1 Division of Gastroenterology, University of Alberta, Edmonton, Alberta, Canada T6G 2C2; and 2 Human Genome Sciences, Rockville, Maryland 20850
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ABSTRACT |
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Stanniocalcin (STC)
is an anti-hypercalcemic glycoprotein hormone previously identified in
the corpuscles of Stannius in bony fish and recently in the human
genome. This study undertook to express human STC in Chinese hamster
ovary (CHO) cells and to determine its effects on calcium and phosphate
absorption in swine and rat intestine. Unidirectional
mucosal-to-serosal
(Jms) and serosal-to-mucosal
(Js
m)
45Ca and
32P fluxes were measured in vitro
in duodenal tissue in voltage-clamped Ussing chambers. Addition of STC
(10-100 ng/ml) to the serosal surface of the duodenum resulted in
a simultaneous increase in calcium
Jm
s and
Js
m
fluxes, with a subsequent reduction in net calcium absorption. This was
coupled with an STC-stimulated increase in phosphate absorption.
Intestinal conductance was increased at the highest dose of STC (100 ng/ml) in swine tissue. The addition of STC to the mucosal surface had
no effect on calcium and phosphate fluxes. STC at doses of
10-1,000 ng/ml had no effect on short-circuit current in any
region of the rat intestine. In conclusion, human recombinant STC
decreases the absorption of calcium and stimulates the absorption of
phosphate in both swine and rat duodenum. STC is a novel regulatory
protein that regulates mammalian intestinal calcium and phosphate
transport.
duodenum
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INTRODUCTION |
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STANNIOCALCIN (STC), a homodimeric glycoprotein, is an anti-hypercalcemic hormone produced by the corpuscles of Stannius (22). It was first identified in bony fish and has more recently been found in mammalian tissue (21, 25). Studies of salmonoid fish have shown that the secretion of STC is positively regulated by extracellular levels of ionic calcium (13, 20, 22); there is a time- and concentration-dependent relationship between calcium levels and measurable hormone release (20, 22).
The primary function of STC in fish is to prevent hypercalcemia, and a rise in serum calcium levels is the primary stimulus for secretion (20). When released into the circulatory system, STC lowers calcium uptake by the gills and intestine and reduces calcium movement from the aquatic environment into the circulatory system, thereby reducing serum calcium levels (16, 17, 22). A second function of STC in fish is the stimulation of phosphate reabsorption by the renal proximal tubules through an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway (16). This renal effect causes increased levels of serum phosphate. The increased phosphate combines with circulating calcium, thus promoting its deposition into bones and scales and reducing serum calcium levels. This combined effect of STC on calcium and phosphate movement results in a synergistic effect in lowering serum calcium levels. Further study has also identified the intestine of marine teleosts as a target organ for STC (18).
Because the corpuscles of Stannius do not exist in mammals, it was long assumed that STC and its physiological effects on calcium and phosphate homeostasis were unique to fish. However, recent immunological evidence of the existence of STC in the human kidney has been demonstrated, and human recombinant STC, like its piscine counterpart, has proved to be a regulator of calcium and phosphate homeostasis in fish (21).
To date, a physiological role for STC in the regulation of mammalian intestinal calcium and phosphate transport has not been identified. Thus the present study examined the role of human recombinant STC as a novel regulatory protein controlling mammalian intestinal calcium and phosphate movement. The data show that STC decreases the absorption of calcium and stimulates the absorption of phosphate in swine and rat duodenum.
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METHODS |
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Animals. Male Sprague-Dawley rats (200-250 g) and male Yorkshire-Landrace swine (15-20 kg) were purchased from the Health Sciences Laboratory Animal Services, University of Alberta. Animals were housed in a temperature-controlled room (21 ± 1°C) on a 12:12-h light-dark cycle at 50% humidity. Rats were fed a standard laboratory diet (Purina Rat Chow) and tap water ad libitum. Pigs were fed a standard pig diet (University of Alberta Farm Feed Mill) and given free access to water. Rats were given an overdose of pentobarbital sodium (240 mg/kg). Swine were premedicated with Torbugesic (0.2 mg/kg), ketamine (11 mg/kg), Rompun (2.2 mg/kg), and Robinul (0.01 mg/kg). After they had reached a tranquilized state, the pigs were given an overdose of pentobarbital sodium (120 mg/kg). Animal care followed the Canadian Council of Animal Care guidelines. Our experimental protocol was approved by the Animal Welfare Committee of Alberta.
Materials. 45Ca (27.8 mCi/mg) and 32P (1 Ci/mM) were obtained from NEN (Boston, MA). The remainder of chemicals were reagent grade and were obtained from Sigma Chemical (St. Louis, MO).
Expression and purification of human recombinant STC. The full-length clone encoding the human homolog of STC was isolated as previously described (17), and stable Chinese hamster ovary (CHO) cell lines expressing the recombinant protein were established (11). To obtain the recombinant protein, cells were seeded into roller bottles in CHO-S-SFM II medium (GIBCO-BRL, Grand Island, NY), supplemented with 1% dialyzed fetal bovine serum and 1% penicillin-streptomycin at a density of 4 × 10 cells/ml in a volume of 250 ml (1 × 10 cells/roller bottle). After 3 days of incubation the cells reached confluence and the tissue culture medium was harvested for purification. Fresh medium was then added to the roller bottles, and after 3 days of further incubation, the medium was harvested again for purification. The harvested medium was filtered through a 0.45-µm filter, and the pH was brought to 5.5 with acetic acid. The medium was then run on a cation exchange resin column (Poros 50, HS; PerSeptive Biosystems, Framingham, MA) and eluted in increasing concentrations of NaCl. Fractions containing STC were pooled and passed through a sizing resin (Sephadex S-200, Pharmacia Biotech, Piscataway, NJ) in 50 mM sodium acetate (pH 6.5) and 150 mM NaCl. Further purification and endotoxin removal employed anion exchange chromatography (Poros 50, HQ, PerSeptive Biosystems) in 20 mM tris(hydroxymethyl)aminomethane · HCl (pH 8). To concentrate the protein, the cation exchange step was repeated. STC was purified to >90% purity as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining (4). In the presence of a reducing agent (2-mercaptoethanol), STC migrates with an apparent molecular mass of ~30 kDa. In the absence of a reducing agent, however, STC shows an apparent molecular mass of 50 kDa. This difference suggests that the CHO cell-expressed STC is secreted in the form of homodimers linked by disulfide bridges (4). The identity of the protein was confirmed by NH2-terminal sequencing (data not shown).
Ussing chamber transport studies. Animals were given an intraperitoneal pentobarbital sodium overdose (240 mg/kg), and in vitro intestinal studies were then performed. The outer muscle layer of segments of duodenum distal to the pylorus was removed, and these segments were mounted in Ussing chambers. The segments were bathed on both sides with a bicarbonate-Ringer solution (in mM: 143 sodium, 124 chloride, 1.1 magnesium, 5.0 potassium, 1.25 calcium, 1.65 HPO4, 0.3 H2PO4, and 25 HCO3) containing 20 mM fructose and circulated with 5% CO2-95% O2 (pH 7.4) at 37°C (9). The spontaneous transepithelial potential difference (PD) and short-circuit current (Isc) were determined for all segments, and tissue conductance was calculated from PD and Isc according to Ohm's law (3).
For the measurement of basal calcium and phosphate fluxes, the tissue was clamped at zero voltage; an appropriate Isc was continuously applied using an automatic voltage clamp (DvC 100 World Precision Instruments, New Haven, CT). For 5-10 s every 10 min, however, the voltage clamp was removed and PD was measured. Tissue pairs were matched for conductance and discarded if conductance varied by >20%. Radioisotopes (10 µCi 32P and 10 µCi 45Ca) were added to either the serosal or mucosal side after mounting, and the tissue was allowed to equilibrate for 20 min, by which time the tissue was electrically stable. Basal fluxes were determined by measuring two 10-min consecutive unidirectional mucosal-to-serosal (JmStatistical analysis. Data are expressed as means ± SE, and statistical analysis was performed using the statistical software SigmaStat (Jandel, San Rafael, CA). Differences between means were evaluated using analysis of variance or paired t-tests where appropriate. Specific differences were calculated using the Student-Newman-Keuls test.
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RESULTS |
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Effect of STC on duodenal calcium transport in swine.
In mammals the absorption of calcium occurs primarily in the upper
small intestine (2). Thus the role of STC in the regulation of calcium
transport was examined in the duodenum of swine. The time course of the
effect of STC on
Jms and
Js
m of calcium in swine duodenum is shown in Fig.
1. After a 20-min equilibration period, two
consecutive 10-min basal fluxes were measured. In all tissues, both
Jm
s and
Js
m
increased from 20 to 40 min, at which time the fluxes stabilized. At 40 min after mounting of the tissue in the Ussing chamber, STC was added
to the serosal surface of the tissue to a final concentration of 100 ng/ml. Control tissue received vehicle only. Three consecutive 10-min
stimulatory fluxes were measured beginning 5 min after the addition of
STC or vehicle. The addition of STC resulted in a significant increase in both
Jm
s and
Js
m
within 10 min of addition. This increase in both
Jm
s and
Js
m was
maintained for the entire 30-min experimental period, with the end
result being an STC-induced reduction in net calcium absorption (Fig.
2). Addition of STC to the mucosal surface
had no effect on net calcium flux (Fig. 2).
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Effect of STC on duodenal phosphate transport.
STC has been shown to stimulate phosphate reabsorption by proximal
tubule epithelium (16). Intestinal phosphate absorption occurs
primarily in the small intestine (2). Thus we examined the role of STC
in the regulation of phosphate transport in the duodenum of swine. The
time course of the effect of STC on
Jms and
Js
m of
phosphate in swine duodenum is shown in Fig. 3. After a 20-min equilibration period, two
consecutive 10-min basal fluxes were measured. In all tissues
Jm
s
increased from 20 to 40 min, at which time the fluxes stabilized. At 40 min after mounting of the tissue in the Ussing chamber, STC was added
to the serosal surface of the tissue to a final concentration of 100 ng/ml. Control tissue received vehicle only. Three consecutive 10-min
stimulatory fluxes were measured beginning 5 min after the addition of
STC or vehicle. The addition of STC resulted in a significant increase
in Jm
s
within 10 min of the addition. This increase in
Jm
s was
maintained for the entire 30-min experimental period.
Js
m
continued to show a small, steady rise over time in both control and
STC-stimulated tissue. Calculation of the net flux demonstrated an
STC-induced increase in net phosphate absorption (Fig.
4). Addition of STC to the mucosal surface
had no effect on net phosphate flux (Fig. 4). In contrast to the
results seen with calcium, the effect of STC on net phosphate transport was not dose dependent in nature between the range of 10 and 100 ng/ml
STC (Table 1).
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STC effect on calcium and phosphate transport preserved across
species.
As shown in Fig. 5 the STC-induced decrease
in net calcium absorption and increase in net phosphate absorption were
also present in rat duodenum. As in the pig, the effects on calcium
were Jsm and on phosphorus
Jm
s. In
contrast to the results seen in the pig, however, there was no
significant effect on
Jm
s of
calcium. In addition, there were no significant differences in PD,
conductance, and
Isc between
control and STC-treated duodenal tissues. Table
2 shows the electrical parameters of
STC-treated tissue (100 ng/ml) and respective controls (vehicle only).
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Effect of STC on electrical responses in rat tissue. In the intestine, Isc is primarily a function of sodium and chloride movement (3). Thus, to determine if STC had any effect on electrogenic ion transport, we examined tissue response to increasing doses of serosal STC (1-1,000 ng/ml) in the rat. STC had no effect on Isc in duodenum, jejunum, ileum, or colon at any concentration (Fig. 6). These findings would suggest that STC does not modulate intestinal electrogenic ion transport.
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DISCUSSION |
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In this study we have demonstrated that human STC significantly decreases net transepithelial intestinal calcium transport and increases net transepithelial phosphate transport in rat and swine. This finding implies that STC may play a significant role in mammalian calcium homeostasis by reducing net calcium absorption and promoting calcium deposition into bones in the presence of increased plasma phosphate levels.
Although most endocrine glands and tissues are preserved across vertebrate groups, the corpuscles of Stannius have in the past been thought to occur only in holostean and teleostean fish. These glands produce and secrete STC, which functions to control plasma calcium through the regulation of gill and intestinal calcium transport (17). Recently, the sequence for STC has been identified in the human genome, and mRNA for STC has been identified in several mammalian tissues, including ovary, prostate, and thyroid (1, 4, 17). In addition, Wagner et al. (21) have demonstrated that human kidney extracts contain an STC-immunoreactive protein that had STC-related effects when injected into fish (21). These reports provide evidence for the presence of STC in mammals and suggest that the hormone may play a more widespread role in the physiological control of calcium and phosphate homeostasis then previously anticipated. However, to date, no direct evidence has been found for STC activity in humans. Indeed, our study is the first to show a direct effect of STC on mammalian intestinal function and also is the first study to utilize mammalian STC.
Both fresh and salt water fish and mammals have absorptive mechanisms for calcium and phosphate in the intestinal mucosa, which are likely to be under the control of calcio- and phosphotropic hormones. In mammals, as in fish, the intestine is constantly exposed to high concentrations of calcium with the highest interluminal concentrations of calcium being present in the duodenum. Intestinal calcium absorption occurs primarily in the duodenum by a saturable, transcellular, and a nonsaturable paracellular pathway (2). Studies have demonstrated that of the total transepithelial calcium flux only ~30% is routed transcellularly, whereas the remainder moves through the paracellular pathway. (14). The addition of STC to swine intestine resulted in an increase in the movement of calcium both in a mucosal-to-serosal direction and in a serosal-to-mucosal direction, resulting in a net decrease in calcium absorption. This simultaneous increase of bidirectional calcium flux as well as the increase in electrical conductance at high doses of STC, strongly suggests that STC enhances calcium flux across the paracellular route in the duodenum. Furthermore, STC was shown to be effective only when added to the serosal side of the tissue, strongly suggesting that STC is mediating its effects through a receptor-mediated process localized in the basolateral membrane. These effects of STC are similar to the concentration-dependent effects of STC in the inhibition of net calcium uptake in Atlantic cod (18), suggesting a conservation of physiological function.
The range of doses used in this study was chosen based on the concentrations found in fish (15, 18) and also on the published results of Wagner et al. (21) and Olsen et al. (17) who have shown that the levels of immunoreactive protein found in the sera of mammals closely matches those seen in fish.
The mechanism by which STC causes an increase in paracellular flux is unknown. STC has been shown to stimulate the release of cAMP in flounder renal proximal tubules (16), and cAMP is known to alter intestinal permeability (6). However, paracellular permeability in the intestine can also be altered by phospholipase C, tyrosine kinases, calcium, and protein kinase C, and the heterotrimeric G proteins (27). The effect of STC on these parameters in the intestine remains to be determined. Interestingly, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] also increases paracellular calcium flux in both directions in mammalian duodenum; however, in contrast, 1,25-(OH)2D3 also stimulates transcellular calcium transport, resulting in an enhancement of net calcium absorption (14). The mechanism behind this effect of 1,25-(OH)2D3 on calcium paracellular flux is currently unknown.
Intestinal phosphate absorption also occurs primarily in the upper
small intestine, involving both an active sodium-independent and a
sodium-dependent process (2). Active phosphate transport appears to be
maximal at low phosphate concentrations, with nonsaturable passive
diffusion occurring at high phosphate levels (24).
1,25-(OH)2D3 has been shown to stimulate active phosphate transport at both the
brush-border and basolateral surfaces, possibly through an effect on
the number and/or affinity of sodium and phosphate carriers (12). In our studies STC stimulated the net absorption of phosphate primarily through an effect on the mucosal-to-serosal movement. However, in contrast to the dose-dependent effects of STC on calcium absorption, there was no clear dose dependence of the effect of STC on
phosphate absorption. This lack of a dose response may be due to the
fact that Pi becomes rapidly
integrated into intracellular metabolic processes on entering the cell.
Therefore, the rate and control of phosphate absorption by the
intestine are not only a function of the transport processes, but are
also dependent on the rate of utilization of intracellular phosphate.
Also in contrast to the effects on calcium flux, STC did not affect the rate of serosal-to-mucosal movement of phosphate. This may be due to
the fact that the presence of negative charges in the paracellular pathway retards the passage of negatively charged
or
at low concentrations of these
ions (2).
Lu et al. (16) demonstrated that STC increased net phosphate flux in flounder renal tubules primarily by decreasing the peritubular-to-luminal transport and had no effect on calcium transport. STC has also been shown to reduce gill calcium transport from the aquatic environment into the bloodstream (22). These effects of STC differ from those seen in the intestine. We found the effect of STC to be primarily on the serosal-to-mucosal movement of calcium, rather than a decrease in the mucosal-to-serosal movement. Although Sundell et al. (18) did not measure unidirectional fluxes in the Atlantic cod, they did observe a dose-dependent decrease in net calcium absorption similar to what we observed in the swine and rat. These findings would suggest that although the end result is the same (i.e., decrease in serum calcium and increase in serum phosphorus), the mechanisms involved in the STC-induced alteration of transport processes may differ among the kidney, gills, and intestine.
The secretion of STC is positively regulated by extracellular levels of ionic calcium (20). Indeed, calcium appears to stimulate STC synthesis from preexisting mRNA, due in part to mRNA stabilization (7). The mechanism of STC mRNA stabilization does not involve ongoing transcription or translation and is possibly mediated by protein-nucleic acid interactions in the cytoplasm.
In conclusion, STC has a concentration-dependent inhibitory effect on mammalian intestinal calcium absorption and a concomitant stimulatory effect on mammalian intestinal phosphate absorption. This is the first demonstration of the physiological effect of STC on intestinal function in mammals. This decreased calcium absorption and increased phosphate absorption in vivo may enhance deposition of calcium and phosphate as hydroxyapatite into bone, an action consistent with the anti-hypercalcemic role of STC.
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FOOTNOTES |
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Address for reprint requests: R. N. Fedorak, Div. of Gastroenterology, Dept. of Medicine, Univ. of Alberta, 519 Newton Research Bldg., Edmonton, Alberta, Canada T6G 2C2.
Received 2 May 1997; accepted in final form 2 October 1997.
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