Porcine Calcitonin Research Articles

Contra-Insular Effect of Calcitonin on Glucose Metabolism

We studied the effects of Russian preparation of porcine calcitonin (Calcitrinum, 1 U/100 g) on the level of glucose and total calcium, glycogen concentration in the liver, and glucose consumption by the muscle and adipose tissues in vivo and in vitro. The basal level of insulin and secretion of insulin in the dynamics of glucose tolerance test were studied after treatment with calcitonin. In addition to hypocalcemic effect, this substance produced significant hyperglycemic effects, decreased glycogen amount in the liver, inhibited insulin-induced glucose consumption by muscular and adipose tissues in vivo and in vitro, slowed down insulin secretion during glucose load, and impaired glucose tolerance. Thus, calcitonin had contra-insular effects on glucose metabolism.

Large Molecule Bioanalysis Using Q-Tof Without Predigestion And Its Data Processing Challenges

Recent developments in LC-MS have turned it into a viable and valid alternative to ligand-binding assays. Large molecule bioanalysis by LC-MS is generally performed by tryptic digestion, purification and detection of one or more small signature peptides. High-resolution MS instruments offer quantification of intact small proteins or peptides and are able to increase the selectivity, while maintaining sensitivity. Unlike multiple reaction monitoring assays, several factors affecting data processing were presented and the optimal parameters to consider during quantification method building were also demonstrated. MUC5AC-13 (MW 1709.8 Da), human hepcidin/LEAP-1 (MW 2797.4 Da), porcine calcitonin (MW 3604 Da) and chicken lysozyme (MW 14.3 kDa) were selected as model compounds and the possibility of intact peptide and small protein quantification, without tryptic digestion, was demonstrated. Selectivity and sensitivity were improved using different scan modes, such as TOF-MS and TOF-MS/MS.

Calcium Channel Blockers Inhibit the Hyperglycemic Effect of Calcitonin

We studied the effect of Russian preparation of porcine calcitonin (calcitrin, 1 U/100 g) and blockers of Ca(2+) channels nifedipine (1 mg/kg, blocker of chemosensitive Ca(2+) channels) and isoptin (5 mg/kg, blocker of voltage-dependent Ca(2+) channels) on glucose metabolism. Calcitonin produced a pronounced hyperglycemic effect in rats. A close negative correlation was found between glucose level and total calcium content. Injection of calcitonin reduced glucose tolerance in rats. Isoptin and nifedipine reduced plasma calcium level and did not affect significantly the blood glucose levels, did not change the pattern of alimentary hyperglycemia in response to glucose load, completely abolished the hyperglycemic effect of calcitonin, and prevented calcitonin-induced impairment of glucose tolerance.

Evidence for a direct anti-inflammatory action of calcitonin: inhibition of histamine-induced mouse pinnal oedema by porcine calcitonin.

Porcine calcitonin (PCT) caused a potent inhibition of histamine-induced vascular leakage in the mouse pinna. These effects were observed at doses of PCT lower than those required to affect plasma calcium concentrations. In contrast PCT did not inhibit responses of the mouse pinna to 5-hydroxytryptamine despite observed falls in plasma calcium concentrations. It would appear that PCT inhibits histamine-induced oedema in the mouse by a direct action which may partially or fully explain the anti-inflammatory action of PCT, observed by other workers, in some acute inflammatory conditions.

Lack of effect of thyrocalcitonin on formation of bone matrix in mice and rats.

The ability of thyrocalcitonin to promote bone formation has been tested in thyroparathyroidectomized mice and rats by measuring the incorporation of 3H-proline into calvarial bone matrix. It is concluded that porcine thyrocalcitonin has little or no direct effect on bone formation in these species.

Genomic organization, expression and evolution of porcine CRSP1, 2, and3

Recently we identified and characterized porcine calcitonin receptor-stimulating peptide (CRSP) 1, CRSP2 and CRSP3 as members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family. In the present study, the genomic sequences and organization of CRSP1, 2, and 3 were determined, and the expression of the genes in the porcine brain was investigated using in situ hybridization. Analysis of 5′-upstream regions of the three CRSPs demonstrated that CRSP1 and CRSP2 have almost identical sequences (>98% similarity) and high sequence similarities including functional transcription binding sites with the corresponding region of human CALCA (CT/αCGRP), whereas CRSP3 retains less similarity with the above genes. RH mapping of CRSPs demonstrated that they resided in a region of swine chromosome 2 (SSC2). The arrangement of the genes in the region was found to be conserved in corresponding human and mouse regions. In situ hybridization demonstrated sense transcripts of the three genes in cerebrum, hippocampus, hypothalamus, pons/midbrain, and thalamus of 3-month-old pigs, and CRSP2 sense transcripts additionally in tractus opticus. The sense transcripts of αCGRP and CALCB (βCGRP) were detected in cerebrum, hippocampus, and pons/midbrain of newborn mice, and to a lesser extent in pons/midbrain of 8-week-old mice. These results taken together with the chromosomal conservation and phylogenetic clustering of CT/CGRP family indicate that CRSP1, 2, and 3 may be functionally different from αCGRP and βCGRP, though they are indicated to have a common progenitor gene.

Regulation of in vitro metabolism of human spongiosa by parathyroid hormone and calcitonin

A method was developed to investigate in vitro metabolism of human trabecular bone and its hormonal regulation. Human and bovine parathyroid hormone 1-34 caused an increase in the release of calcium and magnesium out of human bone. Human, salmon and porcine calcitonin caused a decrease in the release of calcium and magnesium and inhibited the effect of parathyroid hormone.

Comparison of Calcitonin Determinations by Polyclonal and Monoclonal IRMAs

Serum calcitonin (CT) has important diagnostic and prognostic value in patients with medullary thyroid carcinoma (MTC). Although CT can be routinely quantified by a variety of 2-site immunoassays, different assays may yield very different results. This variability, particularly at low CT concentrations, led to the recommendation to use reliable and sensitive assays in the screening of MTC and C-cell hyperplasia (1). To highlight CT variability at high concentrations, we measured 43 serum samples by use of a polyclonal IRMA and by a monoclonal IRMA. The single-step polyclonal assay used 2 different goat antibodies (as the capture and labeled antibodies), which were specific for well-defined regions of the CT molecule, with no cross-reactivity with parathyroid hormone, thyroid-stimulating hormone, CT gene–related peptide, porcine CT, or salmon CT. The reagent set was used according to the manufacturer’s instructions (Calcitonin Assay, Scantibodies Laboratory, Inc.). The …

A novel member of the calcitonin gene-related peptide family, calcitonin receptor-stimulating peptide, inhibits the formation and activity of osteoclasts

We isolated a novel peptide, calcitonin receptor-stimulating peptide-1 (CRSP-1), from porcine brain and found that the administration of this peptide into rats induced a transient decrease in plasma calcium concentration. Therefore, we investigated the effects of CRSP-1 on osteoclastogenesis. Osteoclast-like cells were formed from spleen cells or bone marrow cells by a combination of the receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CRSP-1 dose-dependently inhibited the formation of multinucleated osteoclast-like cells, and a calcitonin receptor inhibitor antagonized in part the inhibition of osteoclast formation by CRSP-1. Furthermore, CRSP-1 destroyed the actin ring that is a typical index of osteoclast resorption activity; it contributed to this action via the signaling pathway of protein kinase A. Our findings indicate that CRSP-1 inhibits osteoclastogenesis by inhibiting the formation and activity of multinucleated osteoclasts. The inhibitory effects of CRSP-1 on osteoclast metabolism were similar in degree to those of porcine calcitonin. CRSP-1 might provide a clue to the development of tools useful in the prevention and treatment of osteoporosis.

Central Effects of Calcitonin Receptor-Stimulating Peptide-1 on Energy Homeostasis in Rats

The CT-R [calcitonin (CT) receptor] is expressed in the central nervous system and is involved in the regulation of food intake, thermogenesis, and behaviors. CT-R-stimulating peptide-1 (CRSP-1), a potent ligand for the CT-R, was recently isolated from the porcine brain. In this study, we first confirmed that porcine CRSP-1 (pCRSP-1) enhanced the cAMP production in COS-7 cells expressing recombinant rat CT-R, and then we examined the central effects of pCRSP-1 on feeding and energy homeostasis in rats. Intracerebroventricular (icv) administration of pCRSP-1 to free-feeding rats suppressed food intake in a dose-dependent manner. Chronic icv infusion of pCRSP-1 suppressed body weight gain over the infusion period. Furthermore, icv administration of pCRSP-1 increased body temperature and decreased locomotor activity. The central effects of pCRSP-1 were more potent than those of porcine CT in rats. In contrast, ip administration of pCRSP-1 did not elicit any anorectic or catabolic effects. Administration icv of pCRSP-1 also induced mild dyskinesia of the lower extremities and decreased gastric acid output. Fos expression induced by icv administration of pCRSP-1 was detected in the neurons of the paraventricular nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, locus coeruleus, and nucleus of solitary tract, areas that are known to regulate feeding and energy homeostasis. Administration icv of pCRSP-1 increased plasma concentrations of ACTH and corticosterone, implying that the hypothalamic-pituitary-adrenocortical axis might be involved in catabolic effects of pCRSP-1. These results suggest that CRSP-1 can function as a ligand for the CT-R and may act as a catabolic signaling molecule in the central nervous system.

Gas-Phase Chemistry of the Negative Ions of Fully-Protected Peptides by High-Resolution Electrospray Ionization Tandem Mass Spectrometry

Fully-protected C-terminal free peptides can be conveniently analyzed by high-resolution electrospray tandem mass spectrometry (ESI-MS/MS) in a quadrupole quadrupole time-of-flight tandem hybrid mass spectrometer, operated in the negative (-) ionizaionization mode. The unusual choice of negative ions in mass spectrometry applications to peptide analysis was needed to obtain exhaustive sequence and structural data. The low-energy collision-induced dissociation (CID) experiments provided, in fact, tandem mass spectra displaying highly diagnostic fragments with a good signal-to-noise ratio. The method is applied to segments of porcine calcitonin (Cal), Cal (1016, 1), Cal (1724, 2) and Cal (2528, 3) whose [M H]- deprotonated molecular ions provided low-energy CID mass spectra which allow the evaluation either of the primary structure of the peptide and of the location of the side-chain protective groups. ESI (+) MS can be conveniently used, in the high resolution mode, to achieve precise information on the elemental composition of the examined peptides.

Calcitonin receptor-stimulating peptide-1 regulates ion transport and growth of renal epithelial cell line LLC-PK 1

Calcitonin receptor-stimulating peptide-1 (CRSP-1) is a peptide recently identified from porcine brain by monitoring the cAMP production through an endogenous calcitonin (CT) receptor in the renal epithelial cell line LLC-PK 1. Here we investigated the effects of CRSP-1 on the ion transport and growth of LLC-PK 1 cells. CRSP-1 inhibited the growth of LLC-PK 1 cells with a higher potency than porcine CT. CRSP-1 enhanced the uptake of 22Na + into LLC-PK 1 cells more strongly than did CT and slightly reduced the 45Ca 2+ uptake. The enhancement of the 22Na + uptake was abolished by 5-( N-ethyl- N-isopropyl) amiloride, a strong Na +/H + exchanger (NHE) inhibitor for NHE1, even at a concentration of 1 × 10 −8 M, although other ion transporter inhibitors did not affect the 22Na + uptake. These results indicate that CRSP-1 enhances the 22Na + uptake by the specific activation of NHE1. Taken together, CRSP-1 is considered to be a new regulator for the urinary ion excretion and renal epithelial cell growth.

Folding analysis of hormonal polypeptide calcitonins and the oxidized calcitonins using electrospray ionization mass spectrometry combined with H/D exchange

Conformational change of calcitonins has been examined by measuring the rate of hydrogen/deuterium (H/D) exchange in amino acids. Time dependent m/z shift caused by H/D exchange was monitored by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT MS). The rate constants of H/D exchange were calculated from apparent first-order kinetics. The time course of H/D exchange exhibited two phases of faster and slower exchange. The smaller rate constant (k2) estimated from the slower H/D exchange was correlated with an alpha-helix content that reflected the folding state. The order of k2 values obtained for human calcitonin (hCT), porcine calcitonin (pCT), salmon calcitonin (sCT), and elcatonin (ECT) was hCT > pCT approximately sCT > ECT. Although the amino acid sequence of sCT is similar to that of ECT, their k2 values were considerably different. The results suggest that ECT is relatively rigid on the N-terminal side cyclic structure in the folded state. Further, the effect of methionine oxidation on k2 has been examined. In the oxidized pCT that possesses similar biological activity with the intact pCT, the k2 values obtained were nearly equal. The k2 of hCT increased via methionine oxidation, and the biological activity was weakened by oxidation. This suggested that methionine oxidation of hCT produced unfolding in the secondary structure and that oxidative unfolding of hCT led to the loss of biological activity. The results indicate that the H/D exchange rate constant may be used as an informative parameter to elucidate the relationship between the folded state and biological activity of polypeptides like calcitonins with secondary structure.

Procalcitonin and the Calcitonin Gene Family of Peptides in Inflammation, Infection, and Sepsis: A Journey from Calcitonin Back to Its Precursors

Calcitonin (CT) is a hormone that received its name because of its secretion in response to induced hypercalcemia and its hypocalcemic effect (1). It was shown to originate from the thyroid gland (2). More specifically, the hormone was revealed to be located within the thyroidal parafollicular cells, interspersed within and about the follicular epithelium (3–5). Subsequently termed C cells, they occur primarily in the central region of each lobe of the human thyroid gland (6, 7). These cells, which have CT-containing secretion granules, are neuroendocrine. Embryologically, they originate from the neural crest and migrate to the ultimobranchial glands (8). In mammals, the ultimobranchial glands fuse with the thyroid gland. It was the demonstration that medullary thyroid cancer (MTC) was a malignancy of the C cells (5, 9) that eventually led to the isolation of human CT from this tumor and the determination of its structure (10, 11). Simultaneously, the amino acid sequence of porcine CT was determined (12). Later, the development of immunoassays of serum CT in humans led to the observation that the level of this hormone was increased in the serum of patients with MTC (13–15) and to the demonstration that these levels were further augmented after iv calcium and/or pentagastrin administration (13, 16, 17). These findings had a great impact on the clinical diagnosis, the evaluation of efficacy of surgical extirpation, and the follow-up monitoring of MTC. Although RET germline mutation testing has replaced CT for the purpose of determining the presence of carriers of this tumor associated with multiple endocrine neoplasia type 2 (18, 19), the measurement of serum CT has become and has remained the classical clinical marker for MTC. Immature and mature CT

Effects of combined Actinobacillus pleuropneumoniae challenge and change in environmental temperature on calcitonin receptor expression levels in growing pigs

We have identified through differential gene expression polymerase chain reaction that porcine calcitonin receptor expression levels could be altered in porcine leukocytes in response to Actinobacillus pleuropneumoniae (App) challenge. This study further investigates the effects of mild pleuropneumonia and changes in environmental temperature, singularly and in combination, on leukocyte expression levels of the calcitonin receptor in domestic pigs. Forty entire male pigs were allocated by weight and temperament at a starting liveweight of 33 0± 5 kg to 4 treatments: control (22°C room temperature); A. pleuropneumoniae challenge (Day 1); varied temperature (15°C for 8 h on Days 0, 1, and 2; 30°C for 24 h on Day 6); and the combined A. pleuropneumoniae challenge and varied temperature treatment. The analysis of the leukocyte expression levels of calcitonin receptor using semi-quantitative reverse transcription PCR revealed that calcitonin receptor was up-regulated in response to the A. pleuropneumoniae challenge (P < 0.001) and the temperature treatment (P < 0.001). In addition, up-regulation of the calcitonin receptor correlated with decreased weight gain, feed intake, and plasma IGF-I. Thus, expression levels of the calcitonin receptor reflect changes in growth performance associated with alteration in ambient temperature and App challenge. In addition, this study indicates that calcitonin receptor expression represents a mechanism through which endocrine and immune systems interact to affect growth.

Identification, structural determination, and biological activity of bovine and canine calcitonin receptor-stimulating peptides

We have recently identified in porcine brain a series of new peptides, designated calcitonin receptor-stimulating peptide-1 (CRSP-1), CRSP-2, and CRSP-3, but failed to find their counterparts in humans and rodents by either database searching or experimental cross-hybridization. In this study, we isolated cDNAs encoding precursors of bovine CRSP-1, canine CRSP-1, and canine CRSP-2 from their thyroid cDNA libraries. Although the deduced mature amino acid sequences of bovine and canine CRSP-1s and canine CRSP-2 showed identity with their respective porcine CRSP counterparts, none of them had a C-terminal amide structure. In LLC-PK 1 cells endogenously expressing the calcitonin (CT) receptor, bovine and canine CRSP-1s enhanced the cAMP production, while canine CRSP-2 did not stimulate it at all. Equine CGRP-I had a high identity in its amino acid sequence with porcine CRSP-1 and stimulated LLC-PK 1 cells at a potency comparable to that of porcine CT. None of these CRSPs or equine CGRP-I stimulated the CT-like receptor, even in the presence of receptor activity-modifying proteins. These results demonstrate that CRSP-1, a new class of biologically active peptide, is present in animals evolutionarily close to pigs and induces its activity through the calcitonin receptor, suggesting a wide existence and common properties of this peptide in mammals.

The extreme N-terminus of the calcitonin-like receptor contributes to the selective interaction with adrenomedullin or calcitonin gene-related peptide

The calcitonin (CT)-like (CL) receptor is a CT gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor when co-expressed with receptor-activity-modifying proteins (RAMP) 1 or 2, respectively. The CL receptor shows 57% overall sequence identity with the CT receptor, but the homology is much lower in the extreme N-terminus. An N-terminal deletion mutant of the human (h) CL receptor (Δ18-hCL) and a chimeric receptor consisting of the N-terminal amino acids of the porcine (p) CT receptor fused to the Δ18-hCL receptor (pCT–hCL) were therefore analyzed. The Δ18-hCL receptor function was abolished when co-expressed with RAMP1 or -2. The pCT–hCL receptor was a fully functional CGRP receptor when co-expressed with RAMP1, but the RAMP2-dependent AM receptor function was impaired. Limited sequence similarities in the N-terminus of the pCT and the hCL receptors rescue CGRP but not AM receptor binding and signalling.

Rapid communication: physical and linkage mapping of the porcine calcitonin (CALC) gene.

Genus and Species. Sus scrofa. Locus. Porcine calcitonin (CALC) gene. Source and Description of Primers. Primers, CCA1F and CCA1R, were designed from canine calcitonin sequence (GenBank Accession no. AJ271090) to amplify genomic porcine DNA. Using sequence obtained from the amplified PCR product, additional pig-specific primers (CIPBF, CIPBR, CIPCF, CIPCR) were designed. Primer Sequences. CCA1F: 5′-CAC TTT GGA TTG GCC GCG C-3′; CCA1R: 5′-ACC AGG GCA GCC AGC AGG A-3′; CIPBF: 5′-AAC TTC CCA CTC TGC ACA CT-3′; CIPBR: 5′-AGA CCA AAC TTC AGC AGG AT3′; CIPCF: 5′-TTC TCC TTC CTC TGC TTC TG-3′; CIPCR: 5′-GCA AAC CCA ATA CAG GCT CT-3′. Method of Detection. A PCR was performed using the CCA1F and CCA1R primers in 10L reactions of the following: 1× PCR buffer, 1.5 mM MgCl, 200 M of each dNTP, 2.5 pmol of each PCR primer, 0.35 units Taq DNA polymerase (Promega, Madison, WI), and 12.5 ng of genomic DNA. Genomic DNA from four individuals for two swine breeds (Hampshire and Yorkshire) was used. The PCR was performed in a Robocycler (Stratagene, La Jolla, CA) under the following thermocycling conditions: initial denaturation at 94°C for 4 min, 40 cycles of 94°C for 45 s, 64°C for 1 min, 72°C for 2:10 min, and a final extension time of 12 min at 72°C. For each breed, the PCR products from the four individuals were pooled. These pools were then directly sequenced using dye terminators and an ABI 377 sequencer (Perkin-Elmer, Foster City, CA) at the Iowa State University DNA Sequencing and Synthesis Facility. The CCA1F and CCA1R primers produced a 1,600-bp fragment covering exons 1, 2, and 3. This fragment’s se-

CGRP receptors mediating CGRP-, adrenomedullin- and amylin-induced relaxation in porcine coronary arteries. Characterization with 'Compound 1' (WO98/11128), a non-peptide antagonist.

1. Calcitonin gene-related peptide (CGRP), amylin and adrenomedullin (AM) belong to the same family of peptides. Accumulating evidence indicate that the calcitonin (CT) receptor, the CT receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) form the basis of all the receptors in this family of peptides. 2. Using reverse transcriptase - polymerase chain reaction the presence of mRNA sequences encoding the CRLR, RAMP1 and RAMP2 were demonstrated in porcine left anterior descending (LAD) coronary arteries, whereas porcine calcitonin (CT) receptor mRNA was not present. The partial porcine mRNA sequences shared 82 - 92% nucleotide identity with human sequences. 3. The human peptides alphaCGRP, betaCGRP, AM and amylin induced relaxation with pEC(50) values of 8.1, 8.1, 6.7 and 6.1 M respectively. 4. The antagonistic properties of a novel non-peptide CGRP antagonist 'Compound 1' (WO98/11128), betaCGRP(8 - 37) and the proposed AM receptor antagonist AM(22 - 52) were compared to the well-known CGRP(1) receptor antagonist alphaCGRP(8 - 37). 5. The alphaCGRP(8 - 37) and betaCGRP(8 - 37) induced concentration-dependent (10(-7) - 10(-5) M) rightward shift of both the alphaCGRP and betaCGRP concentration-response curves. betaCGRP(8 - 37) (10(-6) M) had the same effect as alphaCGRP(8 - 37) (10(-6) M), but with less potent rightward shift of the concentration-response curves for alphaCGRP, AM and amylin. 6. Preincubation with 'Compound 1' (10(-7) - 10(-5) M) and AM(22 - 52) (10(-6) M) had no significant antagonistic effect. 7. In conclusion, the building blocks forming CGRP and AM receptors were present in the porcine LAD, whereas those of the amylin receptor were not. alphaCGRP, betaCGRP, AM and amylin mediated vasorelaxation via the CGRP receptors. No functional response was detected to adrenomedullin via the adrenomedullin receptor.

Receptor activity modifying proteins interaction with human and porcine calcitonin receptor-like receptor (CRLR) in HEK-293 cells.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), two closely related peptides, initiate their biological responses through their interaction with calcitonin receptor-like receptor (CRLR). The CRLR receptor phenotype can be determined by coexpression of CRLR with one of the three-receptor activity modifying proteins (RAMPs). In this report, we characterized the pharmacological properties of the human or porcine CRLR with individual RAMPs transiently expressed in human embryonic kidney cell line (HEK-293). Characterization of RAMP1/human or porcine CRLR combination by radioligand binding ([125I] halphaCGRP) and functional assay (activation of adenylyl cyclase) revealed the properties of CGRP receptor. Similarly characterization of RAMP2/human or porcine CRLR and RAMP3/human or porcine CRLR combination by radioligand binding ([125I] rADM) and functional assay (activation of adenylyl cyclase) revealed the properties of ADM (22-52) sensitive-ADM receptor. In addition, porcine CRLR/RAMP2 or 3 combination displayed specific high affinity [125I] halphaCGRP binding also. Also, co-transfection of porcine CRLR with RAMPs provided higher expression level of the receptor than the human counterpart. Thus the present study along with earlier studies strongly support the role of RAMPs in the functional expression of specific CRLRs.