From Freshwater Research Articles

Altered plasma and pituitary arginine vasotocin and hypothalamic provasotocin expression in flounder ( Platichthys flesus) following hypertonic challenge and distribution of vasotocin receptors within the kidney

Plasma AVT concentration, pituitary AVT content, hypothalamic provasotocin mRNA expression and other osmoregulatory parameters were measured in euryhaline flounder 4, 8, and 24 h after the hypertonic challenge of transfer from fresh water (FW) to seawater (SW). Osmolality and the concentration of major plasma ions, sodium and chloride, were significantly higher in fish transferred to SW by comparison with time matched controls, an effect evident within 4 h. By comparison with time matched controls, pituitary store of AVT was lower while plasma AVT concentration was higher 8 and 24 h after transfer to SW. Higher provasotocin mRNA expression in the hypothalamus was also seen at 4 and 8 h in flounder transferred from FW to SW compared with time matched controls. The lower pituitary store and higher circulating levels imply substantial AVT secretion occurs in the early phase response to this hypertonic challenge. Changes in the regulation of AVT synthesis and secretion appeared quickly following movement to SW, consistent with the rapid osmoregulatory response, including reduced urine production that fish require to accommodate the dehydrative water losses and salt loading on exposure to the new hyperosmotic environment. qPCR measures of whole kidney vasotocin receptor mRNA expression indicated similar levels in SW and FW. Immunohistochemistry for the vasotocin receptor in flounder kidney showed localisation on the afferent and efferent arterioles of the glomerulus and on the capillary bed that extends from the efferent arteriole to the smooth muscle surrounding the collecting duct. Localisation of the vasotocin receptor was comparable in SW and FW fish.

Rapid hyperosmotic coinduction of two tilapia (Oreochromis mossambicus) transcription factors in gill cells.

Gills of euryhaline teleosts are excellent models for studying osmotic-stress adaptation because they directly contact the aquatic environment and are an important effector tissue during osmotic stress. We acclimated tilapia (Oreochromis mossambicus) from fresh water (FW) to seawater (SW); performed suppression subtractive hybridization of gill mRNAs; and identified two transcription factors, osmotic stress transcription factor 1 (OSTF1) and the tilapia homolog of transcription factor II B (TFIIB), that are rapidly and transiently induced during hyperosmotic stress. mRNA levels increase 6-fold for OSTF1 and 4-fold for TFIIB, and they reach maxima 2 h after SW transfer. Protein levels increase 7.5-fold for OSTF1 and 9-fold for TFIIB, and they reach maxima 4 h after SW transfer. Induction of OSTF1 and TFIIB increases gradually with increasing salinity. Induction of OSTF1 and TFIIB is specific for osmotic stress and absent during oxidative stress (1 mM H2O2) or heat shock (+10 degrees C). Bioinformatic analysis of OSTF1 reveals that it is a transcription factor of the TGF-beta-stimulated clone 22/GILZ family. Because some mammalian homologs are strongly induced by glucocorticoids, OSTF1 may represent the molecular link between the SW hormone cortisol and transcriptional regulation of ion transport and cell differentiation in teleost gills. Coinduction of OSTF1 and TFIIB may serve to recruit TFIIB preferentially to OSTF1 target genes during hyperosmotic stress and compensate for reduced rates of transcription resulting from salt-induced chromatin compaction. We conclude that OSTF1 and TFIIB are critical elements of osmosensory signal transduction in euryhaline teleosts that mediate osmotic adaptation by means of transcriptional regulation.

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.

Response to acute changes in salinity of two different muscle type creatine kinase isoforms, from euryhaline teleost (Oreochromis mossambicus) gills

Two CKM isoforms (CKM1 and CKM2) from the gills of tilapia (Oreochromis mossambicus) were obtained after transfer from freshwater (FW) to seawater (SW, 25 ppt). Based on the 5' and 3' RACE, the identity of CKM1 and CKM2 was determined to be 59% in the 5'-untranslated region (5'-UTR) and 41.9% in the 3'-UTR. Using Northern blot hybridization with the CKM1 and CKM2 3'-UTR probes, CKM1 and CKM2 were found to be expressed in muscle, heart and gill. The levels of these two different CK isoforms (CKM1 and CKM2) were shown to be different in FW than after acute SW transfer, showing that CKM isoforms respond to changes in salinity.

Plasma osmolyte concentrations and rectal gland mass of bull sharks Carcharhinus leucas, captured along a salinity gradient

Bull sharks ( Carcharhinus leucas) were captured across a salinity gradient from freshwater (FW) to seawater (SW). Across all salinities, C. leucas were hyperosmotic to the environment. Plasma osmolarity in FW-captured animals (642±7 mosM) was significantly reduced compared to SW-captured animals (1067±21 mosM). In FW animals, sodium, chloride and urea were 208±3, 203±3 and 192±2 mmol l −1, respectively. Plasma sodium, chloride and urea in SW-captured C. leucas were 289±3, 296±6 and 370±10 mmol l −1, respectively. The increase in plasma osmolarity between FW and SW was not linear. Between FW (3 mosM) and 24‰ SW (676 mosM), plasma osmolarity increased by 22% or 0.92% per 1‰ rise in salinity. Between 24‰ and 33‰, plasma osmolarity increased by 33% or 4.7% per 1‰ rise in salinity, largely due to a sharp increase in plasma urea between 28‰ and 33‰. C. leucas moving between FW and SW appear to be faced with three major osmoregulatory challenges, these occur between 0–10‰, 11–20‰ and 21–33‰. A comparison between C. leucas captured in FW and estuarine environments (20–28‰) in the Brisbane River revealed no difference in the mass of rectal glands between these animals. However, a comparison of rectal gland mass between FW animals captured in the Brisbane River and Rio San Juan/Lake Nicaragua showed that animals in the latter system had a significantly smaller rectal gland mass at a given length than animals in the Brisbane River. The physiological challenges and mechanisms required for C. leucas moving between FW and SW, as well as the ecological implications of these data are discussed.

Effect of hypotonic shock on cultured pavement cells from freshwater or seawater rainbow trout gills

The effect of hypotonic shock on cultured pavement gill cells from freshwater (FW)- and seawater (SW)-adapted trout was investigated. Exposure to 2/3rd strength Ringer solution produced an increase in cell volume followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also induced a biphasic increase in cytosolic Ca 2+ with an initial peak followed by a sustained plateau. Absence of external Ca 2+ did not modify cell volume under isotonic conditions, but inhibited RVD after hypotonic shock. [Ca 2+] i response to hypotonicity was also partially inhibited in Ca-free bathing solutions. Similar results were obtained whether using cultured gill cells prepared from FW or SW fishes. When comparing freshly isolated cells with cultured gill cells, a similar Ca 2+ signalling response to hypotonic shock was observed regardless of the presence or absence of Ca 2+ in the solution. In conclusion, gill pavement cells in primary culture are able to regulate cell volume after a cell swelling and express a RVD response associated with an intracellular calcium increase. A similar response to a hypotonic shock was recorded for cultured gill cells collected from FW and SW trout. Finally, we showed that calcium responses were physiologically relevant as comparable results were observed with freshly isolated cells exposed to hypoosmotic shock.

Effect of cortisol on some osmoregulatory parameters of the teleost, Oreochromis niloticus L., after transference from freshwater to seawater

This trial was conducted in order to determine the effects of cortisol on salt water acclimation of tilapia Oreochromis niloticus (L.). Tilapia (n=42) were injected intraperitoneally with cortisol and then were directly transferred from freshwater (FW) to 15‰ salt water (SW). Changes in plasma osmolality, chloride ion concentration (Cl-), plasma level of cortisol and gill Na+, K+-ATPase activity were measured at 6, 12, 24, 48, 72 and 168 hours after transference to 15‰ SW. Plasma osmolality and Cl- increased immediately after transference until 12-24 h. The fish injected with cortisol (F) showed higher plasma levels of cortisol than those from control group (C) that maintained the initial levels during the experiment. Gill Na+, K+-ATPase activity of C fish began to increase at first hours after transference and peak at 48h. The differences between gill Na+, K+-ATPase activity of F and C groups were significant (P<0.05) in FW, which confirm the effect of exogenous cortisol.

Effects of cortisol and prolactin on Na+ and Cl- transport in cultured branchial epithelia from FW rainbow trout.

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of cortisol and prolactin as media supplements. Epithelia were of the double-seeded insert (DSI) type containing both pavement cells (PVCs) and mitochondria-rich cells (MRCs) and were grown in Leibovitz's L15 media on filters allowing exposure to different apical media conditions. Experiments were carried out in two series after 7-9 days symmetrical (L15 apical-L15 basolateral) culture. In both series, 100% L15 was maintained as the basolateral medium throughout and supplemented with physiologically relevant doses of either prolactin (50 ng/ml), cortisol (500 ng/ml), or cortisol + prolactin (500 + 50 ng/ml, respectively). In series 1, epithelia were exposed to progressively diluted apical media (100, 75, 50, 25, 12.5% L15, and FW) at 24-h intervals. The preparations retained integrity [high transepithelial resistance (TER); low ion efflux rates] during this prolonged dilution protocol. Cortisol, or cortisol + prolactin, resulted in a greater TER and reduced ion efflux rates during dilution, likely an effect on junctional permeability of PVCs, but did not promote active Na+ and Cl- uptake from apical FW. In series 2, epithelia were directly exposed to apical FW and ion fluxes measured over the first 6 h. Under these conditions, cortisol or cortisol + prolactin promoted active uptake of both Na+ and Cl- simultaneously from apical FW, probably attributable to actions on the MRCs. However, Na+-K+-ATPase activities were not significantly altered by any of the treatments in either series. Overall, prolactin alone did not appear to promote FW adaptation but exhibited synergism with cortisol. These results provide further support for the cultured DSI epithelium as an in vitro model for ion transport in FW fish.

Development of an enzyme-linked immunosorbent assay for the measurement of plasma growth hormone (GH) levels in channel catfish ( Ictalurus punctatus): assessment of environmental salinity and GH secretogogues on plasma GH levels

We report the development of a sensitive, and specific, competitive, antigen-capture enzyme-linked immunosorbent assay for the measurement of channel catfish ( Ictalurus punctatus) growth hormone (cfGH). The detection limit of the assay (90% binding) was 2.0 ng/ml and the ED 50 value (standard curve range 150–0.59 ng/ml) was 67.3 ng/ml. Recovery of cfGH-spiked plasma samples was determined to be 102%. Dose–response inhibition curves using serially diluted pituitary homogenates and plasma samples consistently showed parallelism with the standard curves using purified cfGH. The GH antibody (rabbit anti-catfish GH) specificity was demonstrated in competitive binding curves employing heterologous hormones and purified channel catfish prolactin (cfPRL). These studies show that there was no significant (0.006%) binding of cfPRL (competitive inhibition of cfGH binding), or heterologous hormones, within the working range of the assay. To physiologically validate the assay, catfish were injected (100 μg/g body weight, 3 injections every 5 days) with either bovine GHRH 1–29-amide or the synthetic hexapeptide GHRP-2 (KP-102: d-Ala- d-β-Nal-Ala-Trp- d-Phe-Lys-NH 2) suspended in corn oil. Following the last injection, half of the animals were sampled for plasma and the remaining transferred from fresh water (FW) to 12 ppt seawater (BW: brackish water). Twenty-four hours after transfer to BW, animals were again sampled for plasma. Plasma GH levels were significantly ( p<0.001) elevated in all the BW groups (control, KP-102, and bGHRH), compared with the FW (fresh water) groups. In addition, plasma GH levels were significantly ( p<0.001) elevated by treatment with either of the GH secretogogues, KP-102 or bGHRH. Our findings demonstrate that two regulatory mechanisms of GH elevation, one which is seen in euryhaline teleosts (salinity-induced GH levels) and another, which has been recently described in teleosts (GHRP-induced GH levels), are present in the stenohaline channel catfish.

Glucocorticoid receptor upregulation during seawater adaptation in a euryhaline teleost, the tilapia ( Oreochromis mossambicus)

Cortisol is an important seawater (SW) osmoregulatory hormone in the Mozambique tilapia ( Oreochromis mossambicus), a highly euryhaline cichlid able to live in environments ranging from fresh water (FW) to salinities well in excess of full-strength seawater. Previous studies indicate that cortisol may promote SW adaptation by increasing gill chloride cell differentiation, Na +/K +-ATPase activity and subsequent excretion of excess salt following seawater acclimation. Despite cortisol’s widely accepted role as a SW-adapting hormone, cortisol receptor regulation during SW acclimation is not well understood. The purpose of these studies was to determine whether the intracellular glucocorticoid receptor (GR) might be regulated in a manner consistent with cortisol’s actions in SW adaptation. Saturation radioligand binding assays were conducted on gill cytoplasm preparations from fish sampled 4 and 24 h and 4 and 14 days after transfer from FW to 2/3 SW or FW (control). Affinity ( K d) of the gill GR remained constant over the timecourse, while numbers of receptors ( B max) in SW fish were significantly elevated compared with controls at 24 h and 4 days after transfer. Plasma osmolality was higher in fish transferred to SW for 24 h, 4 days, and 14 days compared with those animals moved to FW. Plasma cortisol levels and hepatic cortisol binding remained constant between SW and FW fish throughout the timecourse of the salinity challenge. These studies indicate that seawater acclimation is accompanied by a specific upregulation of intracellular GR numbers in gill tissue. The lack of increase in circulating cortisol following SW adaptation may reflect enhancement of clearance of the steroid. It appears that an increase in cortisol receptors, which is closely associated with the rise in blood osmotic pressure that accompanies SW exposure, is an important component of cortisol’s ability to promote SW adaptation in the tilapia.

Reproductive events and associated reduction in the seawater adaptability of brook charr ( Salvelinus fontinalis): evaluation of gill metabolic adjustments

We tested the hypothesis that along with gill Na +K +ATPase activity reduction, branchial energetic metabolism of brook charr ( Salvelinus fontinalis) is altered during sexual maturation. Maturing and sterile fish were transferred from freshwater (FW) to seawater (SW). The activity of gill pyruvate kinase (PK), cytochrome C oxidase (COX), citrate synthase (CS) and lactate dehydrogenase (LDH) was measured at different intervals. Following 1 month in SW, twofold increases in Na +K +ATPase activity were recorded in all groups, while mean metabolic enzyme activities were sharply reduced (COX: 0.051 ± 0.029 to 0.036 ± 0.018; PK: 1.670 ± 0565 to 1.210 ± 0.340 LDH: 2.245 ± 0.690 to 1.642 ± 0.381 μmol mg –1 protein min –1). Interestingly, during this period, no mortality occurred. After 5 months, comparison of Na +K +ATPase:CS and Na +K +ATPase:LDH ratios of mature and sterile fish held either in FW or SW, indicated that the response of Na +K +ATPase largely exceeds the response of the metabolic enzyme apparatus in hyperosmotic conditions. Hence, the reduced iono-osmoregulatory capacity and higher mortality observed in SW-maturing fish during the reproductive season appears to be mainly attributable to Na +K +ATPase activity reduction rather than the alteration of gill metabolic capacity, since no concurrent increase of metabolic enzyme activity with Na +K +ATPase activity occurred.

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout.

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.

Regulation of Drinking Rate in Euryhaline Tilapia Larvae (Oreochromis mossambicus) during Salinity Challenges

Euryhaline tilapia larvae are capable of adapting to environmental salinity changes even when transferred from freshwater (FW) to seawater (SW) or vice versa. In this study, the water balance of developing tilapia larvae (Oreochromis mossambicus) adapted to FW or SW was compared, and the short-term regulation of drinking rate of the larvae during salinity adaptation was also examined. Following development, wet weight and water content of both SW- and FW-adapted larvae increased gradually, while the dry weight of both group larvae showed a slow but significant decline. On the other hand, the drinking rate of SW-adapted larvae was four- to ninefold higher than that of FW-adapted larvae from day 2 to day 5 after hatching. During acute salinity challenges, tilapia larvae reacted profoundly in drinking rate, that is, increased or decreased drinking rate within several hours while facing hypertonic or hypotonic challenges, to maintain their constancy of body fluid. This rapid regulation in water balance upon salinity challenges may be critical for the development and survival of developing larvae.

Medaka ( Oryzias latipes) as a model for hypoosmoregulation of euryhaline fishes

We examined the hypoosmoregulatory ability of a model fish, medaka ( Oryzias latipes), in relation to the gill-chloride cells or mitochondrion-rich (MR) cells, and to cortisol. When the medaka were transferred from freshwater (FW) to 30‰ seawater (SW), muscle water content decreased by 8% after 2 h and normalized within 1 week. Size and density of MR cells in the gill filament increased after 1–2 weeks in SW. Immersion of medaka in FW containing cortisol (10 μg/ml) for 1–2 weeks also doubled the number of MR cells, and abolished the decrease in muscle water content after SW transfer. In SW-adapted medaka, prolactin (PRL; 10 μg/g body weight) injection reduced muscle water content. However, when cortisol (50 μg/g) was injected simultaneously with PRL, cortisol abolished the decrease in muscle water. We concluded that cortisol plays an important role in the SW adaptation of the medaka, whereas PRL may be involved in ion uptake. Medaka seems to be a good model fish useful for the study of osmoregulatory mechanisms in general.

Branchial Na+K+ATPase activity in brook charr (Salvelinus fontinalis): Effect of gonadal development in hypo- and hyperosmotic environments

Changes in gill Na(+)K(+)ATPase activity were examined following the transfer of brook charr (Salvelinus fontinalis) from fresh water (FW) to seawater (SW). Gonadal development was altered at the hatching stage using three doses of ionizing radiation (IR): 6.2, 7.8, and 11.4 Gray (Gy). A non-irradiated control group was also included in the experimental set-up. Following 15 and 19 months of growth in FW, assessment of gill activity in regard to gonadal status (sterile vs. mature) and level of IR exposure was realized by conducting two estuarine challenge tests. A first introduction was performed during June (period of highest osmoregulatory capacities for this species) (summer experiment). A second introduction was conducted during October (period of diminished osmoregulatory capacities) (fall experiment). Gill Na(+)K(+)ATPase activity and water content were measured at different times and two FW control samplings were added in October and January. In the summer experiment (June-December), normal gonadal development of female brook charr was related to reduced gill Na(+)K(+)ATPase activity during the spawning period as compared to sterile fish (4.0+/-1.5 and 7.2+/-1.9 micromole Pi. mg protein(-1). hr(-1)) (P<0.0002). Similar results were not observed in FW conditions, implying that a lack of gonadal growth does not initiate a significant advantage when the osmoregulatory system including the gills are not highly in demand, i.e. in a FW environment. Ionizing radiation exposure of < or =11.4 Gy at the hatching stage had no significant negative or positive effect on Na(+)K(+)ATPase activity either in FW or SW conditions.

Some aspects of osmotic and ionic regulation in Adriatic sturgeon Acipenser naccarii. II: Morpho-physiological adjustments to hyperosmotic environments

Summary A salt water exposure trial with juvenile Adriatic sturgeon, Acipenser naccarii, (5 months old; mean (± SD) weight = 56±28 g) was initiated by direct transfer from freshwater (FW) to brackish water (BW) of medium (310 mOsm.Kg-1= 11 %o) or high salinity (640 mOsm.Kg-1= 23 %o). Survival at 6 weeks and homeostatic regulation of plasma osmolality and ion concentrations demonstrated that A. naccarii of this age and/or size class possess the ability to adapt to hyperosmotic environments. Regulation of the osmotic status of body fluids was associated with differences in the number of mitochondria-rich (MR) cells on the gill lamellae, whereby sturgeon in high salinities exhibited reduced MR cell numbers as compared with those in FW. Measurement of Na+, K+-ATPase activity in crude gill homogenates from the three groups of sturgeon revealed higher activity in sturgeon at salinities of 310 and 640 mOsm.Kg-1 relative to those in FW; significantly higher in the sturgeon at 640 mOsm.Kg-1. There were no differences in H+-ATPase activity amongst the groups, as measured on the same crude homogenates, but there was a significant increase in the ratio of Na+,K+-ATPase to H+-ATPase in the sturgeon in water at 640 mOsm.Kg-1 relative to those in FW. There was a significant negative linear correlation between gill MR cell number and Na+,K+-ATPase activity. Apparently successful adaptation to BW was also indicated by similar low levels of serum Cortisol, and similar rates of resting oxygen consumption, in all groups. Nonetheless, a growth study on triplicate groups of 40 tagged sturgeon in FW or BW at 600 mOsm.Kg-1 (20 %o) revealed that animals in BW grew less well and exhibited less efficient feed conversion. The results indicate that although sturgeon exhibit some morpho-physiological adjustments to hyperosmotic environments and are able thereby to regulate plasma ions and osmolality in BW at 310 and 640 mOsm.Kg“1, they do not grow as well in BW at 600 mOsm.Kg-1 as they do in FW and, in fact, died when disturbed by heavy activity near the tanks.

Effects of environment on tissue norepinephrine concentration in chum salmon (Oncorhynchus keta)

To investigate the effects of environment on tissue norepinephrine (tNE) levels of fish, we examined tNE concentrations in the adult chum salmon (Oncorhynchus keta) from fresh water (FW) and from sea water (SW). Plasma norepinephrine (pNE) and tNE of 15 organs, including the gill, heart, liver, aorta, spleen, stomach, jejunum, colon, brain stem, cerebrum, muscle, bone, sexual organ and kidney (head and body), were determined in river and sea salmon by high performance pressure liquid chromatography with electrochemical detection. tNE concentrations of all organs except the gill of sea salmon were significantly lower in comparison with those in river salmon. The pNE concentration was higher in sea salmon than in river salmon of male but not female. In river salmon, tNE concentrations of 12 organs of females were higher than those of male, whereas in sea salmon, there was no significant difference in tNE concentrations of all organs except the liver between male and female. Plasma osmolality and sodium concentration were higher in sea salmon than in river salmon. These results indicate that tNE concentrations in most organs of salmon in a SW environment are significantly decreased compared with those in a FW environment, and that tNE concentrations are higher in female salmon than in male salmon in a FW environment but not in a SW environment. J. Exp. Zool. 284:107–111, 1999. © 1999 Wiley-Liss, Inc.

Effects of environment on tissue norepinephrine concentration in chum salmon (Oncorhynchus keta)

To investigate the effects of environment on tissue norepinephrine (tNE) levels of fish, we examined tNE concentrations in the adult chum salmon (Oncorhynchus keta) from fresh water (FW) and from sea water (SW). Plasma norepinephrine (pNE) and tNE of 15 organs, including the gill, heart, liver, aorta, spleen, stomach, jejunum, colon, brain stem, cerebrum, muscle, bone, sexual organ and kidney (head and body), were determined in river and sea salmon by high performance pressure liquid chromatography with electrochemical detection. tNE concentrations of all organs except the gill of sea salmon were significantly lower in comparison with those in river salmon. The pNE concentration was higher in sea salmon than in river salmon of male but not female. In river salmon, tNE concentrations of 12 organs of females were higher than those of male, whereas in sea salmon, there was no significant difference in tNE concentrations of all organs except the liver between male and female. Plasma osmolality and sodium concentration were higher in sea salmon than in river salmon. These results indicate that tNE concentrations in most organs of salmon in a SW environment are significantly decreased compared with those in a FW environment, and that tNE concentrations are higher in female salmon than in male salmon in a FW environment but not in a SW environment. J. Exp. Zool. 284:107–111, 1999. © 1999 Wiley-Liss, Inc.

Native Urotensins Influence Cortisol Secretion and Plasma Cortisol Concentration in the Euryhaline Flounder,Platichthys flesus

Adrenocorticotrophic hormone (ACTH) and flounder urotensins I and II (UI and UII) stimulate cortisol secretion of isolated interrenal/head kidney preparations of seawater (SW)-adapted flounder. UI and UII at concentrations of 10−6and 10−7M, respectively, increased cortisol secretion when acting on SW-derived interrenal but did not affect cortisol secretion in tissue derived from freshwater (FW) fish. Combined UI and UII had no synergistic or additive steroidogenic action, but either 10−7M UI or 10−7M UII in combination with ACTH produced a very marked, additive, or synergistic steroidogenic response, most apparent on interrenal derived from FW fish. These results suggest that urotensins enhance the steroidogenic action of ACTH in flounder. In all cases, significant steroidogenesis was apparent within 1 h postperifusion of ligands. In SW-adapted flounder intraarterial infusion of UIIin vivocaused a concentration-dependent increase in plasma cortisol concentration within 1 h after infusion, while after 5 × 10−6M UI infusion a similar trend was evident but this did not achieve statistical significance. The data suggest that the caudal neurosecretory system may control interrenal cortisol secretion, to modulate cortisol secretion independently of the hypophysial axis, perhaps in response to specific stress-induced or osmoregulatory challenge.

Osmoregulation and salinity effects on the expression and activity of Na+,K(+)-ATPase in the gills of European sea bass, Dicentrarchus labrax (L.).

The European sea bass, Dicentrarchus labrax, tolerates salinities ranging from freshwater (FW) to hypersaline conditions. In two experiments, we analysed changes in plasma ions, muscle water content (MWC), gill Na+,K(+)-ATPase activity, and alpha-subunit mRNA expression during the course of acclimation from 15 ppt salt water to FW or high salinity seawater (HSSW). In Experiment 1, fish (6.2 +/- 1.1 g) were acclimated from 15 ppt to either FW, 5, 15, 25, 50, or 60 ppt SW and sampled after 10 days. Gill Na+,K(+)-ATPase activity was stimulated in FW- and in 50 and 60 ppt SW-groups relative to the 15 ppt control group. In Experiment 2, subgroups of fish (89 +/- 7 g) were transferred from 15 ppt SW to FW or 50 ppt SW, and sampled 1, 2, 4, and 10 days later. Plasma osmolality, [Na+] and [Cl-] decreased in the FW-group and increased in the HSSW-group one day after transfer and lasting until day 10. This was accompanied by a pronounced increase in MWC in the FW-group and an insignificant decrease in the HSSW-group. The plasma [Na+]:[Cl-]-ratio increased markedly in the FW-group and decreased slightly in the HSSW-group, suggesting acid-base balance disturbances after transfer. Gill Na+,K(+)-ATPase activity was unchanged in 15 ppt SW but doubled in FW- and HSSW-groups after transfer. In both groups, this was preceded by a 2- to 5-fold elevation of the gill alpha-subunit Na+,K(+)-ATPase mRNA level. Thus increased expression of alpha-subunit mRNA is part of the molecular mechanism of both FW and SW acclimation in sea bass. Gill Na+,K(+)-ATPase Na(+)-, K(+)-, and ouabain-affinity were similar in fish acclimated to FW, 15 ppt, and HSSW, suggesting that identical isoforms of the catalytic subunit of the enzyme are expressed irrespective of salinity.