Endocrine regulation of carbonate precipitate formation in marine fish intestine by stanniocalcin and PTHrP

Marine teleosts sustain an ionic equilibrium with seawater to keep their plasma osmolality within narrow limits. To achieve this, the gills remove excess salts from the body (Evans et al., 2005) in a secretion process that indirectly causes water loss. Therefore, water replacement by drinking becomes essential to sustain ion regulation (Fuentes and Eddy, 1997a). In addition to regulation of the amount of water ingested by endocrine and environmental factors (Fuentes et al., 1996; Fuentes and Eddy, 1997a; Fuentes and Eddy, 1997b; Guerreiro et al., 2004; Guerreiro et al., 2001), the processing of imbibed fluid has a major impact in fish ion regulation. Ingested water is first processed in the oesophagus, where it undergoes selective absorption of NaCl (Hirano and Mayer-Gostan, 1976; Parmelee and Renfro, 1983), which lowers fluid osmolality and is believed to enhance water absorption in the intestine.

Essential to intestinal fluid processing, and thus intestinal water absorption, is the production of luminal divalent carbonate precipitates in marine teleosts (Grosell, 2006; Grosell, 2011; Kurita et al., 2008; Walsh et al., 1991; Wilson et al., 2002). Luminal precipitate formation requires high concentrations of divalent ions and a chemically favourable high pH. The high divalent ion content required for precipitate formation is supplied by the high calcium content of imbibed seawater, which reaches the intestine in a concentration range that varies between 3 and 15 mmol l⁻¹ depending on the species (Fuentes et al., 2006; Genz et al., 2011; Grosell et al., 2001; Wilson et al., 2002). The alkaline pH of the luminal fluid that provides a favourable chemical environment for carbonate precipitation results from high rates of epithelial HCO₃⁻ secretion by the enterocytes (Grosell, 2006; Grosell, 2011)

Mechanisms for epithelial HCO₃⁻ secretion are dependent on luminal Cl⁻ and involve apical Cl⁻/HCO₃⁻ exchangers (Ando and Subramanyan, 1990; Grosell et al., 2005; Wilson et al., 1996), in particular, members of the SLC26 family, that secrete HCO₃⁻ and absorb Cl⁻ (Grosell and Genz, 2006; Grosell et al., 2009b; Kurita et al., 2008). In addition to apical mechanisms, a basolateral Na⁺/HCO₃⁻ co-transporter (NBC), which belongs to the SLC4 gene family, leads to accumulation of cellular HCO₃⁻ to fuel apical secretion (Kurita et al., 2008; Taylor et al., 2010). Furthermore, a bafilomycin-sensitive H⁺ pump participates in the regulation of apical HCO₃⁻ secretion in seawater-adapted rainbow trout and gulf toadfish (Grosell et al., 2009a; Guffey et al., 2011) and is also important for regional specialization of fluid processing in the sea bream intestine (Gregório et al., 2013).

Calcium as well as HCO₃⁻ immobilization in the luminal carbonate precipitates results in reduced fluid osmolality and favours water absorption. Therefore, HCO₃⁻ secretion in marine fish intestine drives not only luminal precipitate formation but also water absorption (Grosell, 2011). In keeping with this suggestion, a recent study in the gulf toadfish demonstrated a role for the HCO₃⁻-sensing soluble adenylyl cyclase (sAC) in intestinal water absorption (Tresguerres et al., 2010), a process also occurring in the sea bream intestine (Carvalho et al., 2012).

The importance of fish intestinal carbonate precipitate production in the ocean carbon cycle has recently been highlighted (Wilson et al., 2009) and brings a new dimension to the ecophysiology of fish and their environment. The endocrine factors that regulate drinking, fluid processing, epithelial HCO₃⁻ secretion and precipitate production require more detailed study. In vitro studies have revealed that the calciotropic hormones STC (stanniocalcin) and PTHrP (parathyroid hormone-related protein) (Fuentes et al., 2010) regulate not only calcium transport but also the epithelial rate of HCO₃⁻ secretion in sea bream. Prolactin (Ferlazzo et al., 2012) and both transmembrane and soluble adenylyl cyclases participate in the regulation of intestinal epithelial HCO₃⁻ secretion (Carvalho et al., 2012), and hypercalcaemic PTHrP regulates drinking rates in vivo in sea bream larvae (Guerreiro et al., 2001) and intestinal calcium transport in vitro (Fuentes et al., 2006). Taken together, the results from in vitro studies support a role for the calciotropic hormones STC and PTHrP in the processing of intestinal fluid in marine fish. Therefore, the objectives of the present study were to (i) establish the relative importance of the calciotropic hormones PTHrP and STC in the regulation of the main molecular mechanisms (SLC26a6, SLC26a3, SLC4a4 and Atp6v1b) involved in epithelial HCO₃⁻ secretion in the intestine of the sea bream Sparus aurata L., and (ii) establish their physiological relevance in intestinal precipitate formation in vivo.

Western blot analysis of sea bream serum with an antibody generated against purified sea bream STC identified a single immunoreactive band (Fig. 1). The apparent size of the immunoreactive band under denaturing reducing conditions is 3 kDa smaller than the purified STC (26 kDa). This apparent disparity in size is probably related to the removal of the 30 amino acid signal peptide in the N-terminus of STC, as previously described for other fish species (Amemiya et al., 2006; Amemiya et al., 2002). Pre-absorption of the sea bream anti-STC sera with a protein extract of isolated Stannius corpuscles ablated the immunoreactive band obtained in western blots of serum and purified STC, and confirms antisera specificity.

STC immunoreaction (Fig. 2) was restricted to the Stannius corpuscles and no immunoreaction was detected in the adjacent kidney (Fig. 2A,D). Moreover, the immunoreaction was restricted to the cell cytoplasm (Fig. 2E). No immunoreaction was obtained when antisera pre-absorbed with extracts of the Stannius corpuscles was used (Fig. 2C,F).

Intraperitoneal injection of PTHrP(1–34) alone, STC antisera alone or a combination of the two treatments induced comparable significant increases in circulating plasma calcium in sea bream juveniles 24 h post-injection (Fig. 3A). Intraperitoneal injection of the PTHrP receptor antagonist PTHrP(7–34) alone or STC alone resulted in absolute plasma calcium levels lower than those of the controls, but the effect was not significant (Fig. 3B). However, intraperitoneal injection of PTHrP(7–34) and STC combined as a single treatment significantly reduced plasma calcium in sea bream juveniles (Fig. 3B).

Intraperitoneal injection of PTHrP(1–34) alone, STC antisera alone or a combination of the two treatments induced comparable significant decreases in the expression of all genes analysed, i.e. the basolateral sodium bicarbonate co-transporter gene SLC4a4, the V-type proton pump β-subunit gene Atp6v1b and the apical anion exchanger genes SLC26a3 and SLC26a6, in the anterior intestine of sea bream juveniles 24 h post-injection (Fig. 4).

Intraperitoneal injection of the PTHrP receptor antagonist PTHrP(7–34) alone or in combination with STC resulted in a significant increase in the expression of SLC4a4, Atp6v1b, SLC26a3 and SLC26a6 in the anterior intestine of sea bream juveniles 24 h post-injection (Fig. 5). However, no significant effects were achieved in response to STC alone, although the general trend was an increase in the expression of all transporters (Fig. 5).

HCO₃⁻ concentration in the intestinal fluid of control fish averaged 45 mequiv l⁻¹ (Fig. 6) and the total carbonate in the precipitate expressed as the average of CaCO₃+MgCO₃ was of the order of 350 nequiv g⁻¹ of body mass (M_b) in control fish. Intraperitoneal injection of PTHrP(1–34) alone resulted in a trend for lower levels of HCO₃⁻ in the intestinal fluid and a significant decrease of total carbonate content in the intestinal precipitate fraction (Fig. 6). In contrast, intraperitoneal injection of the PTHrP receptor antagonist PTHrP(7–34) resulted in a significant increase in HCO₃⁻ in the intestinal fluid and in total carbonate content of the intestinal precipitate (Fig. 6). Additionally, intraperitoneal injection of STC antibodies significantly decreased HCO₃⁻ in the intestinal fluid and total carbonate content of the intestinal precipitate. STC treatment alone was omitted as insufficient purified native sea bream STC was available to treat sea bream juveniles of 20–25 g M_b.

The present study establishes in vivo effects of the calciotropic hormones PTHrP and STC on the mechanisms of carbonate precipitation in the sea bream intestine. This claim is based on the fact that: (1) the hypercalcaemic or hypocalcaemic factors regulate in a reproducible manner key molecular mechanisms involved in the epithelial HCO₃⁻ secretion cascade; (2) the regulatory effect is predictable; and (3) they modify luminal intestinal bicarbonate/carbonate content of both the intestinal fluid and the precipitate fraction.

In a previous study (Fuentes et al., 2010), the in vitro actions of PTHrP(1–34) and STC in the regulation of epithelial HCO₃⁻ secretion in the intestine of the sea bream were revealed. To extend the in vitroresults to an in vivo situation, both PTHrP(1–34) and the receptor antagonist PTHrP(7–34), which has an established bioactivity in sea bream in vivo (Fuentes et al., 2007a), was utilized. The effect of the receptor antagonist PTHrP(7–34) on epithelial HCO₃⁻ secretion in vivo indicates that the effects of PTHrP(1–34) on epithelial HCO₃⁻ secretion in the intestine occur through one of the three previously characterized teleost PTH receptors. The STC receptor(s) sequence(s) remains unidentified, which consequently precludes the design of receptor-specific antagonists. However, a previous study in tilapia (Fenwick et al., 1995) revealed that passive immunization with antibodies against fish STC is a useful tool to uncover the physiological actions of STC in fish calcium physiology. For this reason, specific polyclonal antibodies raised against the purified native sea bream STC (Fuentes et al., 2010) that recognize STC in western blotting (Fig. 1) and immunohistochemistry (Fig. 2) were used in the present study. A significant characteristic of this antibody is its ability to modify plasma calcium levels in the expected direction (increase) within 24 h of administration as a single intraperitoneal injection to sea bream juveniles (Fig. 3). This effect is similar to the action of PTHrP(1–34) when administered alone or in combination with STC antibodies. Together, these results support the idea that manipulation of plasma calcium can be achieved by administering PTHrP to potentiate hypercalcaemia, or by inhibition of the hypocalcaemia-blocking agent by administration of STC antibodies. The notion that PTHrP and STC have counter-effects on plasma calcium regulation was consolidated by the demonstration that the PTH/PTHrP receptor antagonist PTHrP(7–34) or purified sea bream STC administered alone failed to significantly decrease plasma calcium. In contrast, co-administration of PTHrP(7–34) and STC significantly decreased plasma calcium levels in vivo.

The PTHrP/STC endocrine regulatory system(s) provides alternative mechanisms for the control of plasma calcium levels, and probably regulates other putative downstream actions of PTHrP and STC in fish. In this context, PTHrP and STC have opposite effects on the regulation of HCO₃⁻ secretion in the intestine of marine fish in vitro (Fuentes et al., 2010). In this intestinal model, the process of apical HCO₃⁻ secretion relies on luminal Cl⁻, suggesting the involvement of Cl⁻/HCO₃⁻ exchangers (Grosell et al., 2009b; Grosell et al., 2005; Wilson et al., 1996). Amongst the members of the SLC26 family, SLC26a6 and SCL26a3 were targeted in the present study as they exhibit Cl⁻/HCO₃⁻ exchange activity (Mount and Romero, 2004). For example, the mefugu SLC26a6 shows a high capacity for HCO₃⁻ transport when expressed in Xenopus oocytes (Kurita et al., 2008), and its expression is salinity dependent in both mefugu (Kurita et al., 2008) and the sea bream (Gregório et al., 2013). There is a limited body of literature describing the endocrine control of anion exchangers in vertebrates and the present study contributes to this by demonstrating that SLC26a6 and SCL26a3 expression is regulated by different combinations of hypercalcaemic or hypocalcaemic factors in the anterior intestine of the sea bream. Thus, hypercalcaemic factors [e.g. PTHrP(1–34) and passive immunization with STC antisera] down-regulate the expression of both transporters. Further studies will be required to establish why STC administration failed to modify SLC26a6 and SCL26a3 expression but it may be related to the dose of STC given or the already high levels of STC detected in fish plasma (Mayer-Gostan et al., 1992).

A proton pump is present in the marine fish intestine (Gregório et al., 2013; Guffey et al., 2011) and is probably associated with intestinal fluid processing via modulation of epithelial HCO₃⁻ secretion. In the intestine of the gulf toadfish, the proton pump constitutes a functional metabolon with the SLC26a6 anion exchanger, and drives intestinal Cl⁻ uptake against a steep electrochemical gradient (Grosell et al., 2009b) and enables epithelial competence for HCO₃⁻ secretion. In light of this idea, it was unsurprising that the regulation of expression of Atp6v1b (proton pump β-subunit) in the anterior intestine of the sea bream paralleled the response to endocrine regulation described for SLC26a6 (Figs 4, 5).

In fish intestine, SLC4a4 is located in the basolateral membrane of the enterocyte and is highly expressed in seawater fish (Kurita et al., 2008). SLC4a4 is a high capacity HCO₃⁻ supply that drives transcellular secretion at the apical membrane of the enterocyte and is suggested to act as a limiting step of HCO₃⁻ secretion in the intestine of marine fish (Chang et al., 2012; Taylor et al., 2010). The capacity of calcitropic factors to modulate the expression of SLC4a4 in the anterior intestine of the sea bream (Figs 4, 5) provides evidence that reinforces the relationship between epithelial calcium movements and HCO₃⁻ secretion in the intestine of marine fish. However, the present study reveals that the antagonistic action of PTHrP and STC goes beyond calcium regulation and that they also affect key mechanisms of the intestinal HCO₃⁻ secretion cascade. In particular, the calcitropic hormones had a similar effect on the transcript abundance of SLC26a3, SLC26a6, SLC4a4 and Atp6v1b transporters in the sea bream intestine and this contrasts with the ex vivo effect of prolactin (Ferlazzo et al., 2012), where only SLC4a4 transcript abundance was modified in the intestine. The divergence between PTHrP, STC and prolactin action may arise from the experimental approach (in vivo versus ex vivo) or may simply reflect the physiological roles and selectivity of these hormones.

The intestinal molecular response to hypercalcaemic/hypocalcaemic treatment established an important feature; the molecular mechanisms targeted by the two calcitropic hormones in the formation of carbonate precipitates are the same. In light of this result the relative importance, if any, of such regulation in normal physiology could only be accessed in vivo. The intestinal fluid and the precipitate fraction were treated as different compartments, as the fluid acts as an intermediate step between epithelial HCO₃⁻ secretion and the final precipitation in the form of intestinal carbonate precipitates. The precipitated fraction represented the values as total carbonate to account for the magnesium and calcium content of the precipitates (Perry et al., 2011; Woosley et al., 2012). The results revealed parallel endocrine effects (Fig. 6) in the two fractions, clearly demonstrating the endocrine regulation of intestinal carbonate precipitation. Hypercalcaemic factors reduced the amount of carbonate precipitate, e.g. PTHrP(1–34) treatment reduced precipitated carbonates to about 50%, while immunization with STC antibodies reduced precipitates by about 65%. In contrast, hypocalcaemic factors increased precipitate output: the PTH/PTHrP receptor antagonist PTHrP(7–34) induced about a 3-fold increase in the precipitate fraction present in the intestinal lumen.

Fish intestinal carbonate precipitate production has a significant (Wilson et al., 2009) impact in the ocean carbon cycle. Consequently, the characterization of the endogenous endocrine response to the predicted ocean acidification will be essential to model and establish the contribution of fish in a changing environment. The present study demonstrates the predictable effect of the calciotropic hormones STC and PTHrP on the regulation of intestinal mechanisms related to epithelial HCO₃⁻ secretion, and exposes the end result on precipitate formation. Assuming that intestinal water absorption and epithelial HCO₃⁻ are functionally associated (Carvalho et al., 2012; Grosell, 2011; Tresguerres et al., 2010), the endocrine regulation of epithelial HCO₃⁻ secretion may have a central role in this process. The endogenous modulation of carbonate precipitation in the intestine highlights a novel physiological role for calcium-regulating factors and probably impacts the general osmoregulatory process in marine fish, and may have a global impact in the ocean carbon cycle.

In vivo and in vitro PTHrP(1–34) and PTHrP(7–34) bioactivity has previously been reported in fish (Canario et al., 2006; Fuentes et al., 2006; Fuentes et al., 2007a; Fuentes et al., 2007b; Guerreiro et al., 2001; Rotllant et al., 2005). STC was purified from the sea bream Stannius corpuscles and bioactivity has been confirmed using in vitro Ussing-type chambers (Fuentes et al., 2010). All chemicals were of the highest grade and obtained from Sigma-Aldrich unless stated otherwise.

Sea bream (Sparus aurata) juveniles were obtained from commercial sources (CUPIMAR SA, Cádiz, Spain) and maintained in open-seawater circuits under natural conditions of water temperature (18–20°C), photoperiod and salinity (37 ppt) at a density <5 kg m⁻³. For maintenance, fish were fed twice daily to a final ration of 2% of M_b, with a commercial sea bream diet (Trow España SA, Cojóbar, Burgos, Spain). All fish were fasted for 24 h before experimental manipulations.

All animal manipulations were carried out in compliance with the Guidelines of the European Union Council (86/609/EU) and Portuguese legislation for the use of laboratory animals. All animal protocols were performed under a Group-1 licence from the Direcção-Geral de Veterinária, Ministerio da Agricultura, do Desenvolvimento Rural e das Pescas, Portugal.

Antibodies were commercially produced in rabbits (Genemed Synthesis, Sigma-Aldrich, Madrid, Spain) with a triple boost of 1 mg purified native sea bream STC (Fuentes et al., 2010). All antibody procedures were performed with the neat antiserum and no purification attempt was made. For tissue and serum collection, sea bream (N=4, 250 g M_b) were anaesthetized (2-phenoxyethanol 1:10,000; Sigma-Aldrich); blood was obtained by puncture of the caudal peduncle, left to clot on ice and serum was then obtained by centrifugation (10,000 rpm for 5 min); this was stored at −20°C for later use. Stannius corpuscles were dissected out after decapitation of the sea bream, and either fixed in Bouin’ fixative or flash-frozen in liquid N₂ and stored at −20°C for later use.

Stannius corpuscles were homogenized in extraction buffer (240 mmol l⁻¹ Tris; 48% glycerol; 13.5% SDS) and total protein in the extract quantified (Nanodrop 1000, Thermo Scientific, Waltham, MA, USA). Serum samples (2 μl per lane) and purified sea bream STC (800 ng per lane) were separated by SDS-PAGE on 15% polyacrylamide gels under reducing conditions at 100 V and blotted onto enhanced chemiluminescence (ECL) membranes (Hybond-P PVDF ECL, Amersham Biosciences) in a vertical tank transfer system (Mighty Small Hoefer, Amersham Pharmacia) for 1 h at 250 A. The membranes were incubated with blocking solution [3% (w/v) BSA, 0.1% (v/v) Tween 20 in Tris-buffered saline (TBS)] overnight at 4°C. Subsequently, membranes were washed in TBS-T and incubated for 2 h at room temperature with constant agitation with either the diluted STC primary antisera (1:50,000 in TBS-T) or antisera pre-absorbed with an extract of Stannius corpuscles. Antibody pre-absorption was performed overnight at 4°C with an antibody dilution of 1:1000 in TBS-T in the presence of an extract of sea bream Stannius corpuscles (see above) with 1 μg ml⁻¹ total crude protein. After incubation with primary antibody, the excess antibody was removed, and the membranes were washed several times in TBS-T and incubated with a secondary antibody (anti-rabbit IgG-peroxidase conjugate 1:35,000, Sigma-Aldrich) for 1 h at room temperature. Excess secondary antibody was removed and the membranes were washed several times in TBS-T and incubated with streptavidin–horseradish peroxidase conjugate (GE Healthcare, Amersham) diluted 1:50,000 in TBS-T at room temperature for 1 h. Excess streptavidin was removed, and membranes were washed several times in TBS-T and developed using the ECL Prime western blotting reagents kit (GE Healthcare, Amersham) following the manufacturer's protocol.

After fixation with Bouin's fixative, kidney tissue containing the Stannius corpuscles was rinsed in PBS, dehydrated and embedded in wax, and serial sagittal sections (5 μm thickness) were prepared and mounted onto poly-l-lysine-coated slides for subsequent immunohistochemical analysis. Prior to antibody incubation, sections were washed with a solution of PBS (1×), (0.1%) Triton X-100 and 10% sheep serum (Sigma-Aldrich) for 1 h at room temperature. Slides were incubated overnight at 4°C with rabbit polyclonal STC antibody (1:2500 dilution). After washing with PBS (1×, 0.1% Triton X-100) the sections were incubated overnight at 4°C with a secondary antibody (goat anti-rabbit IgG H-R Hyligth-488, 1:400 dilution; AnaSpec, Fremont, CA, USA) and then washed in PBS (1×, 0.1%Triton X-100), and mounted with VectaShield (Vector Labs, Burlingame, CA, USA). Fluorescence microscopy images were captured using a Zeiss Z2 Axioscope fluorescence microscope coupled to a Zeiss HR camera. Preliminary testing was conducted to optimize anti-STC dilution. None of the negative controls (normal host sera, omission of primary antibody) gave a signal.

Unfed sea bream (N=80, body mass 2.5–3.5 g) were anaesthetized in 2-phenoxyethanol (1:10,000; Sigma-Aldrich) and weighed to the nearest 0.1 g.

In the first experiment, fish received a single intraperitoneal injection of pre-immune rabbit antiserum (5 μl g⁻¹), PTHrP(1–34) (0.5 μg g⁻¹), rabbit STC-Ab (5 μl g⁻¹) and a combination of PTHrP(1–34) (0.5 μg g⁻¹) + STC-Ab (5 μl g⁻¹). In the second experiment, fish were injected with 0.9% NaCl (5 μl g⁻¹), PTHrP(7–34) (0.5 μg g⁻¹), purified sea bream STC (5 μg g⁻¹), or a combination of PTHrP(7–34) (0.5 μg g⁻¹) + purified sea bream STC (sbSTC, 5 μg g⁻¹). Final injection volumes were of 5 μl g⁻¹ in all treatments. Dosages for PTHrP(1–34) and PTHrP(7–34) (receptor antagonist) used in the present study were based on those previously used in sea bream juveniles (Fuentes et al., 2007a).

After injection, fish were transferred to 60 l tanks with flowing seawater for recovery. Twenty-four hours after injection, fish were terminally anaesthetized with 2-phenoxyethanol (1:1000) and blood samples were collected into heparinized glass capillaries from the caudal peduncle after severing the tail. Plasma was obtained by centrifugation of whole blood (10,000 rpm for 5 min), and stored at −20°C for later analysis.

After decapitation of fish, samples from the anterior intestine, which corresponds to a section 1–1.5 cm in length caudal to the point of insertion of the pyloric caeca, were collected from individual fish and incubated overnight in RNALater (Ambion, Austin, TX, USA) at 4°C and stored at −20°C until utilized for RNA extraction (within 2 weeks).

Plasma calcium levels were determined in duplicate using a commercial kit (Spinreact, Girona, Spain) with a Benchmark Microplate Reader (Bio-Rad, Hercules, CA, USA).

Total RNA was extracted from samples of the anterior intestine with Total RNA Kit I (E.Z.N.A., Omega Bio-tek, Norcross, GA, USA) and the quantity and quality of RNA assessed (Nanodrop 1000, Thermo Scientific, Waltham, MA, USA). Prior to cDNA synthesis, RNA was treated with DNase using a DNA-free kit (Ambion) following the manufacturer's instructions. Reverse transcription of RNA into cDNA was carried out using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Thermo Scientific) with 500 ng of total RNA in a reaction volume of 20 μl.

Primers and reaction conditions followed those previously described (Ferlazzo et al., 2012; Gregório et al., 2013). Table 1 shows primer sequences and predicted amplicon sizes. Real-time qPCR amplifications were performed in duplicate in a final volume of 10 μl with 5 μl SsoFast EvaGreen Supermix (Bio-Rad) as the reporter dye, 20 ng cDNA, and 0.5 pmol μl⁻¹ of each forward and reverse primer. Amplifications were performed in 96-well plates using the One-step Plus sequence detection system (Applied Biosystems, Foster City, CA, USA) with the following protocol: denaturation and enzyme activation step at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. After the amplification phase, a temperature-determining dissociation step was carried out at 65°C for 15 s, and 95°C for 15 s. For normalization of cDNA loading, all samples were run in parallel using 18S rRNA (18S). To estimate efficiencies, a standard curve was generated for each primer pair from 10-fold serial dilutions (from 10 to 0.001 ng) from a cDNA pool that included all samples. Standard curves represented the cycle threshold value as a function of the logarithm of the number of copies generated, defined arbitrarily as one copy for the most diluted standard. All calibration curves exhibited correlation coefficients R²>0.98, and the corresponding real-time PCR efficiencies were >99%.

This experiment was designed to test the effect of PTHrP(1–34), PTHrP(7–34) and the polyclonal sea bream antibody STC-Ab in the luminal fluid contents of the sea bream. STC was not tested as insufficient quantities were available.

Sea bream juveniles (N=36, 20–25 g M_b) were anaesthetized in 2-phenoxyethanol (1:10,000) weighed to the nearest 0.1 g and received a single intraperitoneal injection of 0.9% NaCl (5 μl g⁻¹, control), PTHrP(1–34) (0.5 μg g⁻¹), PTHrP(7–34) (0.5 μg g⁻¹) or STC-Ab (5 μ g⁻¹). Individual control groups (0.9% NaCl injected) were run in parallel with each hormone treatment. After injection, fish were transferred for recovery to 150 l tanks supplied with well-aerated running seawater. Twenty-four hours after treatment, fish were anaesthetized with 2-phenoxyethanol (1:1000) and decapitated. The intestinal fluid of individual fish was collected from the excised intestinal tract clamped (from pyloric caeca to anal sphincter) with two mosquito forceps, and emptied into pre-weighed vials and centrifuged (12,000 g, 5 min) to separate fluid from precipitate.

The intestinal fluid was transferred to pre-weighted vials and the volume measured to the nearest 0.1 μl (0.1 mg assuming a density of 1). Intestinal fluid titratable alkalinity (HCO₃⁻ + CO₃²⁻) was manually measured using the double titration method and a combination semi-micro pH electrode (HI1330B, Hanna Instruments) attached to a pH-meter (PHM84, Radiometer, Copenhagen, Denmark) as follows: 50 μl of the intestinal fluid samples were diluted in 10 ml NaCl (40 mmol l⁻¹), gassed with CO₂⁻-free gas for 30 min to remove CO₂ and titrated to pH 3.8 with 10 mmol l⁻¹ HCl; an additional gassing period of 20 min was applied to remove any remaining CO₂ and the sample was back titrated to its original pH with 10 mmol l⁻¹ NaOH. The volume difference between added acid and base in the two titrations and titrant molarities were used to calculate total HCO₃⁻ equivalents (mequiv l⁻¹). Intestinal precipitates were re-suspended in 400 μl of triple-distilled water, homogenized in a glass homogenizer and a 100 μl aliquot was double titrated as described for the intestinal fluid, normalized by fish mass and expressed as nequiv g⁻¹.

All results are shown as means ± s.e.m. After assessment, homogeneity of variance and normality statistical analysis of the data were carried out using, as appropriate, Student's t-test or one-way analysis of variance followed by the post hoc Bonferroni test to establish differences from control groups. All statistical analyses were performed using GraphPad Prism version 5.00 for Macintosh (GraphPad Software, San Diego, CA, USA). Groups were considered significantly different for P<0.05.

The authors acknowledge Ramalhete Marine Station (CCMar, University of Algarve, Portugal) for fish maintenance and Professor J. M. Mancera (University of Cadiz, Spain) for access to sea bream fry.