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Endocrinology Vol. 144, No. 2 559-566
Copyright © 2003 by The Endocrine Society

ARTICLE

Environmental Salinity Regulates Receptor Expression, Cellular Effects, and Circulating Levels of Two Antagonizing Hormones, 1,25-Dihydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3, in Rainbow Trout

Dennis Larsson, Ilka Nemere, Lage Aksnes and Kristina Sundell

Fish Endocrinology Laboratory (D.L., K.S.), Department of Zoology/Zoophysiology, Göteborg University, SE-405 30 Göteborg, Sweden; Department of Nutrition and Food Sciences and the Biotechnology Center (D.L., I.N.), Utah State University, Logan, Utah 84322-8700; and Department of Pediatrics (L.A.), University of Bergen, Haukeland Hospital, N-5021 Bergen, Norway

Address all correspondence and requests for reprints to: Dennis Larsson, Fish Endocrinology Laboratory, Department of Zoology/Zoophysiology, Göteborg University, Box 463, SE-405 30 Göteborg, Sweden. E-mail: dennis.larsson{at}zool.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
In freshwater-adapted rainbow trout, intestinal cells (enterocytes) possess receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the basolateral membrane, and respond to treatment with 1,25(OH)2D3 with increased intracellular calcium concentrations. No receptors are found for the antagonizing hormone 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] at the enterocyte basolateral membrane, and it has no effect on enterocyte calcium homeostasis. After acclimation to seawater, however, the enterocyte membrane receptors for 1,25(OH)2D3 are down-regulated and specific binding for 24,25(OH)2D3 appears, which is further up-regulated with time spent in seawater. This shift in receptor expression is concurrent with an increased sensitivity of the enterocytes to 24,25(OH)2D3 and a decreased sensitivity to 1,25(OH)2D3. This results in a partial inhibition of intracellular calcium uptake, which would be beneficial when inhabiting a calcium-rich environment like seawater.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
TELEOST FISH ACCOUNT for the greatest number of vertebrate species (~23,000) and inhabit marine, brackish, and freshwater (FW) environments. The ion composition of body fluids of marine and FW teleosts differs from that of the environment. A marine fish is constantly at risk of losing water and gaining excess salts, whereas an FW fish risks gaining too much water and losing salts. To maintain homeostasis of body fluids, an intricate pattern of physiological processes in specialized tissues and organs has evolved. The activity and coordination of the specialized tissues involved in ion and water regulation are under hormonal control.

One of the most extensively studied hormone systems in calcium regulation is the vitamin D endocrine system. Of the 37 different metabolites of vitamin D described in vertebrates, most attention has been focused on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] (1, 2, 3, 4, 5). In FW fish, within minutes of administration, 1,25(OH)2D3 increases intestinal calcium transport and activates protein kinase C (6, 7, 8). In the marine Atlantic cod (Gadus morhua), 24,25(OH)2D3 decreases intestinal calcium transport by inhibiting enterocyte calcium uptake through L-type calcium channels (9). However, in the same species, 1,25(OH)2D3 does not affect intestinal calcium uptake (9, 10, 11, 12). Rapid effects of steroid hormones are probably mediated by membrane receptors because there is only a short interval between the steroid treatment and its observed effects (seconds to minutes), and the effects are initiated at the plasma membrane (4, 5, 13, 14, 15). Comparison of specific binding of different vitamin D metabolites to enterocyte basolateral membrane (BLM) from a marine fish (Atlantic cod), an FW fish (carp, Cyprinus carpio), and a terrestrial vertebrate (chicken, Gallus gallus) has demonstrated that marine fish show specific binding only for 24,25(OH)2D3, whereas FW fish and chickens show specific binding for both 1,25(OH)2D3 and 24,25(OH)2D3 (8, 12, 16, 17). Marine fish live in a calcium-rich environment ([Ca2+] {approx} 10 mM), whereas FW fish and terrestrial vertebrates live in calcium-poor environments ([Ca2+] 0 to {approx} 2 mM). The differences in rapid effects and specific binding of 1,25(OH)2D3 and 24,25(OH)2D3 to enterocyte BLM suggest either a divergent evolution of the vitamin D endocrine system or an adaptation of the system to calcium concentrations in the external environments.

This study investigated the balance between the two main antagonizing endocrine regulators of intestinal calcium transport, 1,25(OH)2D3 and 24,25(OH)2D3, in response to environmental salinity. The euryhaline teleost species, the rainbow trout (Onchorhynchus mykiss), adapted to FW and acclimated to seawater (SW) for different lengths of time, was used, and the expression of specific binding proteins for 1,25(OH)2D3 and 24,25(OH)2D3 in enterocyte BLM, the effects of these two metabolites on enterocyte calcium homeostasis, and circulating levels of the two metabolites were analyzed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Experimental animals
Rainbow trout of both sexes (body weight 200–450 g) were purchased from a local hatchery (Antens laxodling AB, Alingss, Sweden). The fish were placed in a holding tank (3 m3) and were acclimated to partly recirculated, filtered, and aerated FW at 12 C for 20 d before the start of the experiments. The fish were fed a commercial diet (Biomare AS, Myre, Norway) ad libitum every other day, in the holding tank as well as in the experimental tanks throughout the experiments. The experiments were performed during spring 1999, and the photoperiod was 12 h of light and 12 h of darkness. At the start of the experiment (d 0) 8 fish were killed and blood plasma and intestines were sampled. Five groups of eight fish were placed in each of five tanks (1.5 m3) supplied with the same FW as the holding tank. Another five groups of eight fish were placed in five separate tanks (1.5 m3) supplied with recirculated, filtered, and aerated SW (25{per thousand}) at 12 C. The photoperiod was kept at 12 h of light and 12 h of darkness for all groups. One group of fish from each water type was sampled at 1, 2, 3, 7, and 30 d after the start of the experiment and was assessed for specific binding of [3H]1,25(OH)2D3 and [3H]24R,25(OH)2D3 to enterocyte BLM and circulating concentrations of 1,25(OH)2D3 and 24R,25(OH)2D3 (see below). Effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on enterocyte calcium uptake were carried out in isolated enterocytes from FW-adapted rainbow trout and rainbow trout acclimated to SW for 30 d (see below).

All experiments and animal care were approved by the Göteborg University ethical committee according to Swedish law.

Chemicals
The 24R,25(OH)2D3 was provided by Kureha Chemical Co. Ltd. (Tokyo, Japan). Pluronic F-127 and 1,25(OH)2D3 were purchased from Calbiochem (La Jolla, CA), and fura-2/AM from Molecular Probes, Inc. (Leiden, The Netherlands). All other chemicals were bought from Sigma (St. Louis, MO) and were of analytical grade.

Preparation of BLM and saturation analyses of [3H]1,25(OH)2D3 and [3H]24,25(OH)2D3 binding to membranes
BLM from fish was prepared by differential centrifugation and sucrose gradient centrifugation as previously described by Nemere et al. (8). The protein concentrations of the different membrane preparations were measured according to Lowry et al. (18).

Saturation analyses of [3H]1,25(OH)2D3 binding were performed in BLM from rainbow trout adapted to FW or acclimated to SW for 1, 3, or 7 d. Membranes from two fish were pooled together in one sample and adjusted to 50 µg protein/200 µl TED buffer (10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4). Triplicate aliquots were incubated (0 C, overnight) with 0.5, 1, 2, or 4 nM [3H]1,25(OH)2D3 in the absence of unlabeled steroid (total binding) or presence of a 200-fold molar excess of unlabeled hormone, also in triplicate, to determine nonspecific binding. Protein-bound radioactivity was precipitated with perchloric acid in the presence of carrier bovine {gamma} globulin (16). The precipitate was collected by centrifugation (10,000 x g), dissolved in 6 M guanidine-HCL, and transferred to vials for liquid scintillation counting.

Saturation analyses of [3H]24,25(OH)2D3 binding were performed in BLM from rainbow trout adapted to FW or acclimated to SW for 1, 2, 3, 7, or 30 d. Membranes from two fish were pooled together in one sample and adjusted to 50 µg protein/200 µl TED buffer. Triplicate aliquots were incubated (0 C, overnight) with 4, 8, 16, or 32 nM [3H]24,25(OH)2D3 in the absence of unlabeled steroid (total binding) or presence of a 200-fold molar excess of unlabeled hormone, in duplicate, to determine nonspecific binding. Protein-bound radioactivity was separated from free radioactivity by the hydroxylapatite technique (12). The hydroxylapatite-bound receptor (with ligand) was pelleted at 1500 x g for 4 min, the supernatant was decanted, and the pellet washed three times with 0.5% Triton X-100 in TED. The pellet was treated with ethanol to extract ligand, supernatant transferred to scintillation vials, ethanol evaporated, and amount of [3H]24,25(OH)2D3 in each sample assessed by liquid scintillation counting.

Enterocyte preparation and measurement of free intracellular Ca2+ concentrations
The method of enterocyte isolation was modified from Larsson et al. (19). The fish were killed by a blow to the head and the proximal two thirds of each intestine was dissected out, everted, and rinsed with 0.9% NaCl. To reduce the possibility of contamination by excitable cells, the everted intestinal segments were ligated in both ends. The ligated intestinal segment from one fish was placed in 96 mM NaCl, 1.5 mM KCl, 8 mM KH2PO4, 5.6 mM Na2HPO4, and 2 mM citrate at pH 7.3 and vigorously shaken for 10 min. Free cells and the remaining mucosal segment were sedimented by centrifugation at 700 x g for 10 min. The pellet was resuspended in 154 mM NaCl, 10 mM Na2HPO4, 1.5 mM EDTA, and 0.5 mM dithiothreitol at pH 7.3 and shaken for another 10 min. Free cells were then sedimented by centrifugation at 700 x g for 10 min; washed twice with 154 mM NaCl and 10 mM Na2HPO4 at pH 7.3; and finally resuspended in 120 mM NaCl, 20 mM HEPES-Tris, 10 mM glucose, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 2 mM CaCl2 at pH 7.3. All preparative and centrifugation steps were performed at 4 C.

Cell viability was determined by trypan blue exclusion in combination with phase contrast microscopy and cell suspensions showing viability greater then 95% were used in the experiments.

The fura-2/AM loading was performed as described by Thomas and Delaville (20). Briefly, freshly dissected enterocytes were incubated for 45 min in Hanks’ balanced salt solution (HBSS; 120 mM NaCl, 20 mM HEPES-Tris, 10 mM glucose, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2 at pH 7.3), with fura-2/AM (5 µM), pluronic F-127 (0.025%), and albumin (0.5%) at 37 C. The cells were washed three times with HBSS by centrifugation at 700 x g for 10 min, and finally resuspended in HBSS.

Measurements of intracellular Ca2+ concentrations ([Ca2+]i) in fura-2/AM loaded rainbow trout intestinal cells (5 x 105 cells ml-1), were performed in a Ratio Master ratio fluorescence spectrometer (model C-44, Photon Technology International Inc., Lawrenceville, NJ) at a 340/380 nm excitation ratio, with an emission wavelength of 510 nm (21). Three milliliters of the cell suspension were placed in a quartz cuvette and stirred slowly at a constant temperature of 10 C. According to the different experimental protocols (described below), stock solutions of the two hormones (10 µl) and Ca2+ (100 µl) were added directly to the cuvette to give the final concentration stated under the appropriate experimental protocol. Ethanol served as vehicle for 1,25(OH)2D3 and 24R,25(OH)2D3. In the control groups, the same type and volume of vehicle were used as for the corresponding stock solution of the treatment substances.

Recordings of [Ca2+]i were performed every second during a time period of 300 sec. Fluorometric calibrations were made by addition of digitonin (100 µg/ml) to lyse the cells and obtain the maximum fluorescence intensity of Ca2+-saturated fura-2, followed by addition of 15 µl 400 mM EGTA/3 M Tris to measure the intensity of Ca2+-free fura-2. [Ca2+]i was calculated according to Grynkiewicz et al. (22) and a dissociation constant (Kd) for fura-2 of 362 nM, at 10 C, was used (21).

The measurements of enterocyte [Ca2+]i were conducted for 2–4 h after the loading of fura-2. Control experiments revealed that the cell viability remained above 95% for at least 4 h after fura-2 loading. Furthermore, the registration of basal [Ca2+]i throughout the experiments served as an internal control because viability tests (trypan blue exclusion and phase contrast microscopy) in combination with fluorospectrophotometry show that an increase in the basal [Ca2+]i are coupled to an increased cell death.

Effects of 1,25(OH)2D3 on enterocyte [Ca2+]i
Enterocytes from either FW or SW adapted rainbow trout were incubated for 300 sec in HBSS. The basal [Ca2+]i was recorded for 150 sec, and then vehicle (n = 7) or 1,25(OH)2D3 (to reach a final concentration of 100 or 500 pM; n = 4 for each concentration tested) was added to the cuvette and the [Ca2+]i was recorded for another 150-sec period. Data obtained were compared for the maximal increase in [Ca2+]i immediately after addition of vehicle or 1,25(OH)2D3, as percent change from the mean basal [Ca2+]i.

Effects of 24R,25(OH)2D3 on enterocyte Ca2+ uptake
Enterocytes from FW- and SW-adapted rainbow trout were acclimated in a Ca2+-free HBSS in the presence of vehicle (n = 7) or 24R,25(OH)2D3 (final concentration of 5 nM or 20 nM; n = 4 for each concentration tested) for 300 sec before the start of the experiment. The basal [Ca2+]i was recorded for 150 sec, then Ca2+ (to reach a final concentration of 10 mM) was added to the cuvette, and the [Ca2+]i was recorded for another 150-sec period. Data obtained were compared for the velocity of calcium entry as percent from the mean basal [Ca2+]i during the first 10 sec after addition of 10 mM calcium.

Plasma levels of 1,25(OH)2D3 and 24,25(OH)2D3
Plasma from eight trout in each treatment group and each sampling point were collected and analyzed for 1,25(OH)2D3 and 24,25(OH)2D3 content according to the method of Larsson et al. (9). Briefly, 1.5 ml plasma was mixed with 2 ml acetonitrile, vortexed, and centrifuged at 10,000 x g for 10 min to remove proteins. The supernatant was collected and 3.5 ml 0.1 M K2HPO4, pH 10.5, was added to the sample. The sample was applied to a C-18 hydroxy vacuum column (pressure of 5 in. Hg; Varian Inc., Middelburg, The Netherlands), washed with 5 ml distilled H2O, followed by a second wash with 5 ml methanol:H2O (70:30, vol/vol), after which the steroids were eluted with hexane:isopropanol (95:5, vol/vol), and the collected samples were evaporated with nitrogen gas. The sample was dissolved in 250 µl hexane:isopropanol:ethanol (95:2.5:2.5, vol/vol), and the vitamin D metabolites were separated on a Supelcosil silica column (15 cm x 4.6 mm, 3 µm; Supelco Inc., Bellefonte, PA) by HPLC. The fractions containing the specific metabolites were evaporated with nitrogen gas. The 1,25(OH)2D3 was quantified by radioreceptorassay, using the 1,25(OH)2D3 receptor from calf thymus (23). The 25(OH)D3 and 24,25(OH)2D3 were quantified by radioreceptorassay, using human vitamin D binding protein from blood plasma (23).

Statistics
Specific 1,25(OH)2D3 and 24R,25(OH)2D3 binding to membranes was tested by nonlinear regression. The concentration of the labeled hormone was plotted against the amount of specifically bound labeled hormone (femtomoles per milligram protein), and the data were fitted to a three-parameter sigmoid equation:

The specific bindings were further examined for allosteric binding by Hill analyses, in which the Hill coefficient (napp) was calculated from the slope in the Hill plot (not shown). In the saturation and Hill analyses, a one-way ANOVA with F-statistics was used to gauge the contribution of the independent variable to predict the dependent variable. The adjusted coefficient of variation (adjR2), was used as a measure of how well the regression models described the data. Values represent mean ± SEM for n = 3–4 experiments. P < 0.05 was considered statistically significant.

F-statistics (24) together with a sequentially rejective Bonferroni test (25) were used to test for differences, evoked by exposure time to SW, in specific binding for 1,25(OH)2D3 or 24R,25(OH)2D3 to BLM in rainbow trout enterocytes. The testing used was two tailed, and the significance level was set at P < 0.01.

A two-way ANOVA followed by a Student-Newman-Keuls post hoc test was used to test for statistical differences on enterocyte [Ca2+]i between administered doses of the 1,25(OH)2D3 in FW- and SW-acclimated trout and significant differences between FW- and SW-acclimated trout for each dose. The testing used was two tailed, and the significance level was set at P < 0.05. Data are presented as mean ± SEM.

Dose-dependent effects on enterocyte [Ca2+]i between administered doses of the 1,25(OH)2D3 in FW-acclimated trout was tested by linear regression. P < 0.05 was considered statistically significant. Scatchard analyses was performed to calculate an EC50 value of the 1,25(OH)2D3-evoked effect on enterocyte [Ca2+]i.

A two-way ANOVA followed by a Student-Newman-Keuls post hoc test was used to test for statistical differences on enterocyte Ca2+ uptake velocities between administrated doses of 24R,25(OH)2D3 in FW- and SW-acclimated trout and significant differences between FW- and SW-acclimated trout for each dose. The testing used was two tailed, and the significance level was set at P < 0.05. Data are presented as mean ± SEM.

Dose-dependent effects on enterocyte Ca2+ uptake velocities between administrated doses of 24R,25(OH)2D3 in FW-acclimated trout was tested by linear regression. P < 0.05 was considered statistically significant. Scatchard analyses was performed to calculate an EC50 value of the 24,25(OH)2D3-evoked effect on the enterocyte Ca2+ uptake velocity.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The specific binding of 1,25(OH)2D3 and 24,25(OH)2D3 to BLM changed with time spent in SW (Figs. 1Go and 2Go). Rainbow trout adapted to FW expressed receptors for 1,25(OH)2D3 (Fig. 1AGo) but not 24,25(OH)2D3 (Fig. 2AGo). Twenty-four hours after transfer to SW, specific binding for 1,25(OH)2D3 was still detectable but displayed a lower affinity for the hormone (Kd increased from 1.3 ± 0.12 to 2.6 ± 1.1 nM; Fig. 1BGo). After 3 and 7 d of exposure to SW, the saturable component of the specific binding for 1,25(OH)2D3 disappeared (Fig. 1Go, C and D). This loss of saturable binding with prolonged acclimation to SW clearly indicated a down-regulation of 1,25(OH)2D3-receptor expression (Fig. 1Go, C and D). No saturable component of the specific binding for 24,25(OH)2D3 was present in FW-adapted rainbow trout (Fig. 2AGo). One day after transfer to SW, specific, saturable binding with high affinity for 24,25(OH)2D3 appeared (Kd = 11.2 ± 4.1 nM; Fig. 2BGo). The high affinity remained and the number of receptors increased further with time in SW (Fig. 2Go, C–F). Thus, exposure of FW-adapted rainbow trout to SW resulted in a clear shift in membrane-bound receptors for the vitamin D3 metabolites so that 1,25(OH)2D3 receptors were down-regulated and 24,25(OH)2D3 receptors up-regulated. These findings support the hypothesis that rapid effects evoked by different metabolites of the vitamin D endocrine system are regulated by the calcium concentration in the external environment (11, 17).



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  		Figure 1. Saturation analyses of [3H]1,25(OH)2D3 binding in BLM from rainbow trout adapted to FW (A) or acclimated to SW for 1 d (B), 3 d (C), or 7 d (D). Membrane aliquots were incubated with 0.5, 1, 2, or 4 nM [3H]1,25(OH)2D3 in the absence of unlabeled steroid (total binding) or presence of a 200-fold molar excess of unlabeled hormone to determine nonspecific binding. Specific [3H]1,25(OH)2D3 bindings to BLM were fitted to a three-parameter sigmoid function and tested by nonlinear regression. Bmax, Maximum binding capacity. Values represent mean ± SEM for three experiments. P < 0.05 was considered statistically significant.

 


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  		Figure 2. Saturation analyses of [3H]24,25(OH)2D3 binding in BLM from rainbow trout adapted to FW (A) or acclimated to SW for 1 d (B), 2 d (C), 3 d (D), 7 d (E), or 30 d (F). Membrane aliquots were incubated with 4, 8, 16, or 32 nM [3H]24,25(OH)2D3 in the absence of unlabeled steroid (total binding) or presence of a 200-fold molar excess of unlabeled hormone to determine nonspecific binding. Specific [3H]24,25(OH)2D3 bindings to BLM were fitted to a three-parameter sigmoid function and tested by nonlinear regression. Bmax, Maximum binding capacity. Values represent mean ± SEM for three to four experiments. P < 0.05 was considered statistically significant.

 
The putative receptor proteins for 1,25(OH)2D3 and 24,25(OH)2D3 binding in plasma membranes, investigated in several animal species as well as tissues, exhibit allosteric binding as a common characteristic (8, 12, 17, 26, 27). In the present study, Hill analyses of the specific binding to BLM from FW- and SW-acclimated trout support these earlier findings; both 1,25(OH)2D3 and 24,25(OH)2D3 showed a positive cooperative binding to their respective membrane receptors (Figs. 1Go and 2Go). Allosteric binding to putative membrane receptors is not unique to 1,25(OH)2D3 and 24,25(OH)2D3. The steroid hormones estradiol and progesterone share this characteristic (4), indicating that allosteric binding is a common feature among steroid membrane receptors.

Enterocytes from FW-adapted rainbow trout, loaded with the fluorescent calcium chelator fura-2, responded to 1,25(OH)2D3 with an immediate increase in intracellular calcium concentrations. The response was dose dependent, and an EC50 of 2.0 nM was calculated by Scatchard analyses (Fig. 3CGo). In contrast, enterocytes isolated from trout acclimated to SW for 30 d showed no response to 1,25(OH)2D3 (Fig. 3Go, A–C). Pretreatment of enterocytes from SW-acclimated trout with 24,25(OH)2D3 resulted in a partial inhibition of enterocyte calcium uptake, when calcium was added to the external medium (Fig. 4Go, B and C). The inhibitory effect of 24,25(OH)2D3 was dose dependent, revealing an EC50 of 0.44 nM (Fig. 4CGo). In rainbow trout adapted to FW, on the other hand, 24,25(OH)2D3 did not inhibit calcium uptake (Fig. 4Go, A and C). These results from FW-adapted trout are in agreement with earlier studies on FW and terrestrial animals, in which 1,25(OH)2D3 triggered increased calcium uptake (2, 4). Furthermore, the response to 24,25(OH)2D3 in enterocytes from rainbow trout adapted to SW is similar to that demonstrated in enterocytes from the marine Atlantic cod (9, 17). The sensitivity of the response to 1,25(OH)2D3 and 24,25(OH)2D3 shifted in accordance with the shift in expression of enterocyte plasma-membrane receptors for the two vitamin D metabolites during exposure to different environmental salinities. This gives support for the hypothesis that rapid effects of the vitamin D system are regulated by the external calcium concentration in the animal’s environment. The physiologically important shift in enterocyte responsiveness and expression of specific membrane receptors could be due to regulation of vitamin D metabolite receptor expression by calcium-sensing receptors in the apical membrane of the enterocytes. Calcium receptors have been demonstrated in enterocytes (28, 29, 30), although investigations on potential interactions between calcium receptors and endocrine factors regulating calcium transport in enterocytes are lacking.



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  		Figure 3. Effects of 1,25(OH)2D3 on changes in [Ca2+]i were investigated in enterocytes from rainbow trout adapted to FW (A and C) and SW (B and C). Rainbow trout enterocytes were isolated by calcium chelation, loaded with fura-2, washed, and resuspended in HBSS. Aliquots of enterocytes (106 ml-1) in HBSS, containing 10 mM calcium, were removed to a cuvette for determination of baseline fluorescence before the addition (t = 150 sec) of ethanol, 100 pM 1,25(OH)2D3 or 500 pM 1,25(OH)2D3. Fluorescence measurements were continued for an additional 150 sec. Data obtained were compared for the maximal increase in [Ca2+]i immediately after addition of vehicle or 1,25(OH)2D3, as percent change from the mean basal [Ca2+]i. A two-way ANOVA followed by a Student-Newman-Keuls post hoc test was used to test for statistical differences between administered doses of the vitamin D metabolite in FW and SW acclimated trout and significant differences between FW and SW acclimated trout for each dose. An EC50 value of the 1,25(OH)2D3 evoked effect on enterocyte [Ca2+]i in FW-adapted trout was calculated by Scatchard analyses (C). Different letters indicate significant differences between groups. Data are expressed as mean ± SEM for four to seven experiments, and the level of significance was set at P < 0.05.

 


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  		Figure 4. Effects of 24R,25(OH)2D3 on calcium entry were investigated in enterocytes from rainbow trout adapted to FW (A and C) and SW (B and C). Rainbow trout enterocytes were isolated by calcium chelation, loaded with fura-2, washed, and resuspended in HBSS. Aliquots of enterocytes (106 ml-1) in calcium-free HBSS were removed to a cuvette for determination of baseline fluorescence (t = 150 sec) with vehicle, 5 nM 24R,25(OH)2D3, or 20 nM 24R,25(OH)2D3 present from the start of the experiment. At t = 150 sec, calcium (final concentration 10 mM) was added to the cells and fluorescence measurements were continued for an additional 150 sec. Data obtained were compared for the velocity of calcium entry as percent from the mean basal [Ca2+]i during the first 10 sec after addition of 10 mM calcium. A two-way ANOVA followed by a Student-Newman-Keuls post hoc test was used to test for statistical differences between administrated doses of the vitamin D metabolite in FW- and SW-acclimated trout and significant differences between FW- and SW-acclimated trout for each dose. An EC50 value of the 24,25(OH)2D3-evoked effect on the enterocyte Ca2+ uptake velocity in SW-acclimated trout was calculated by Scatchard analyses (C). Different letters indicate significant differences between groups. Data are expressed as mean ± SEM for four to seven experiments, and the level of significance was set at P < 0.05.

 
Circulating levels of 1,25(OH)2D3 and 24,25(OH)2D3 also changed during acclimation of rainbow trout from FW to SW (Fig. 5Go). Plasma levels of 1,25(OH)2D3 increased 1 d after transfer to SW but decreased with prolonged exposure (Fig. 5AGo). The 24,25(OH)2D3 levels increased 2 d after transfer to SW and then slowly declined throughout the acclimation period (Fig. 5BGo). After binding to its plasma-membrane receptor, the 1,25(OH)2D3-receptor complex has been shown to be internalized, and an increased number of receptor complexes are found in the nucleus (31). Binding of the vitamin D metabolite to its membrane receptor thus results in its clearance from the circulation, an occurrence that has been documented for other hormonal systems acting via membrane-bound receptors (32). Consequently, plasma levels of the vitamin D metabolites are the result of the turnover rate, and therefore changes in circulating levels can be due to alterations in the rates of either synthesis or clearance. Even though increased plasma levels of 1,25(OH)2D3 or 24,25(OH)2D3 are generally interpreted as the result of increased synthesis, they could also be explained by a down-regulation of the corresponding receptor and a reduced clearance.



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  		Figure 5. Levels of 1,25(OH)2D3 (A) and 24,25(OH)2D3 (B) in blood plasma from rainbow trout adapted to FW or 1, 2, 3, 7, or 30 d in SW. Plasma from eight trout in each treatment group were collected and analyzed for 1,25(OH)2D3 and 24,25(OH)2D3 content as described in Materials and Methods.

 
Circulating levels of 1,25(OH)2D3 and 24,25(OH)2D3 were low, compared with the calculated Kd values for the membrane receptors of each metabolite. A possible explanation for the observed discrepancies is the presence of allosteric binding of 1,25(OH)2D3 and 24,25(OH)2D3 to enterocyte BLM, as demonstrated in the present and previous investigations (8, 12). One interpretation of these findings is that there may exist different membrane receptor populations for 1,25(OH)2D3 and 24,25(OH)2D3 in enterocytes. It can be several receptor proteins with different origins or the same receptor protein forming a complex with different effector proteins. The measured Kd would then be the sum of the Kd values for the different receptor populations for each metabolite. Thus, subpopulations of membrane receptors with a high affinity for each metabolite are probably present in the enterocytes. Calculated EC50 values for the effects of 1,25(OH)2D3 and 24,25(OH)2D3 on enterocyte calcium homeostasis, were in the same range as the circulating metabolite levels and thus also low, compared with the Kd values for the membrane receptors. This further supports the presence of several receptor populations for the two metabolites.

Earlier studies on the marine Atlantic cod (12) and the SW-acclimated rainbow trout (17) also suggests more than one receptor population for the 24,25(OH)2D3 membrane receptor. In these two teleost species, the isoform 24R,25(OH)2D3 is more effective in inhibiting enterocyte calcium uptake, whereas 24S,25(OH)2D3 is more effective in triggering an intracellular calcium release. Another explanation for a Kd that is higher then the corresponding circulating metabolite level is that intestinal cells can produce 1,25(OH)2D3 and 24,25(OH)2D3 (33, 34, 35). The metabolism of 25(OH)D3 to 24,25(OH)2D3 has been suggested to be a catabolic step to decrease the production of 1,25(OH)2D3 and thereby decreasing circulating calcium concentrations. However, in light of the observations that 24,25(OH)2D3 regulates enterocyte calcium entry (9, 12) and intestinal calcium absorption (10, 11), the metabolization of 24,25(OH)2D3 by intestinal cells rather suggest a paracrine function. A local production of 24,25(OH)2D3 would result in not only a decreased intestinal calcium uptake because of decreased levels of 1,25(OH)2D3 but also a decreased intestinal calcium uptake because of active inhibition by 24,25(OH)2D3. Both mechanisms would aim at decreasing circulating calcium concentrations.

In conclusion, the findings of this study enable us to put forward a plausible model of the regulatory events occurring in the vitamin D endocrine system during acclimation from FW to SW. Increased salinity, and thus increased calcium concentrations in the environment, could be sensed by a calcium receptor (36), resulting in a down-regulation of 1,25(OH)2D3 membrane receptors (Fig. 1Go, A–D) and a lower clearance rate of the hormone. This would result in a transient increase in circulating levels of 1,25(OH)2D3 (Fig. 5AGo), which in turn would provide negative feed-back on the 1-{alpha}-hydroxylase activity (37), leading to a sustained decrease in 1,25(OH)2D3 synthesis. This would explain the reduced circulating levels of 1,25(OH)2D3 seen at 2, 3, 7, and 30 d after transfer to SW (Fig. 5AGo). For 24,25(OH)2D3, the pattern would be reversed. Increased environmental salinity results in increased expression of 24,25(OH)2D3 membrane receptors, within 1 d (Fig. 2BGo), probably corresponding to the concurrent small decrease in circulating 24,25(OH)2D3 levels (Fig. 5BGo). After 48 h, plasma levels of 24,25(OH)2D3 increases (Fig. 5AGo). This probably indicates an increased synthesis of 24,25(OH)2D3 because of stimulation of the 24-hydroxylase activity by 1,25(OH)2D3 (38). Thereafter, circulating levels of 24,25(OH)2D3 decline slowly, in parallel with the gradual increase in 24,25(OH)2D3 receptor density seen during prolonged SW exposure (Fig. 2Go, C–F). This proposed model of regulatory events suggests a transition of the vitamin D endocrine system from the role of promoting intestinal calcium uptake in FW (governed by the metabolite 1,25(OH)2D3) to the role of inhibiting intestinal calcium uptake in SW (governed instead by 24,25(OH)2D3). The changes in the vitamin D endocrine system and its effects on intestinal calcium uptake take place both during FW and SW adaptation. The end result is to keep plasma and intracellular calcium concentrations constant. The physiological benefit of this adaptive system is that the animal can quickly regulate its net uptake of calcium independent of the calcium load in the environment.


   Acknowledgments
 
[3H]24R,25(OH)2D3 and 24R,25(OH)2D3 were generously supplied by Kureha Chemical Co. Ltd. (Tokyo, Japan).


   Footnotes
 
This work was supported by the Göteborgs Universitets Jubileumsfond (to K.S. and D.L.), Helge Ax:son Johnsons Foundation (to D.L.), C. F. Lundströms Foundation Stockholm (to D.L.), Swedish Foundation for International Cooperatation in Research and Higher Education 99/820 (to D.L.), National Research Initiative Competitive Grants Program/USDA (to I.N.), and Utah Agricultural Experiment Station (to I.N.), approved as paper no. 7464.

Current address for D.L.: Utah State University, Department of Nutrition and Food Sciences, Logan, Utah 84322-8700. E-mail: dennis.larsson{at}zool.gu.se or larsson{at}cc.usu.edu.

Abbreviations: adjR2, Adjusted coefficient of variation; BLM, basolateral membrane; FW, freshwater; HBSS, Hanks’ balanced salt solution; Kd, dissociation constant; napp, Hill coefficient; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 24,25(OH)2D3, 24,25-dihydroxyvitamin D3; SW, seawater; TED buffer, Tris, EDTA, and dithiothreitol.

Received July 29, 2002.

Accepted for publication October 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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