The effects of calcitonin on plasma calcium levels and bone metabolism in the fresh water teleost Channa punctatus

Administration of salmon calcitonin (sCT) caused significant reduction in total and ultrafiltrable plasma calcium content in the plasma of a fresh water female teleost Channa punctatus. A time-bound analysis on the effect of sCT showed a highly significant short duration reduction in total and ultrafiltrable plasma calcium content in fish kept in normal tap water and low-calcium water and a moderate hypocalcemia in fish kept in high-calcium water. Sexually immature adult fish showed a greater response than the sexually mature ones. Using tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) activities in plasma and hydroxyproline (HYP) excretion in urine, the effect of sCT on the inhibition of bone calcium resorption were examined. In both sexually mature and immature adult fish, kept in normal tap water, sCT significantly suppressed TRACP and ALP activities in plasma and excretion of HYP in urine within 2-6 h with a maximum at 4 h after injection. Salmon CT treatment to sexually immature adult fish caused significant increase in skeletal bone calcium concentration. Taken together, all this information indicates that CT in a fresh water female teleost is an effective regulator of plasma calcium levels, and its action, at least in part, operates through inhibition of bone calcium resorption.     

Calcitonin induces hypercalcemia in grey mullet and immature freshwater and sea-water adapted rainbow trout

1. Changes in plasma calcium levels, in response to salmon calcitonin injections, were studied in freshwater and sea-water adapted trout (Salmo gairdnerii) and in grey mullet (Chelon labrosus). 2. Low doses (0.1 ng sCT/100 g body weight) elicited hypercalcemia in the two species studied. 3. High doses (0.5 microgram) provoked hypocalcemia only in freshwater and sea-water adapted trout. 4. An hypercalcemic response appears as the primordial effects of CT injections, higher doses of CT leading to hypocalcemic effects.     

Biologically potent analogues of salmon calcitonin which do not contain an N-terminal disulfide-bridged ring structure

The disulfide bridge formed between the cysteine residues at positions 1 and 7 of salmon calcitonin (sCT) is not required for biological activity. The analogues [Ala1,7]sCT,[AcmCys1,7]sCT and [AmcCys1,Ala7]sCT (AcmC = S-acetamido-methylcysteine) are linear sequences which retain full hypocalcemic activity in the intact rat and ability to activate adenylate cyclase of rat renal membranes. The secondary structure of these peptides in aqueous solution in the presence or absence of lipid is not greatly perturbed by the opening of the disulfide ring. In contrast with salmon calcitonin, substitution of Cys by AcmCys in human calcitonin results in greatly reduced hypocalcemic activity but no loss in the ability of the peptide to activate renal adenylate cyclase. Thus in vitro activation of adenylate cyclase by human calcitonin analogues is not always correlated with in vivo hypocalcemic potency.     

Ability of calcitonins to alter food and water consumption in the rat

Synthetic salmon calcitonin (sCT) given SC (30 MRC U/kg) or ICV (1.4 U, 300 ng) inhibited 24-hr food consumption in the rat by 50–100%. Furthermore, ICV doses of sCT ranging from 75 ng (0.35 U) to 300 ng (1.4 U) lowered both food and water intake in a dose-dependent manner. Use of various forms of CT give an apparent order of potency of salmon>porcine≥human with sCT being at least 40 fold more potent than the mammalian forms. Measurement of food and water intake over a brief (30–90 min) period showed that ICV sCT was effective in reducing food and water consumption regardless of whether it had been given 1, 12, or 23 hr previously. Daily administration of sCT for 5 days caused marked suppression of food and water intake for 2 days; thereafter, consumption returned toward normal, becoming equivalent to vehicle injected controls by day 3 or 4 despite continued daily injections of sCT. The results show that CT can act centrally to modify certain types of behavior and are of special interest since CT-like peptides have been described in the pituitary and hypothalamus and since CT receptors have been reported in hypothalamic and other brain regions.     

Characterization and regional distribution of calcitonin binding sites in the rat brain

Binding sites for calcitonin (CT), as assayed by the displacable binding of [125-I] iodo salmon CT ([125-I]sCT), were found on a membrane fraction prepared from rat brain. The half times of association varied between 23 and 7 min as a function of the temperatures used in the incubation medium, ranging from 6° to 37°C. Salmon CT in amounts as low as 10^?10 M inhibited the binding of [125-I]sCT to the membranes, whereas the virtually biologically inactive free acid of human CT and human CT sulfone did not affect the binding. The specific binding of [125-I]sCT to the membranes was directed to structural and/or conformational features in the COOH-terminal half of salmon CT. 133 to 8,900 times higher amounts of porcine CT and human CT and analogues thereof were required to achieve an inhibition of binding equal to that produced by salmon CT. Sixty-seven percent of specific binding of labeled hormone was not dissociable, even after 6 h of incubation with an excess of unlabeled hormone. [125-I]sCT extracted from the membranes was not degraded, as judged by gel permeation chromatography, and retained binding activity. Specific binding was highest in the hypothalamus, followed by the brainstem. It was intermediate in the midbrain-thalamus and the striatum, lower in the cortex and negligible in the hippocampus, and cerebellum and the spinal cord.     

Action of calcitonin gene-related peptide at the calcitonin receptor of the T47D cell line

Some effects of calcitonin (CT) can also be produced by calcitonin gene-related peptide (CGRP), an alternative product of the calcitonin gene. This might be mediated by interaction of CGRP at the CT-receptor site. The human breast cancer cell line T47D possesses well characterized CT-receptors (KD = 2.3 x 10(-10) M for 125I salmon CT). 50% inhibition of 125I-sCT binding was achieved with 10(-9) M sCT, 5 x 10(-6) M rat CGRP and 10(-5) M human CGRP. Half maximal cAMP production in T47D cells was seen with 6 x 10(-10) M sCT, 5 x 10(-6) M rCGRP and 10(-5) M hCGRP. Binding and displacement capacity as well as the biological activity of CT and CGRP seems to correlate well. These findings suggest that CGRP in pharmacological doses acts via the CT-receptor. This could be explained by the homology and conformational similarities between CT and CGRP.     

Amylin receptors mediate the anorectic action of salmon calcitonin (sCT)

The teleost salmon calcitonin (sCT), but not mammalian CT, shows similar biologic actions in the skeletal muscle as amylin and calcitonin gene-related peptide (CGRP). The peptides have also been shown to reduce food intake in rams. Because sCT, but not amylin, binds irreversibly to amylin binding sites, the aim of the present study was to compare the anorectic potency of both peptides. To determine whether sCT reduces food intake through interaction with amylin binding sites, we also tested whether appropriate antagonists (CORP 8-37, AC 187) attenuate the anorectic effect of sCT. Finally, we wanted to know whether rat calcitonin (rCT) and sCT reduce food intake to the same extent. Peptides were injected intraperitoneally at dark onset in 24 h food-deprived rats. At doses of 5 or 0.5 microg/kg, the anorectic effect of sCT was more potent and lasted much longer (e.g. 5 microg/kg: sCT > 10 h; amylin approx. 2 h) than that of amylin. Both CORP 8-37 and AC 187 (10 microg/kg) markedly reduced the anorectic action of sCT (0.5 microg/kg). In contrast to sCT, rCT (0.5 microg/kg) had no effect on food intake. It is concluded that sCT s anorectic effect is partly mediated by amylin receptors. Irreversible binding of sCT to amylin receptors may lead to a stronger and prolonged effect in comparison to amylin due to a sustained activation of the binding sites. Similar to other actions of CTs, the anorectic potency of sCT in rats was higher than that of mammalian (rat) CT. This agrees with binding profiles of amylin, sCT, and rCT at amylin binding sites as observed in in vitro studies.     

Application of the yeast‐surface‐display system for orally administered salmon calcitonin and safety assessment

High manufacturing costs and oral delivery are the constraints in clinical application of calcitonin. We selected surface‐displayed Saccharomyces cerevisiae as a low‐cost and safe carrier for oral delivery of salmon calcitonin (sCT). The sCT DNA fragment, optimized according to the codon preference of S. cerevisiae, was synthesized and cloned into the plasmid M‐pYD1 to yield recombinant yAGA2‐sCT, which was induced to express sCT by galactose for 0, 12, and 24 h. sCT expression was detected on the cell surface by indirect immunofluorescence and peaked at 12 h. About 65% recombinants expressed sCT on flow cytometry. The in vivo and in vitro activity of recombinant sCT was determined by detecting bioactivity of antiosteoclastic absorption on bone wafers and orally administering yAGA2‐sCT to Wistar rats, respectively. For safety assessment of yAGA2‐sCT, we observed abnormalities, morbidity, and mortality and determined body weight, serum chemistry parameters, hematological parameters, and organ weight. In vitro bioactivity of the recombinant sCT was similar to that of commercial sCT, Miacalcic; oral administration of 5 g/kg yAGA2‐sCT induced a long‐term hypocalcemic effect in Wistar rats and no adverse effects. This study demonstrates that yAGA2‐sCT anchoring sCT protein on a S. cerevisiae surface has potential for low‐cost and safe oral delivery of sCT. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Preparation and Characterization of Salmon Calcitonin–biotin Conjugates

This study was performed to prepare and characterize the biotinylated Salmon calcitonin (sCT) for oral delivery and evaluate the hypocalcemic effect of biotinylated-sCTs in rats. Biotinylated sCTs was characterized by using high performance liquid chromatography (HPLC) and MALDITOF-MS. The effect of biotinylation on permeability across Caco-2 cell monolayers was examined. Their hypocalcemic effect was determined in rats. Mono- and di-bio-sCTs were separated by reverse phase HPLC. The molecular weights of mono-bio-sCT and di-bio-sCT were determined to be 3,660.5 and 3,900.2 Da, respectively. The permeability of biotinylated-sCTs across Caco-2 cell monolayers was observed with a significant enhancement compared with sCT. Intrajejunal (ij) administration of mono-bio-sCT and di-bio-sCT resulted in sustained reduction in serum calcium levels, with a maximum reduction (% max(d)) of 21.6% and 30% after 4 h and 6 h of application, respectively. The biotin conjugation of sCT may be a promising strategy for increasing the oral bioavailability of sCT and achieving sustained calcium-lowering effects.     

Anti-hypercalcemic effect of orally administered recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing salmon calcitonin on hypercalcemic rats

Oral delivery of salmon calcitonin (sCT) to rats via a recombinant Saccharomyces cerevisiae was assessed. A synthetic sCT gene was cloned and expressed in S. cerevisiae yAGA2-sCT. Recombinant salmon calcitonin (rsCT) expression was detected by flow cytometry. The resorption activity of osteoclasts was inhibited by 3 × 10⁻⁶ M rsCT. Oral administration of 5 g lyophilized yAGA2-sCT/kg to hypercalcemic rats decreased serum calcium from 2.8 ± 0.02–2.7 ± 0.02 mM.     

Paradoxical acute hypercalcemic effect of salmon calcitonin in patients having Paget's disease of bone after treatment with dichloromethylene diphosphonate

The hypocalcemia following administration of calcitonin may be an index to disease activity in Paget's disease of bone. Therefore, we assessed the effect of a single injection of 100 MRC units of salmon calcitonin (SCT) on plasma calcium in 28 patients with active Paget's disease before and after 6 months of treatment with dichloromethylene diphosphonate (Cl2MDP) at a dose of 400 mg/day (3 patients), 800 mg/day (8 patients), 1.600 mg/day (9 patients) or 2.600 mg/day (8 patients). The mean SCT-induced hypocalcemia was reduced by Cl2MDP and there was a significant positive correlation between the decrease of serum calcium induced by SCT and bone resorption evaluated by the number of osteoclasts on bone biopsy taken in pagetic iliac crest. After Cl2MCP treatment, 5 patients manifested a paradoxical hypercalcemic response to SCT injection ranging from +0.3 mg/dl to +0.5 mg/dl, which was sustained over the 9 hours following injection. As these patients had a dramatic inhibition of bone resorption induced by Cl2 MDP, it is suggested that the hypercalcemic response to SCT might reflect persistence or exaggeration of the early hypercalcemic effect of CT which reportedly precedes the hypocalcemic response to SCT.     

Overexpression, purification and characterization of recombinant salmon calcitonin, a therapeutic protein, in Streptomyces avermitilis

Calcitonin (CT) is a peptide hormone produced by the parafollicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Salmon calcitonin (sCT), which is more potent and longer lasting than human CT, has been used widely for the treatment of osteoporosis, paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients. sCT is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated sCT in recombinant Streptomyces avermitilis. With this approach the utilization of expensive peptide synthesis can be circumvented.     

Plasma calcium and calcitonin in the marine teleost, Gadus morhua

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

人降钙素类似物的合成及活性研究

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Glycosylation is important for binding to human calcitonin receptors

Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied after transient expression in monkey kidney COS-1 cells. Tunicamycin, administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.     

Conformational flexibility and biological activity of salmon calcitonin

We have assessed the biological activity of salmon calcitonin I (sCT) using an in vivo biological assay of hypocalcemic activity in rats. The changes in biological activity observed are explained on the basis of changes in the conformational properties of the hormone analogues. Helical content in the presence and absence of lipids and detergents was assessed by using circular dichroism, and the section of the molecule that folds into a helix was predicted on the basis of the helix-coil transition theory of Mattice and co-workers. In the amino acid sequence of sCT, residue 8 is valine and residue 16 is leucine. The synthetic calcitonin derivatives [Gly8]sCT and [Ala16]sCT have higher biological activity than the native hormone although they have a lower helical content. The increased biological activity of these derivatives is ascribed to an increase in their conformational flexibility resulting from the substitution of amino acid residues with less bulky side chains and less tendency to form helical structures. The derivative [Met8]sCT has less substitution than sCT on the beta-carbon at position 8, but it has increased helix-forming potential in the region of residues 8-12. These two factors affect conformational flexibility in opposite ways, resulting in the biological activity of [Met8]sCT being slightly higher than that of sCT. However, increased conformational flexibility does not always increase biological activity. Substitution of the L-arginine at residue 24 for a D-arginine has little effect on the conformational properties or biological activity of sCT. However, [Gly8, D-Arg24]sCT is less active than sCT, [Gly8]sCT, or [D-Arg24]sCT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood pressure, electrolyte composition and renal studies of pike, Esox lucius, following administration of salmon calcitonin

Pike were adapted to both fresh water and brackish water (salinity 2.2‰) conditions prior to measurements of blood pressure, electrolyte composition and renal function both before and after administration of either 3.75 μg calcitonin kg⁻¹ body weight or acetate saline. This protocol was designed to test the ability of calcitonin to affect significantly the fluid and electrolyte status of fish exposed to varying salinity. Whilst in freshwater-adapted animals the plasma content of calcium and magnesium was significantly depressed following calcitonin injection, no corresponding effect was seen following 2.2‰ salinity brackish water-adaptation. Moreover, no significant changes were apparent in renal function of pike adapted to either medium. It is concluded from these experiments that calcitonin in pike may play some part in extra-renal control of plasma calcium and magnesium content in fresh water but that it is unable to affect significantly the measured plasma ion content of brackish water-adapted animals.     

Effect of calcitonin on Ca-ATPase activity of plasma membrane in liver of rats.

The effect of calcitonin (CT) on Ca-ATPase activity in the plasma membrane fraction of rat liver was investigated. CT (80 MRC mU/100 g BW) administered subcutaneously to rats, caused a significant decrease in serum calcium, while increasing liver calcium. The administration of CT produced a rapid decrease of Ca-ATPase activity in the plasma membrane fraction of liver, whereas CT did not cause a significant alteration of p-nitrophenyl phosphatase activity. The maximal response of CT was obtained with 80 MRC mU/100 g BW. Meanwhile, the administration of imidazole (30 mg/100 g BW) which has a hypocalcemic effect, like CT, produced a significant increase in liver calcium and a corresponding fall in Ca-ATPase activity of the plasma membrane fraction. The reduction of Ca-ATPase activity produced by imidazole was significantly potentiated by the simultaneous administration of CT, and the rise in liver calcium was enhanced slightly. The present results suggest that the action of CT on liver calcium involves the decrease of Ca-ATPase activity in the plasma membrane of rat liver.     

Extracellular ionic and acid-base adjustments of Atlantic salmon presmolts and smolts in fresh water and after transfer to sea water: the effects of ovine growth hormone on the acquisition of euryhalinity

In order to better understand the basis for the acquisition of euryhalinity by juvenile salmon and the role of endogeneous stimuli, experiments have been carried out to examine the dynamics of ionic and acid-base adjustments in fresh water (FW) and after direct transfer to full salinity (32 g l⁻¹) sea water (SW) (1) on Atlantic salmon smolt during the natural period of smoltification in spring, (2) on presmolt salmon in autumn, after intraperitoneal implantation of pellets containing ovine growth hormone (oGH). During parr-smolt transformation in FW, gill Na⁺/K⁺ ATPase activity gradually rises, the plasma osmolality (Posm) is unaffected and the total CO₂ of the plasma decreases significantly while whole blood pH fluctuates slightly. Direct transfer of smolt from FW to SW provokes only a slight increase in Posm and emphasizes the acid-base balance disruptions shown in FW. An oGHimplant in a presmolt stimulates gill Na⁺/K⁺ ATPase activity in FW, and affects the acid-base balance. After SW transfer (12 days after implantation), oGH treatment prevents the increase of osmotic pressure and the restoration of the acid-base, ionic equilibrium was faster for oGH-implanted fish than for sham-operated fish. These observations show that in FW smelting salmon develop most of the systems they need for migration and growth in SW and that oGH implants induce the development of physiological characteristics of smolts in a non-natural period of smolting.