specker 1991) may be due to changed intestinal function, in addition to changed branchial function. The duration of these cortisol implants and their longer term effects on growth, survival, and disease resistance are not currently known, but warrant further consideration (see Pickering 1993). We suggest that our non-stressful cortisol implants could be used to facilitate transfer of salmonids to seawater at times of the year, and at developmental stages, outside of the springtime smolt phase.
189 Acknowledgements This publication is the result of research sponsored by NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant #NA89AA-D-SG082 (J.L.S.). The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon. The authors thank Dr. Nathan Collie, Texas Tech University, for his expert advice; Dr. Penny Swanson, National Marine Fisheries Service, for generously supplying salmon GH; Dr. Jerry White, Eastern Washington University, for stimulating discussions; Ms. Rochelle Webster for her expert assistance; and Mr. Kenneth Davignon for graphics.
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