1,25-Dihydroxyvitamin D3 in the Atlantic cod: plasma levels, a plasma binding component, and organ distribution of a high affinity receptor.

Physiological studies of the Atlantic cod, Gadus morhua, have suggested a role for the vitamin D3 system in this marine teleost similar to that in other vertebrates. Accordingly, the present study was carried out to assess the plasma concentrations of vitamin D3, 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in this fish. Additionally, the presence of binding proteins in plasma and target-specific tissue receptors for these vitamin D3 metabolites was studied in organs normally associated with calcium regulation. Plasma levels of 25-OHD3 (undetectable to 148 pg/ml; n = 5) were comparatively low (20-30 ng/ml), whereas the levels of vitamin D3 (approximately 30 ng/ml) and 1,25-(OH)2D3 (approximately 50 pg/ml) were comparable to levels reported in higher vertebrates. Cod plasma contained a protein that bound both 25OHD3 and 1,25-(OH)2D3. This plasma binding protein revealed low affinity for 25OHD3, did not bind G-actin, and had a sedimentation coefficient of 3.4S. High affinity 1,25-(OH)2D3 receptors [Kd, 1.02 +/- 0.36 (n = 6), 1.02 +/- 0.3 (n = 5), and 0.95 +/- 0.51 (n = 5) nM; mean +/- SEM] were found in high salt cytosols from intestine, liver, and gills, respectively, and had sedimentation coefficients (3.6-3.8S in 0.3 M KCl sucrose gradients) similar to those in higher vertebrates. No specific 1,25-(OH)2D3 binding was found in kidney, ultimobranchial glands, corpuscles of Stannius, or bone. The finding of significant quantities of 1,25-(OH)2D3 in the plasma, the presence of plasma binding proteins that bind this seco-steroid, and the localization of specific high affinity receptors for this metabolite in calcium regulatory tissues in teleosts are all consistent with a physiological role for the vitamin D3 system in the calcium regulation of the cod.

17 beta-estradiol increases plasma calcitonin levels in salmonid fish.

The functional role of calcitonin in teleost fish is in question. Data on the role of calcitonin in calcium regulation are inconsistent, and while a participation in some aspects of sexual maturation has been strongly indicated, the exact function is not known. To establish if there exists a functional relationship between 17 beta-estradiol (E2) and calcitonin in salmonid species, rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and coho salmon (Oncorhynchus kisutch) were injected ip with a single or repeated doses of E2. It is concluded that E2 treatment increases plasma calcitonin levels directly or indirectly, and that it is possible that E2 is responsible for the rise in calcitonin levels during late sexual maturation of female salmonids. In accord with earlier studies, no correlation was found between changes in calcitonin levels and free plasma calcium levels. It seems clear that changes in free plasma calcium levels are not the primary cause of the plasma calcitonin changes in teleost fish. It is possible that calcitonin is involved in mobilizing calcium or directing its mobilization by protection of certain calcium pools during vitellogenesis. However, the increase in calcitonin occurs so close to ovulation that a reproductive role other than a calcium regulatory one is likely. The possibility of transfer of calcitonin itself to the developing oocytes and a subsequent role in embryonic development must also be considered.

Growth hormone increases predation exposure of rainbow trout.

The energetic state of an animal strongly influences decisions that balances feeding against predation risk. Growth hormone increases the metabolic demands, which should elevate the feeding motivation of an animal. This, in turn, may increase the willingness to risk exposure to predators during feeding. To test this hypothesis, we studied the effect of growth hormone on the behavioural response of rainbow trout (Oncorhynchus mykiss) to simulated attacks from a model heron. After attacks, growth hormone treated trout foraged closer to the water surface, resumed feeding earlier, and ate more food than did control trout. Such behaviour should increase the susceptibility to aerial predation. Thus, predation may select against high endogenous growth hormone secretion in wild fish. Furthermore, genetic manipulations to increase growth hormone levels, intended to improve growth performance in aquaculture, may result in individuals with substantially altered behavioural patterns. In light of the increasing potential for interactions between farmed and wild fish, growth hormone transgenic fish may pose a threat to wild fish populations.

Regulation of cartilage glycosaminoglycan synthesis in the rainbow trout, Oncorhynchus mykiss, by 3,3',5-tri-iodo-L-thyronine and IGF-I.

The actions of 3,3',5-tri-iodo-L-thyronine (T3) and recombinant human IGF-I (rhIGF-I) as well as their interaction on cartilage growth in rainbow trout (Oncorhynchus mykiss) were examined. Uptake of 3H-methyl thymidine and 35S-sulfate by isolated branchial cartilage was measured as a marker for chondrocyte proliferation and sulfated glycosaminoglycan synthesis respectively. When T3 (1.0 microgram/g) was injected intraperitoneally, plasma T3 levels reached a transient peak after 1 day and decreased rapidly thereafter. Sulfate and thymidine uptake were not affected by T3 at 1 and 3 days post-injection, but at 6 days post-injection both were significantly higher in T3-injected fish than those in controls. The stimulatory effects of a T3 injection on sulfate and thymidine uptake were dose-dependent over the range of 0.01, 0.1 and 1.0 micrograms/g. In vitro exposure of cartilage to T3 (0.075, 0.75, 7.5, 75 and 750 nM) for 6 days resulted in dose-dependent stimulation of sulfate uptake, with a maximum response at 7.5 nM and higher. T3 exposure (7.5 nM) for 2 or 3 days also increased sulfate uptake, but only slightly. Thymidine uptake was not clearly affected by T3. In vitro addition of rhIGF-I (0.075, 0.75 and 7.5 nM) increased sulfate uptake, but not thymidine uptake, dose-dependently. Compared with T3, rhIGF-I induced a greater maximum level of sulfate uptake: at 7.5 nM rhIGF-I increased the uptake 17-fold whereas T3 increased the uptake fourfold. When T3 (0.075, 0.75 or 7.5 nM) and rhIGF-I (0.1 or 1.0 nM) were added together, stimulative actions of T3 on sulfate uptake were largely additive to those of rhIGF-I. The results indicate that T3 as well as IGF-I are important modulators of sulfated glycosaminoglycan synthesis in rainbow trout cartilage.

The involvement of calcitonin in the reproductive physiology of the rainbow trout.

Plasma from rainbow trout (Salmo gairdneri) of both sexes was sampled every month throughout an annual reproductive cycle in order to elucidate possible relationships between plasma calcitonin and free and protein-bound calcium and magnesium. This was then studied in greater detail around the time of ovulation in the female fish. The plasma levels of the parameters studied were stable in males during the whole annual cycle and were similar to the levels found in females during at least 6 months of the cycle. Plasma levels of protein-bound calcium and magnesium as well as calcitonin were raised in the females for 6, 4 and 3 months respectively. These increases occurred concomitantly during the months before and after ovulation, but no correlations between the protein-bound ion and calcitonin levels were found during this period. With the exception of a decrease observed in December, the free plasma levels of calcium and magnesium were stable in both males and females throughout the cycle. This decrease was attributed to the high production rate of the yolk-protein precursor vitellogenin, which binds both calcium and magnesium. In the detailed study on the ovulating females, plasma calcitonin levels were high 4 weeks before, and continued to increase until the time of ovulation, when a sharp decrease towards normal was noted. The free plasma calcium and magnesium levels were not affected, while the protein-bound levels of calcium and magnesium were found to decline towards normal for the duration of the experiment, without any obvious correlation with the time of ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of direct regulation of prolactin cells by estradiol-17 beta in rainbow trout (Oncorhynchus mykiss).

The effects of estradiol-17 beta (E2) implants on plasma prolactin (PRL) concentrations, pituitary PRL content and pituitary PRL mRNA levels were examined in rainbow trout (Oncorhynchus mykiss). Intact immature fish treated with 1 mg estradiol-17 beta did not show significant changes in both PRL mRNA levels and pituitary PRL content after 3 days of treatment. In a similar experiment, no changes were observed in plasma PRL levels followed during 7 days. Similarly, lack of estradiol-17 beta effect on plasma PRL levels and on final PRL pituitary content was observed in ovariectomized female rainbow trout treated during 48 days with 25 mg estradiol-17 beta and in mature male fish over a 3-week treatment period. Localization of estradiol receptor (ER) mRNAs in the pituitary was carried out by Northern blot analysis using a full-length rainbow trout estrogen receptor (rtER) cDNA as a probe. The rostral pars distalis of the pituitary which contained mostly PRL cells showed the lower amount of rtER mRNA when compared to other parts of the pituitary. Moreover, two mRNAs of different size (3.5 and 1.4 kb) were detected in different parts of the pituitary. Further hybridization experiments using probes containing part of the rtER cDNA (E domain or C and D domains) indicated that the small-sized mRNA (1.4 kb) probably encodes a truncated ER protein lacking hormone binding domain or an ER-related protein. Thus, only the 3.56 kb mRNA appeared to be involved in the regulation of pituitary function by estradiol. In situ hybridization analysis allowed a more precise localization of this rtER mRNA in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromogranin A is present in and released by fish endocrine tissue.

Chromogranin A (CgA) is a protein that is present in many mammalian endocrine cells and co-secreted with their resident hormones. We have demonstrated the presence of CgA by immunohistology in the ultimobranchial glands and corpuscles of Stannius of rainbow trout. CgA was also detected by radioimmunoassay in the medium of incubated coho salmon ultimobranchial glands. Our observations demonstrate the presence of CgA in endocrine glands of evolutionarily divergent species. These observations are consistent with the hypothesis that CgA participates in the secretory process of a wide variety of hormones.

Cortisol inhibits glycosaminoglycan synthesis in cultured rainbow trout cartilage.

In vitro actions of corticosteroids (cortisol, 11-deoxycortisol, cortisone, and corticosterone) as well as interaction between cortisol and triiodothyronine (T3) or recombinant human insulin-like growth factor-I (rhIGF-I) on cartilage glycosaminoglycan (GAG) synthesis in rainbow trout (Oncorhynchus mykiss) were examined. Uptake of [35S]sulfate by isolated branchial cartilage was measured as a marker for GAG synthesis. In vitro exposure of cartilage to cortisol at concentrations of 10, 100, and 1000 nM for 6 days dose-dependently inhibited sulfate uptake, while exposure to 0.1 and 1 nM cortisol had no effect. Corticosterone and 11-deoxycortisol at concentrations of 10 and 100 nM inhibited sulfate uptake slightly but not dose-dependently. Cortisone (1, 10, and 100 nM) had no effect. When cortisol (1, 10, and 100 nM) and T3 (0.075 and 0.75 nM) were simultaneously added to the culture, the T3-induced sulfate uptake was dose-dependently reduced by the presence of 10 and 100 nM cortisol. When cortisol (1, 10, and 100 nM) and rhIGF-I (0.1 and 1 nM) were added together, the sulfate uptake induced by 0.1 nM rhIGF-I was only slightly inhibited by 100 nM cortisol, but 1 nM rhIGF-I completely masked the inhibitory effect of cortisol. These data suggest that GAG synthesis in the rainbow trout cartilage is controlled by multiple interactions among stimulative hormones, such as T3 and IGF-I, and inhibitory hormones, such as cortisol.

Plasma calcium and calcitonin in the marine teleost, Gadus morhua.

Total, free and ionic plasma levels of calcium as well as total plasma magnesium and inorganic phosphate levels were studied in the Atlantic cod, Gadus morhua, in relation to salmon calcitonin injections. Plasma ion levels and endogenous levels of calcitonin were studied during environmentally induced hypercalcemia. It is concluded that no apparent relationship between calcitonin and calcium levels was found, and it is implied that calcitonin in fish may have a physiological function not related to blood calcium regulation.

Effects of vitamin D3, 25(OH) vitamin D3, 24,25(OH)2 vitamin D3, and 1,25(OH)2 vitamin D3 on the in vitro intestinal calcium absorption in the marine teleost, Atlantic cod (Gadus morhua).

The role of vitamin D3, 25(OH) vitamin D3, 24,25(OH)2 vitamin D3, and 1,25(OH)2 vitamin D3, in the regulation of calcium absorption across the intestine in the marine teleost, Gadus morhua, was investigated. The intestine was perfused, in vitro, both vascularly and through the intestinal lumen, and the calcium influx was measured using 45Ca. Vitamin D3 and its metabolites were tested in perfusate concentrations of 10 ng.ml-1.25(OH)D3 increased the intestinal calcium uptake by 65%, while 24,25(OH)2D3 decreased it by 36%. Vitamin D3 and 1,25(OH)2D3, on the other hand, did not affect the calcium influx across the intestinal mucosa. This indicates that 25(OH)D3 and 24,25(OH)2D3 may be active regulators of calcium transport across the intestine of Atlantic cod.

Renal and extra-renal excretion of calcium in the marine teleost, Gadus morhua.

The calcium turnover, with particular emphasis on the routes and rates of excretion, was investigated in the Atlantic cod, Gadus morhua. A very rapid internal redistribution of injected 45Ca was demonstrated, with the decline in the plasma concentration conforming to the power function f(t) = 8.83% X t-0.585. The clearance for 45Ca and [3H]inulin were both linearly correlated to urine flow and showed a ratio (Cca/CIn) of 2.6:1. Likewise a linear correlation between the renal and extra-renal excretion of 45Ca could be demonstrated with a ratio of 1:0.47, indicating that the renal route of excretion is of major importance in this species. The renal excretion of calcium was estimated to be 4.2 mumol X kg-1 X h-1 and the total calcium excretion to be 6.2 mumol X kg-1 X h-1. Calculations are presented, suggesting that the balance between intake and excretion of calcium can be explained in terms of growth of the animal, which would allow for an increase in the amount of body calcium of approximately 5 g X yr-1. It is also concluded that because about two-thirds calcium is excreted via the renal route, the kidneys may be the most important site for the regulation of calcium excretion in the cod.

Effects of hypophysectomy on the plasma ionic and osmotic balance in rainbow trout, Salmo gairdneri.

The effects of hypophysectomy on the plasma ionic and osmotic balance in juvenile rainbow trout (Salmo gairdneri) kept in 1/3 sea water for various periods were studied. Hypophysectomy caused a decrease in the plasma levels of ionized calcium and chloride within a week after the operation. At 2-5 weeks after hypophysectomy, the plasma levels of ionized calcium and chloride were still significantly lower in hypophysectomized fish when compared to sham-operated controls. Hypophysectomy had no effects on the plasma levels of sodium, total calcium, total magnesium, inorganic phosphate, plasma proteins, or plasma osmolality. When 1/3 sea water adapted (1-4 weeks), hypophysectomized and sham-operated fish were exposed to fresh water for 2 weeks, the plasma levels of ionized calcium, chloride, sodium, and plasma osmolality were significantly lower in hypophysectomized fish when compared to sham-operated fish, while the plasma levels of total calcium, total magnesium, and proteins were higher. No effects were noted on the plasma levels of potassium and inorganic phosphate. The presented data indicate that the pituitary gland plays a significant role in the regulation of plasma calcium and chloride levels in S. gairdneri, while its role in the regulation of plasma magnesium and sodium is less clear. The pituitary gland seems to play a minimal role in the plasma regulation of inorganic phosphate and potassium.

Growth hormone inhibits growth hormone secretion from the rainbow trout pituitary in vitro.

Growth hormone (GH) secretion in salmonids and other fish is under the control of a number of hypothalamic factors, but negative feed-back regulation by circulating hormones can also be of importance for the regulation of GH secretion. Mammalian studies show that GH has a negative feed-back effect on its own secretion. In order to elucidate if GH levels present a direct ultra-short negative feedback loop at the pituitary level GH secretion was studied in intact pituitaries from 50 g fish in an in vitro perifusion system. Following an initial equilibrium period pituitaries were exposed to five increasing concentrations (1-1,000 ng ml(-1)) of ovine GH (oGH) in 20-min steps, before being returned to a GH-free perifusion. Ovine GH caused a significant dose-dependent inhibition of GH secretion and it is concluded that GH can exert a direct negative feedback control on GH secretion at the pituitary level.

Chum salmon (Oncorhynchus keta) stanniocalcin inhibits in vitro intestinal calcium uptake in Atlantic cod (Gadus morhua).

Chum salmon (oncorhynchus keta) stanniocalcin was purified, partially identified and tested for bioactivity in an assay on the intestinal calcium uptake in a marine teleost (Gadus morhua). Basic ethanol extraction, ion exchange chromatography, gel filtration and reverse-phase high-performance liquid chromatography resulted in the isolation of a homogeneous glycoprotein that appears as a 46-kDa product under nonreducing conditions and as a 23-kDa product under reducing conditions after sodium dodecylsulphate-polyacrylamide gel electrophoresis. The glycoprotein is likely to be a homodimer composed of two subunits of 23 kDa each. Further characterization indicates homology to Australian eel, sockeye salmon, coho salmon and rainbow trout stanniocalcin, and the glycoprotein is thus concluded to be stanniocalcin. Stanniocalcin-like immunoreactivity was demonstrated in the corpuscles of Stannius of the Atlantic cod, with a specific antiserum raised against purified chum salmon stanniocalcin. The physiological importance and the biological activity of chum salmon stanniocalcin was tested by evaluating its effect on intestinal calcium uptake by the Atlantic cod in vitro. The intestine was perfused, both vascularly and through the intestinal lumen, and the calcium mucosa-to-serosa flux was measured using 45Ca2+ as a tracer. Stanniocalcin decreased the intestinal calcium uptake in a dose-related manner by 13.5% and 22.4% at doses of 2.2 and 10.9 nM stanniocalcin, respectively. The results establish the intestine as a target organ for stanniocalcin in marine teleosts.

Estradiol-17ß-induced calcium uptake and resorption in juvenile rainbow trout, Oncorhynchus mykiss.

Juvenile rainbow trout, Oncorhynchus mykiss, were injected with estradiol-17ß (E2) in order to study the source of extra calcium needed during vitellogenesis. E2-treatment increased the calcium uptake from the external medium as well as calcium mobilization from muscle and scale. Judged by the increase in plasma protein-bound calcium levels, the E2-induced increase in calcium uptake is an apparent over-mobilization of calcium, i.e., the calcium uptake of the fish is in excess of what is found bound to plasma proteins. As the calcium excretion and calcium space (calculated from free plasma calcium levels) were unaffected, the excess calcium is suggested to be incorporated into internal calcium stores. This implies that the systems regulating vitellogenesis and calcium balance are integrated on the mechanistic or endocrine level, and that E2 causes calcium mobilization of a magnitude geared to the needs of the sexually maturing female.

Dominance, nutritional state, and growth hormone levels in rainbow trout (Oncorhynchus mykiss).

This study addressed three questions concerning interactions between physiology and dominance in juvenile rainbow trout: (1) the validity of a model predicting a time-dependent effect of fasting on competitive ability (i.e., the ability to obtain contested food items) was tested in a series of dominance trials between fed and progressively more fasted trout, as was (2) the association between fasting and plasma growth hormone levels. (3) The relationship between plasma growth hormone levels and the competitive ability of individual trout was also studied. The main results were as follows: (1) The predictions of the time-dependent model were supported by the fasting-dominance experiment. After 3 days, fasted fish were dominant over fed fish, whereas after 6 and 9 days, the competitive ability of fed and fasted fish was similar. After 12 days, there was a tendency for fed fish to be dominant over their fasted competitors. (2) Sampling of plasma from fed and fasted trout, after 3, 6, 9, and 12 days, demonstrated that plasma growth hormone levels increases in food-deprived rainbow trout after more than 6 days of fasting, which is consistent with previous work. (3) No difference in plasma growth hormone levels was found between paired dominant and subordinate trout. Possible interactions between nutritional state, growth hormone levels, and dominance, and their implications are discussed.

Low temperature limits photoperiod control of smolting in atlantic salmon through endocrine mechanisms.

We have examined the interaction of photoperiod and temperature in regulating the parr-smolt transformation and its endocrine control. Atlantic salmon juveniles were reared at a constant temperature of 10 degrees C or ambient temperature (2 degrees C from January to April followed by seasonal increase) under simulated natural day length. At 10 degrees C, an increase in day length [16 h of light and 8 h of darkness (LD 16:8)] in February accelerated increases in gill Na(+)-K(+)-ATPase activity, whereas fish at ambient temperature did not respond to increased day length. Increases in gill Na(+)-K(+)-ATPase activity under both photoperiods occurred later at ambient temperature than at 10 degrees C. Plasma growth hormone (GH), insulin-like growth factor, and thyroxine increased within 7 days of increased day length at 10 degrees C and remained elevated for 5-9 wk; the same photoperiod treatment at 2 degrees C resulted in much smaller increases of shorter duration. Plasma cortisol increased transiently 3 and 5 wk after LD 16:8 at 10 degrees C and ambient temperature, respectively. Plasma thyroxine was consistently higher at ambient temperature than at 10 degrees C. Plasma triiodothyronine was initially higher at 10 degrees C than at ambient temperature, and there was no response to LD 16:8 under either temperature regimen. There was a strong correlation between gill Na(+)-K(+)-ATPase activity and plasma GH; correlations were weaker with other hormones. The results provide evidence that low temperature limits the physiological response to increased day length and that GH, insulin-like growth factor I, cortisol, and thyroid hormones mediate the environmental control of the parr-smolt transformation.

Dopaminergic innervation of the rainbow trout pituitary and stimulatory effect of dopamine on growth hormone secretion in vitro.

To elucidate which factors regulate growth hormone (GH) secretion in rainbow trout, dopaminergic innervation of the rainbow trout pituitary along with the action of dopamine in vitro, were studied. Brains with attached pituitaries were double-labeled for putative dopaminergic neuronal fibers and somatotropes, using fluorescence immunohistochemistry. A direct dopaminergic innervation to the proximal pars distalis (PPD) with dopaminergic fibers terminating adjacent to somatotropes was demonstrated. Growth hormone secretion from whole pituitaries was measured in perifusate using a homologous GH-RIA. Dopamine (DA; 10(-7)-2x10(-6) g ml(-1)) increased basal GH secretion, with the GH secretion normalizing again after the DA exposure was halted. When pituitaries were pre-treated with somatostatin-14 (SRIF-14; 10(-12)-10(-9) g ml(-1)), before being exposed to different doses of DA, there was an inhibition of GH secretion which was not reversed after treatment of SRIF-14 was halted, unless DA was added. It is concluded that dopamine can function as a GH secretagogue in the rainbow trout pituitary gland.

1,25(OH)2 vitamin D3 increases ionized plasma calcium concentrations in the immature Atlantic cod Gadus morhua.

Intraperitoneal injections of 1,25(OH)2 vitamin D3 (1,25(OH)2D3) produced hypercalcemia in the marine teleost the Atlantic cod (Gadus morhua) with increased plasma concentrations of ionized calcium (CaI) while the total plasma calcium concentrations (CaT) were unaffected. A single injection of 10 micrograms kg bw-1 of 1,25(OH)2D3 increased CaI concentrations from 1.74 +/- 0.03 mM to 1.8 +/- 0.01 mM after 24 hr in Experiment 1 and from 1.67 +/- 0.03 mM to 1.82 +/- 0.06 mM after 72 hr in Experiment 2. This hypercalcemic effect was sustained by daily injections for 5 but not 7 days. Daily injections of a lower dose of 1,25(OH)2D3, 1 microgram kg bw-1, caused hypercalcemia after 5 days (CaI increased from 1.68 +/- 0.01 mM to 1.76 +/- 0.02 mM). Plasma calcium concentrations were not affected by any of the other seco-steroids (vitamin D3, (25(OH)), vitamin D3 (25(OH)D3), or 24,25(OH)2 vitamin D3 (24,25(OH)2D3), except for a decrease in CaT concentrations after 7 daily in injections of 25(OH)D3 (2 micrograms kg bw-1). Plasma phosphate concentrations were not changed by any of the seco-steroids. It is concluded that 1,25(OH)2D3 is hypercalcemic in the marine Atlantic cod.