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Am J Physiol Regul Integr Comp Physiol 291: R1499-R1506, 2006. First published June 8, 2006; doi:10.1152/ajpregu.00892.2005
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Parathyroid hormone-related protein regulates intestinal calcium transport in sea bream (Sparus auratus)

Juan Fuentes, Joana Figueiredo, Deborah M. Power, and Adelino V. M. Canário

Centro de Ciências do Mar, CIMAR Laboratório Associado, Universidade do Algarve, Campus de Gambelas, Faro, Portugal

Submitted 20 December 2005 ; accepted in final form 1 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Parathyroid hormone-related protein (PTHrP) is a factor associated with normal development and physiology of the nervous, cardiovascular, immune, reproductive, and musculoskeletal systems in higher vertebrates. It also stimulates whole body calcium uptake in sea bream (Sparus auratus) larvae with an estimated 60% coming from intestinal uptake in seawater. The present study investigated the role of PTHrP in the intestinal calcium transport in the sea bream in vitro. Unidirectional mucosal-to-serosal and serosal-to-mucosal 45Ca fluxes were measured in vitro in duodenum, hindgut, and rectum mounted in Ussing chambers. In symmetric conditions with the same saline, bathing apical and basolateral sides of the preparation addition of piscine PTHrP 1–34 (6 nM) to the serosal surface resulted in an increase in mucosal to serosal calcium fluxes in duodenum and hindgut and a reduction in serosal to mucosal in the rectum, indicating that different mechanisms are responsive to PTHrP along the intestine. In control asymmetric conditions, with serosal normal and mucosal bathed with a saline similar in composition to the intestinal fluid, there was a net increase in calcium uptake in all regions. The addition of 6 nM PTHrP 1–34 increased net calcium uptake two- to threefold in all regions. The stimulatory effect of PTHrP on net intestinal calcium absorption is consistent with a hypercalcemic role for the hormone. The results support the view that PTHrP, alone or in conjunction with recently identified PTH-like peptides, counteracts in vivo the hypocalcemic effects of stanniocalcin.

fish; intestine; hypercalcemia

IN HIGHER VERTEBRATES, calcium homeostasis is controlled largely by the interplay of the hypocalcemic hormone calcitonin, produced in the C cells of the thyroid gland, and the hypercalcemic parathyroid hormone (PTH), produced in the parathyroid gland. The parathyroid gland first appears as an isolated and functional gland in amphibians (6), and its formation is controlled by the transcription factor Gcm-2. In fish, Gcm-2 controls internal gill bud formation (18, 28), and its coexpression with PTH transcripts has led to the suggestion that the parathyroid gland derived from the fish gill (28). However, coexpression of PTH and Gcm-2 has not been confirmed (17).

Early studies (29) suggested that the pituitary gland of fishes contains a hypercalcemic factor related to, but distinct from, PTH. Furthermore, immunohistochemical studies indicated the presence in the pituitary and other tissues of several teleosts of parathyroid hormone-related protein (PTHrP) (7, 20), a factor that shares with PTH hypercalcemic activity and a common receptor (25). The first fish PTHrP gene and cDNA were cloned in the puffer fish, Takifugu rubripes (31), and in the sea bream, Sparus auratus (10), respectively. PTHrP was present at its highest concentration in fish pituitary (36), and the fish NH2-terminal PTHrP 1–34 was shown to have hypercalcemic effects in sea bream larvae, promoting whole body calcium uptake and a decrease in calcium efflux (15). Both observations are consistent with a possible endocrine function for PTHrP.

In higher vertebrates, both PTH and PTHrP bind to PTH receptor 1 (PTH1R) (25), but in teleost fish they also share a receptor designated as PTH3R, encoded by a separate gene and closely related to PTH1R (37, 38). In sea bream, enterocyte PTHrP binds PTH3R, activating the adenylate cyclase/protein kinase A intracellular signaling pathway. In other sea bream tissues that have been studied, PTHrP activates PTH1R only or both receptors (33, 35).

Fish obtain their calcium from two main sources, the diet and the surrounding water. In freshwater species, a large part of the calcium transport takes place through the gills (11) and, at least in some species, partly through the skin (26, 27). In freshwater, virtually all the calcium incorporated via the intestine is of dietary origin, because fish drink very little and freshwater calcium content is also low. Thus, when dietary calcium is low or absent, calcium requirements can be compensated by uptake via the gills from the surrounding medium (19). In contrast, if dietary calcium content becomes high, there is a substantial decrease in branchial calcium uptake (2). In seawater (which contains 10–12 mM calcium), fish ingest large amounts of water to compensate for renal and branchial osmotic water loss. Intestinal intake from seawater has been estimated to be 20% in tilapia (39), 40% in cod, Gadus morhua (40), 70% in the flounder, Paralichthys lethostigma (16), and 90 and 40%, respectively, in juveniles (13) and larvae (14) of sea bream. Whether some of the variability in intestinal calcium intake reflects species or specific experimental differences is not clear. The present study was designed to characterize the calcium transport in the intestinal epithelium in vitro and how it is regulated by PTHrP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peptides and Chemicals

The PTHrP 1–34 from puffer fish was synthesized by Genemed Synthesis (San Francisco, CA). All chemicals were of the highest grade and obtained from Sigma-Aldrich (Madrid, Spain).

Animals

Sea bream (Sparus auratus) were obtained as juveniles from a commercial fish farm (Viveiros Vilanova, Vilanova de Milfontes, Portugal) and kept at the Ramalhete Experimental Marine Station (University of Algarve) for at least 2 wk before experimentation. Fish were held in 1,000-liter tanks (maximal density 7 kg/m3) supplied with well-aerated seawater (salinity 38{per thousand}; water temperature 18–24°C) under natural photoperiod and were fed once a day (10:00–11:00 AM) with 1% body weight commercial dry pellets (Provimi Portugal). Food was withheld at least 48 h before the experiments to ensure emptiness of the intestinal canal.

Intestinal Fluid and Blood Plasma Composition in Sea Bream

The blood plasma and intestinal fluid composition were analyzed (Table 1) to establish a proximate composition for saline to be used in the in vitro experiments. Fish (300–400 g body weight, n = 7) were anesthetized with 2-phenoxyethanol (1:10,000 vol/vol) in seawater, bled, killed by decapitation, and placed ventral side up for dissection. The skin covering the abdomen was carefully cut to expose the intestine, and two mosquito forceps were placed, one immediately after the stomach and the second on the anal sphincter. The isolated intestine was removed, and the intestinal fluid was emptied into a 1.5 ml vial. Blood and intestinal fluid were centrifuged (5 min, 10,000 g), and the supernatants (plasma and fluid free of debris) were frozen in liquid nitrogen and stored at –20°C for ion analysis.


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  		Table 1. Ionic composition and osmolality of plasma and intestinal fluid in unfed sea bream

 
Total calcium, phosphate, chloride, and magnesium in intestinal fluid were measured by colorimetric assay using Sigma-Aldrich procedures no. 587, 360, 461-M, and 596, respectively. Total sodium was measured using sodium-sensitive electrodes (MI-425; Microelectrodes, Bedford, MD) with an Ag-AgCl electrode (MI-409; Microelectrodes) as external reference. All determinations were performed in triplicate, and data are expressed as millimolar concentrations. Osmolality (mosmol/kgH2O) was measured using a Camlab, Roebling microosmometer. The compositions of the blood plasma and intestinal fluid are detailed in Table 1.

In Table 2, basolateral and in vivo-like luminal saline correspond to the ionic composition of sea bream plasma and intestinal fluid composition, respectively. Symmetric experiments were performed using basolateral saline in both the basolateral and apical sides of the Ussing chambers, and asymmetric experiments were performed with basolateral saline in the basolateral side of the preparation and in vivo-like luminal saline in the apical side of the preparation. Glucose (5 mM) was added to the apical and basolateral sides of the preparation to maintain glycolysis.


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  		Table 2. Ionic composition of saline used for in vitro calcium transport in sea bream intestine

 
In Vitro Calcium Transport in Intestinal Regions

Fish were killed by decapitation, and the duodenum, hindgut, and rectum were carefully removed and transferred to freshly prepared and gassed (99:1 O2/CO2) saline. The intestinal portions were defatted, cleaned with fresh saline, and opened longitudinally to produce a flat sheet, the mucosal crypts were separated to create a homogeneous surface of exchange, and the tissue was mounted and oriented (apical and basolateral sides identified) in an Ussing half-chamber. The Ussing chamber (1 cm2 opening) was assembled with the two half-chambers separated by the tissue and 4 ml of saline in each half-chamber. Gas tubing (99:1 O2/CO2) was connected to each half-chamber to provide oxygenation, good mixture by gas lift, and pH control to 7.8. Temperature was maintained between 21 and 22°C throughout the experiments. The preparations were left to stand for 30 min; the saline was replaced with fresh saline and left to mix for 15 min. For measurement of calcium uptake or calcium efflux, 45Ca (0.2 µCi CaCl2; NEN Life Sciences Products) was added to either the apical or basolateral side, respectively, and left to stand for 15 min after addition of radioactivity. Saline (100 µl) from the opposite half-chamber was collected and replaced with 100 µl of fresh saline (time 0) followed by similar procedures at 30-min intervals for the duration of the experiments. Samples (100 µl) were also collected from the 45Ca-labeled saline at time 0 and at the end of the experiments for calculation of specific activities. All radiotracer experiments were performed in open circuit. Quality control of experiments was double checked on the basis of, first, initial velocity and linearity of flux measurements, and second, physical integrity at the end of the experimental manipulation. The volume of one of the half-chambers was removed, and those preparations in which fluid moved from the full to the empty half-chamber were discarded.

Unidirectional fluxes were calculated according to the following equations. For calcium uptake

Formula

where {Delta}[45Ca]Bl represents the increase in radioactivity on the basolateral half-chamber and SAAp represents the apical side specific activity (cpm/nmol). For calcium efflux

Formula

where {Delta}[45Ca]Ap represents the increase in radioactivity on the apical half-chamber and SABl represents the basolateral side specific activity (cpm/nmol). For calcium net flux

Formula

A series of experiments were carried out to characterize in vitro calcium transport in control and PTHrP-treated preparations.

Transport linearity and regional calcium transport. To assess whether the experiments were performed at initial velocity, the time-response relationship in symmetric conditions (using the same saline in both sides of the preparation) in the duodenum, hindgut, and rectum was determined and net calcium transport in the three regions was measured. These experiments were performed for uptake and efflux with fish averaging 300–400 g of body weight.

Dose-response effect of PTHrP in calcium uptake in duodenum. After two consecutive control periods of 30 min were measured, PTHrP was added to the basolateral side of the preparation to give final doses of 5, 25, and 125 ng/ml (1.2, 6, and 30 nM), and calcium uptake was calculated for two additional 30-min periods.

Effect of PTHrP on regional calcium transport. Uptake and efflux in duodenum, hindgut, and rectum were measured in symmetric conditions. These experiments were performed with two consecutive control periods of 30 min and two additional 30-min periods in the presence of basolaterally added PTHrP (6 nM). These series of experiments were performed for uptake and efflux with fish averaging 300–400 g of body weight

Effect of symmetric and asymmetric saline on calcium transport in duodenum, hindgut, and rectum. These experiments were performed with intestinal epithelia from 100- to 150-g fish, maintaining the basolateral saline constant and changing the composition of the apical saline (Table 2).

Bioelectrical Measurements

The intestinal flat sheets from discrete intestinal fragments were collected and prepared as described. Epithelia were lightly stretched to minimize the unstirred boundary layer, pinned over the circular aperture (1 cm2) of the chamber with the perimeter area lightly greased to minimize edge damage, and mounted in a recirculation vertical Ussing chamber (4-ml half-chamber volume). Measurement of bioelectrical variables was performed only in symmetric conditions by using the basolateral saline composition described in Table 2 under voltage clamp to 0 mV. Epithelial resistance Rt ({Omega}·cm2) was automatically calculated from voltage deflections after bipolar 200-ms pulses of 50 µA were injected every minute. Rt and short-circuit current (Isc; µA/cm) were monitored by means of Ag-AgCl electrodes connected to the chambers by 3-mm bore agar bridges (3 M KCl in 3% agar). Clamping of epithelia to 0 mV and recording of variables was performed using a microcomputer-controlled voltage-current Clamp Electronic with automatic correction for fluid resistance and electrode voltage asymmetry (KMSCI, Aachen, Germany).

Statistics

Results are shown as means ± SE unless otherwise stated. After normality and homogeneity of variance were checked, comparisons between groups were performed as appropriate using one-way ANOVA, paired Student’s t-test, or one-way repeated-measures ANOVA followed by the Bonferroni multicomparison test to identify significantly different groups (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Basal Calcium Transport in Intestine

Linearity of calcium transport in symmetric conditions, i.e., absorptive and secretory, as a function of time was demonstrated in the three regions studied: duodenum, hindgut, and rectum (Fig. 1). The preparations that did not comply with the criterion of linearity (i.e., initial velocity) were considered defective and excluded from the data pool (<5% of the preparations).


Figure 1
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  		Fig. 1. Linearity of in vitro intestinal calcium transport in symmetric conditions in 3 different intestinal regions of the sea bream (300–400 g); i.e., duodenum, hindgut, and rectum mounted in 1-cm2-opening Ussing chambers. Results are means ± SE (n number of preparations = 9–12 individuals for each point). Transport from mucosa to serosa (influx) is shown as positive values, whereas transport from serosa to mucosa (efflux) is shown as negative values.

 
PTHrP and Intestinal Calcium Transport

Increasing concentrations of PTHrP applied to the basolateral side of the duodenum preparations resulted in parallel increases in calcium uptake transport (Fig. 2), with statistical significance obtained at 6 and 30 nM PTHrP (P < 0.05, one-way ANOVA). A concentration of 6 nM was chosen for further experiments.


Figure 2
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  		Fig. 2. Dose-response to parathyroid hormone-related protein (PTHrP) of calcium uptake in vitro in duodenum collected from fish with body weight of 300–400 g. Results (means ± SE of 4 fish) are shown as percentages of the control (basal uptake is 18.2 ± 2.01 nmol·cm–2·h–1). *P < 0.05, significant difference from controls (assessed using 1-way repeated-measures ANOVA).

 
Net Intestinal Calcium Fluxes in Response to PTHrP 1–34

In the absence of PTHrP and in symmetric conditions, both the duodenum and the rectum showed positive inside net calcium transport, whereas the rectum showed negative (i.e., outside directed) net calcium transport (Fig. 3). The effect of 6 nM PTHrP 1–34 applied to the basolateral side of the preparation was different according to the intestinal region. It increased the already positive net calcium transport two- to threefold in the duodenum, had no effect on the net calcium transport in the hindgut, and caused a reversion of the outside-directed net calcium flux in the rectum, making it serosal (positive).


Figure 3
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  		Fig. 3. Net calcium transport in isolated duodenum, hindgut, and rectum of the sea bream measured in symmetric conditions. Preparations were assayed in control conditions for 1 h and for an additional hour in the presence of 6 nM PTHrP 1–34 added to the basolateral side. Values are means ± SE (n = 9–12 individuals; body weight, 300–400 g).

 
It should be noted that the application of the same concentration of hormone to the apical side of the preparations had no effect in the three regions (n = 7, data not shown). All further experimentation was carried out with basolateral addition of peptide.

Symmetric vs. Asymmetric Conditions in Intestinal Calcium Transport

In the three regions, duodenum, hindgut, and rectum, there was a significant increase in calcium uptake in response to switching from basolateral to apical medium (Fig. 4), giving a good indication of the importance of the paracellular vs. transcellular transport of calcium. In addition, although some preparations showed a small but significant increase in response to 6 nM PTHrP 1–34 in symmetric conditions (duodenum and hindgut), the maximal stimulation of calcium uptake was obtained in asymmetric conditions in the presence of PTHrP 1–34 in all three regions (Fig. 4, A–C).


Figure 4
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  		Fig. 4. Calcium transport in isolated regions of the sea bream intestine in controls and in response to 6 nM PTHrP 1–34. Influx was measured in duodenum (A), hindgut (B), and rectum (C). Results are shown as means ± SE (n = 10–12; fish body weight, 100–150 g). *P < 0.05, significant difference between symmetric and asymmetric transport conditions (1-way ANOVA). §P < 0.05, significant difference between controls and PTHrP-treated preparations (paired t-test).

 
There was a significant decrease (P < 0.05, one-way ANOVA) in calcium efflux in response to apical vs. basolateral saline in the duodenum, whereas no significant alterations were observed in the hindgut and rectum (Fig. 5). The addition of 6 nM PTHrP 1–34 to the basolateral side of the same preparations resulted in different responses according to the region under study. Whereas there were no significant changes in calcium efflux in the duodenum and hindgut in response to PTHrP (Fig. 5, A and B), calcium efflux was significantly decreased in the rectum (P < 0.05, paired t-test) in both asymmetric and symmetric conditions (Fig. 5C). In symmetric conditions, only the duodenum showed positive net calcium uptake (Fig. 6A), whereas hindgut and rectum showed net calcium efflux (Fig. 6, B and C). In contrast, when the separate regions were assayed in asymmetric conditions, all indicated positive net calcium uptake. The addition of PTHrP to symmetric preparations resulted in higher serosal (positive) calcium net flux in the duodenum, a more negative calcium net flux in the hindgut, and a mucosal, instead of serosal, directed net flux in the rectum. In asymmetric conditions, all three regions substantially increased the inside-directed net calcium flux to different degrees. There was a fourfold increase in net calcium uptake in the duodenum (Fig. 6A), a twofold increase in net calcium uptake in the hindgut (Fig. 6B), and a nearly threefold increase in the rectum (Fig. 6C). The increase in net calcium uptake was achieved in different ways in the different regions. Thus, whereas there was an increase of calcium movement in both directions in the duodenum and hindgut, the increase in net calcium transport in the rectum was achieved by the combination of two processes, i.e., the increase of calcium uptake (Fig. 4) and the decrease of calcium efflux (Fig. 5).


Figure 5
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  		Fig. 5. Calcium transport in isolated regions of the sea bream intestine in controls and in response to 6 nM PTHrP 1–34. Efflux was measured in duodenum (A), hindgut (B), and rectum (C). Results are shown as means ± SE (n = 10–12; fish body weight, 100–150 g). *P < 0.05, significant difference between symmetric and asymmetric transport conditions (1-way ANOVA). §P < 0.05, significant difference between controls and PTHrP-treated preparations (paired t-test).

 

Figure 6
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  		Fig. 6. Net calcium transport in duodenum (A), hindgut (B), and rectum (C) of the sea bream intestine (fish body weight, 100–150 g) in symmetric and asymmetric conditions in controls and in response to 6 nM PTHrP 1–34. *P < 0.05, significant difference between symmetric and asymmetric transport conditions. §P < 0.05, significant difference between controls and PTHrP-treated preparations (1-way ANOVA).

 
When comparing fish of different sizes, in large fish (300–400 g) in the absence of both osmotic gradient and PTHrP, the duodenum and hindgut exhibit positive net calcium uptake (Fig. 3), whereas in smaller fish (100–150 g), the net calcium transport is close to zero in the duodenum and is outside-directed in the hindgut (Fig. 6). This suggests that the relative capacity and/or importance of the intestine for calcium transport may change with size/age.

Bioelectrical Measurements in Sea Bream Intestine

In control conditions, the Isc increases from the duodenum toward the rectum. In contrast, Rt decreases from the duodenum to the rectum (Fig. 7). The basolateral addition of 6 nM PTHrP 1–34 resulted in a heterogeneous response of the Isc according to the region (Fig. 7). There was a significant increase of Isc in response to both 6 and 30 nM PTHrP 1–34 in the duodenum, no change in the hindgut, and a significant decrease in the rectum (P < 0.05, one-way repeated-measures ANOVA). The addition of 30 nM PTHrP 1–34 resulted in small but significant reduction of epithelial resistance in all intestinal regions (Fig. 7).


Figure 7
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  		Fig. 7. Effect of treatment with 6 and 30 nM PTHrP 1–34 on tissue short-circuit current (Isc; top) and epithelial resistance (Rt; bottom) in duodenum, hindgut, and rectum of sea bream (fish body weight, 300–400 g). Each column corresponds to averaged values from 1-h consecutive measurement periods. Results are means ± SE (n = 10–12). Differences between periods were assessed using 1-way repeated-measures ANOVA followed by the Bonferroni multicomparison test. a,bP > 0.05, treatments sharing the same letter are not significantly different.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study establishes for the first time a physiological role for PTHrP in the regulation of fish intestinal calcium transport and is consistent with the recent demonstration of a PTHrP-activated PTH3R receptor in sea bream enterocytes (34). These results imply that PTHrP plays an important role in fish calcium homeostasis by increasing net calcium absorption and making more calcium available for physiological requirements, such as deposition into bone and scales.

The short-term increase in net calcium uptake in the sea bream in an in vitro system is consistent with the in vivo actions of a hypercalcemic factor. These results are also in good agreement with effects of PTHrP in chicks, where 50–200 pM PTHrP 1–40 stimulates calcium transport in the perfused intestine (43). However, the concentration of PTHrP needed to evoke increases in calcium transport in sea bream intestine was one order of magnitude higher than in chicks. Although we do not have a good explanation for this difference, we also observed that circulating levels of sea bream at close to 1 nM (36) are one order of magnitude higher than in terrestrial vertebrates at the low picomolar range (22, 23). Because fish receptors appear to be of similar sensitivity to those described in higher vertebrates (37, 38), the recent finding of multiple PTH and PTHrP peptides in fish (4, 8, 12) could imply a cooperative action between PTH peptides in fish, a possibility that has not as yet been tested.

The regional distribution of calcium transport in fish has been little studied, mainly because of limitations of intestinal size. In mammals the duodenum takes up a large proportion of the calcium ingested, and calcium absorption is tightly controlled, especially during increased calcium requirement (3). In our in vitro study in the sea bream, net calcium absorption occurred along the intestinal tract when different intestinal regions were subjected to in vivo-like conditions, i.e., asymmetric conditions. Nevertheless, in the absence of an osmotic gradient with the same saline bathing both sides of the Ussing chamber, some of the preparations indicated negative net calcium balance. This indicates that the total transepithelial calcium flux is routed via both transcellular and (mainly) paracellular pathways and that, to a certain extent, calcium uptake would rely on high calcium concentrations in the intestinal lumen. However, members of the transient receptor potential vanilloid (TRPV) (30) family, such as CaT1 (TRPV6) and/or ECaC (TRPV5), may mediate apical entry of calcium to drive the increase in transcellular calcium uptake in response to PTHrP; it has been shown that ECaC mRNA is expressed in fish intestine (32).

Sea bream intestinal fluid contains 7.7 mM calcium, resulting from a combination of at least three different processes: calcium absorption, calcium secretion, and water absorption. It is not possible, at this stage, to determine the relative contribution of each process in that little is known about water absorption in this species. Considering that a large amount of fluid (probably in the range of 70–90% of the imbibed water) is absorbed (13) and that water calcium concentration is in the range of 10–12 mM, a substantial amount of calcium is taken up by the intestine. This situation was reflected in vitro, and calcium transport in isolated intestinal regions when analyzed in asymmetric conditions, i.e., in vivo like, always indicated net positive calcium balance.

At concentrations within the higher range found in vivo, 6 nM PTHrP 1–34 was shown to be effective only when added to the serosal side of the tissue, strongly suggesting that its effects are mediated through a receptor localized in the basolateral membrane. The action of PTHrP 1–34 in intestinal calcium transport was region dependent. The peptide increased the movement of calcium in both directions, i.e., mucosal-to-serosal and serosal-to-mucosal, and in both the duodenum and hindgut, still resulting in a net increase in calcium absorption. A similar situation has been described in mammalian intestine in response to vitamin D derivates (21) and in swine intestine in response to stanniocalcin (24). The ion-transporting mechanisms underlying these effects remain unknown. cAMP is known to alter intestinal permeability (9), and PTHrP stimulates the release of cAMP in sea bream enterocytes (34). However, paracellular permeability in the intestine can also be altered by other factors such as phospholipase C, tyrosine kinases, calcium, protein kinase C, and the heterotrimeric G proteins (41). Thus the simultaneous increase of bidirectional calcium flux suggests that PTHrP 1–34 enhances calcium flux across the paracellular route in the duodenum and hindgut.

In contrast to the duodenum and hindgut, the increase in net calcium balance observed in response to PTHrP in the rectum is a combination of both an increase in the mucosal-to-serosal calcium movement and a decrease in the mucosal-to-serosal calcium movement. Furthermore, when rectum preparations were in negative calcium balance, the addition of PTHrP 1–34 reversed the net flux to positive values.

The regional differences in the action of PTHrP 1–34 in calcium transport seem to suggest that although the end result is the same (i.e., more calcium available to the plasma), the mechanisms involved in the PTHrP-dependent calcium transport differ among intestinal regions. This is supported by the differential response to PTHrP in Isc along the intestinal tract. Thus, whereas the duodenum shows a small increase in Isc, the rectal tissue responds in the opposite direction at both doses of PTHrP tested. One possibility is that more than one receptor could be involved in the different regions. At present, there is only evidence for a single PTH receptor (PTH3R) in sea bream duodenum (34), but the hindgut and rectum regions have not been specifically analyzed for the presence of PTH receptors. In addition, or alternatively, the active signaling pathways present along the intestine may be different, triggering a variety of ion transport mechanisms in response to PTHrP. For example, 1) in duodenum enterocytes with PTH3R, PTHrP stimulated adenylate cyclase but not phospholipase C (34); 2) in scales with PTH1R, both enzymes were activated (35); and 3) in interrenal cells with both receptors present, the two signaling pathways were also activated by PTHrP (33).

It is generally accepted that seawater is an endless source of calcium for marine fish. However, a substantial amount of the imbibed calcium from drinking water may be precipitated as calcium carbonate along the intestine, which can function as an efficient route for excreted HCO3– and excess calcium removal from the intestinal fluid (42). Thus it has been shown that experimentally increased calcium in the luminal side of fish intestinal preparations is able to modulate HCO3– secretion (42). In light of this, it would be tempting to speculate on a putative relationship among PTHrP, HCO3– secretion, and calcium processing in the intestine. Thus the region-dependent control of calcium movements by PTHrP in the sea bream intestine would in turn regulate HCO3– secretion to the intestinal fluid.

Assuming that a proportion (possibly variable between species) of the calcium is immobilized in the form of calcium carbonate, the intestine has to be treated as a three-compartment system with regard to calcium, i.e., calcium carbonate precipitates, calcium in solution, and calcium in the epithelia. The proportion of calcium available becomes lower as the fluid advances in the gut. In seawater, the calcium concentration is in the range of 10–12 mM, and in the sea bream, calcium in the intestinal fluid is 7.7 mM. Two processes could account for the reduction of calcium concentration, either its precipitation in the form of calcium carbonate or epithelial uptake. In the European flounder (Platichthys flesus), 30–40% of the calcium imbibed is deposited as carbonate aggregates (42); this proportion remains unknown for the sea bream. However, we have shown that from the fraction of freely available calcium in the intestinal fluid, the intestine is able to extract enough to exhibit serosal (positive) net calcium uptake. In addition, a varying proportion of the calcium imbibed has been demonstrated to be absorbed in the intestine of sea water fish in vivo. Thus, in the sea bream, a substantial amount (in the order of 80–90%) of calcium is absorbed in the intestine (13) as shown from dual labeling with 51Cr-EDTA and 45Ca, and estimates of 20% have been described for the European flounder (P. flesus) (42). Probably in vivo, the basal intestinal calcium uptake and the increase observed in response to PTHrP would certainly be dependent on the fraction of calcium present in the luminal fluid, i.e., calcium in solution.

The action of PTHrP in calcium uptake in the intestinal tract demonstrated in this study is of significance for in vivo calcium metabolism. In a previous report (15) in sea bream larvae, we characterized the whole body effects of NH2-terminal PTHrP and assumed a constant intestinal contribution to the whole body calcium uptake, thus shifting the hypercalcemic effect of PTHrP to the extraintestinal component of calcium uptake. In the present study we have shown that PTHrP increases the intestinal net calcium uptake between two- and fourfold depending on the intestinal region (Fig. 6) when transport is measured in in vivo-like conditions. This indicates that in addition to the branchial component, the intestinal contribution to whole body calcium metabolism is significant and prone to be a target for calcium-regulating hormones, i.e., PTHrP. Despite the demonstrated actions at whole body (15) and intestinal levels (this study), effects of PTHrP in renal calcium transport remain unknown in fish. However, if the effects are consistent with the hypercalcemic effects shown for PTHrP, we would expect actions leading to decreased urinary calcium.

Until recently, the endocrine control of calcium balance in fish was thought to be effected by a hypocalcemic (or antihypercalcemic) factor. The regulatory factor proposed was stanniocalcin, a hormone first discovered and biologically tested in fish and believed to be a fish-specific endocrine factor but now shown to be present in other vertebrates (reviewed in Ref. 5). The current evidence for increased intestinal calcium transport stimulated by PTHrP presented, together with previous data showing a consistent action on whole body calcium fluxes in sea bream larvae (15) and a positive correlation between plasma free calcium and circulating PTHrP levels in sea bream juveniles (1), strongly points toward PTHrP as a hypercalcemic factor controlling extracellular calcium in fish, counteracting the action of stanniocalcin. However, this model requires reappraisal in the light of recent findings of PTH peptides in fish, one of which appears to also have strong calciotropic activity (4).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research has been carried out with the financial support of the Commission of the European Union Quality of Life and Management of Living Resources specific research, technological development, and demonstration program (Q5RS-Q5RS-2001-02904) and FCT, Ministry of Science, Portugal (POCTI/BIA/13174/2002).


   ACKNOWLEDGMENTS
 
We acknowledge Adriana Silva for fish maintenance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Fuentes, Centro de Ciências do Mar, CIMAR Laboratório Associado, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal (e-mail: jfuentes{at}ualg.pt)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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