Genomic and expression analysis of canine calcitonin receptor-stimulating peptides and calcitonin/calcitonin gene-related peptide

Calcitonin receptor-stimulating peptides (CRSPs) are new members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified in pigs, dogs and other domestic animals, and CRSP-1 is an active ligand for the CT receptor (CT-R). We recently sequenced porcine CRSP genes (Crsps) and found similarity with the CT/CGRP gene (Ct/Cgrp) in sequence and genomic organization. In this study, we identified five Crsps, Crsp-1 to Crsp-5, in dogs. Crsp-1 has five exons with an exon-intron organization identical to that of porcine Crsp-1 or Crsp-2, while Crsp-2 and Crsp-3 have additional CT-2- and CT-3-coding exons like Ct/Cgrp. Crsp-2 was renamed as Ct-2/Crsp-2 because both CRSP-2 and CT-2 mRNAs were tissue-specifically expressed. Crsp-4 and Crsp-5 are presumably generated by retrotransposition. We postulate that Crsps were generated from the gene duplication of Ct/Cgrp, and gained their diversity during mammalian evolution. Among the canine CTs and CRSPs, CRSP-1, CT-1 and CT-2 are active ligands for the CT-R, but CRSP-2 and others are inactive. Canine CRSP-1 and CT-2 are expressed in the central and peripheral systems, while CT-1 is localized in the thyroid gland. These findings indicate that dogs can be used for an experimental model as analysing the physiological roles of the CT/CGRP/CRSP family.     

Tumour calcitonin. Interaction with specific calcitonin receptors.

The human epidermoid bronchial carcinoma (BEN) cell line has been shown to have specific membrane binding sites for calcitonin and to secrete high-molecular-weight forms (ranging from 40000 to 10000) of immunoreactive calcitonin. Synthetic salmon and human calcitonins and a thyroid extract of porcine calcitonin have been shown to displace 125I-labelled salmon calcitonin from the receptors in a dose-related fashion. The binding to these receptors of calcitonins derived from the BEN cell line and a medullary thyroid carcinoma with molecular weights ranging from 28000 to 3500 (both separated by gel-filtration chromatography) has been investigated. Neither major peaks of BEN-cell-line calcitonin showed receptor binding activity. Only one form of medullary thyroid carcinoma calcitonin, that which co-eluted with synthetic calcitonin monomer on gel-filtration chromatography, caused any significant displacement of labelled hormone from the receptors.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (C-IR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

F.9 embryonal carcinoma cell calcitonin autocrine system: correlation between immunoreactive calcitonin secretion and calcitonin receptor number

Mouse teratocarcinoma cells in culture offer an in vitro system to study the initial steps of embryogenesis. It has been suggested that, at such early stages, cell functions are regulated by an autocrine process in which embryonic cells produce factors that in turn act on themselves. F.9 cells possess specific membrane receptors for calcitonin (CT) (120 fmol/mg of protein, Ka, = 3.5 X 10(8) M-1). These cells produce CT detected by heterologous radioimmunoassay in serum-free culture-conditioned medium (75 pg/10(7) cells/12 h). When F.9 cells are incubated in serum-free medium, CT binding and secretion concomitantly drop by 50% within the first 2 h, then increase progressively to an upper plateau after the sixth hour. Preincubation with 10(-6) M CT leads to disappearance of CT receptors and CT secretion in the culture medium up to 6 h. Avoiding accumulation of CT in the medium by a continuous flow rate for 6 h leads to a progressive decrease of CT receptors. In addition, retinoic acid treatment of cells induces a parallel progressive decrease of CT receptor number and of total CT synthesis. These results suggest a reciprocal regulation of CT receptors and CT secretion, or a close relationship between their regulations.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

Summary In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (CIR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Biological actions of human and rat calcitonin and calcitonin gene-related peptide

We assessed the central and peripheral biological actions of human and rat calcitonin and calcitonin gene-related peptide (CGRP). After intravenous administration, human and rat calcitonin, but neither human nor rat CGRP significantly decreased plasma calcium and phosphorus concentrations in awake, freely moving rats. After intracerebroventricular as well as after intravenous administration, human and rat calcitonin and human and rat CGRP significantly inhibited gastric acid secretion in conscious rats. Intracerebroventricular administration of rat calcitonin did not alter plasma calcium and phosphorus concentrations. Linear, partially protected CGRP and calcitonin did not exhibit any biological effects. These studies indicate that calcitonin, but not CGRP, affects calcium and phosphorus homeostasis while both peptides decrease gastric acid secretion similarly. Furthermore, these studies support the hypothesis that the calcium and phosphorus lowering effects of calcitonin are peripheral while the gastric inhibiting actions of the calcitonin and CGRP are mediated by the central nervous system.     

Stimulation of calcitonin secretion in the pig by calcitonin gene-related peptide

Pig thyroid glands were surgically isolated in situ and perfused with autologous blood to which was added known concentrations of calcitonin gene-related peptide (αCGRP). When thyroids were perfused with measured concentrations of CGRP within the range of 0.6–600 nM, the secretion rate of calcitonin (CT) was stimulated while the release of T₃, T₄, and somatostatin remained unchanged. Specific binding of ¹²⁵I-CGRP to pig thyroid plasma membranes was demonstrated, and binding was inhibited by unlabelled CGRP but not by CT or by other peptides unrelated structurally to CGRP. The findings indicate that the pig thyroid gland contains plasma membrane binding sites for CGRP and that CGRP is capable of stimulating the secretion of CT.     

Development of an avian calcitonin radioimmunoassay using synthetic chicken calcitonin as immunogen

1. A sequential double antibody radioimmunoassay (RIA) has been developed using synthetic chicken calcitonin (CT) as antigen, tracer and standard. 2. The immunoassay has a minimum detection limit of 0.5 ng and effective dose (ED50) of 7 ng. Serial dilutions of chicken and turkey plasma were parallel to serial dilutions of CT standard. Extracts of chicken and turkey ultimobranchial glands caused parallel displacement of tracer similar to synthetic CT. 3. Primary antisera (anti-chicken CT) was raised in guinea pigs immunized with RIBI: animals treated with Freund's complete adjuvant failed to respond. 4. Chicken CT was determined to have a half-life of 60 sec in the turkey hen. Development of a homologous RIA for avian CT will allow studies to elucidate the role of this hormone in birds.     

Monoclonal antibodies against calcitonin

Summary Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dotblot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde-or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu^1,7]-eel CT by bot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.Presented in part at the International Symposium on Biotechnology in Clinical Medicine, BIOTECH RIA '87, 13th–15th April, 1987, Rome, Italy     

Modulating calcitonin fibrillogenesis: an antiparallel alpha-helical dimer inhibits fibrillation of salmon calcitonin

We have investigated the prefibrillar state of salmon (s) and human (h) calcitonin (CT). Size exclusion chromatography at pH 3.3 and 7.4 indicates that sCT is present in solution as a dimer, whereas hCT elutes as a monomer at pH 3.3 and as monomer-dimer at pH 7.4. Guanidine hydrochloride unfolding experiments show that dimerization is stabilized by hydrophobic interactions. We investigated the dimeric structure by multidimensional nuclear magnetic resonance spectroscopy and calculations by using an sCT mutant (LAsCT) in which Pro23 and Arg24 were substituted for Leu23 and Ala24. As indicated by the Leu9-Tyr27 and Leu12-Leu19 contacts, the mutated hormone forms a head-to-tail dimer whose basic unit is an alpha-helix in the region Leu12-Tyr22. The solution behavior of LAsCT is identical to that of sCT, so the dimeric structure can safely be extended to sCT: we believe that such a structure inhibits fibril maturation in sCT. No stable dimer was observed for hCT, which we attributed to the absence of a defined helical structure. However, we suggest that intermolecular collisions of short ordered regions (for example, a sequence of turns) in hCT favors intermolecular contacts, and specific orientation can be obtained through hydrogen bond formation involving Tyr12, Phe16, and Phe19, with the aromatic ring acting as an acceptor. Taken together, our results indicate that hCT fibrillation can be reduced by favoring a helical dimer, obtainable by replacing the three aromatic amino acids with leucines.     

Deletions of genes encoding calcitonin/alpha-CGRP, amylin and calcitonin receptor have given new and unexpected insights into the function of calcitonin receptors and calcitonin receptor-like receptors in bone

It has been suggested that skeletal nerves fibers may play important roles in neuro-osteogenic interactions. This view is partly based upon information obtained from immunohistochemical studies, chemical and surgical denervation experiments and clinical observations in patients with stroke and spinal cord injury, indicating the presence of a network of nerve fibers in the skeleton and that defective signalling in skeletal nerve fibers affects remodelling of bone. This view is also supported by data showing that functional receptors for signalling molecules in skeletal nerve fibers are expressed in bone cells and that activation of these receptors leads to profound effects on bone forming osteoblasts and bone resorbing osteoclasts. Convincing evidence for a role of neuronal signalling in bone metabolism has been provided by gene deletion approaches in which it has been shown that leptin-sensitive and neuropeptide Y-sensitive receptors in hypothalamus are important for bone remodelling in mice. Recently, gene deletion experiments have shown that calcitonin gene-related peptide (CGRP), one of the neuropeptides present in skeletal nerve fibers, is an important physiological regulator of bone formation at the level of osteoblast activity. CGRP belongs to the calcitonin (CT) family of peptides also including CT, amylin and adrenomedullin, as well as the recently described intermedin and calcitonin receptor-stimulating peptide. These peptides utilize two seven transmembrane G protein-coupled receptors - the calcitonin receptor (CTR) and the calcitonin receptor- like receptor (CRLR) - which can dimerize with three different single transmembrane proteins, making up the RAMP family. Associations between RAMPs and either CTR or CRLR give rise to seven distinct, molecularly characterized, receptors for CT, CGRP, amylin and adrenomedullin. Deletions of the genes for ligands in the CT family of peptides and for one of the receptors have revealed unexpected findings that have changed our view on the role of these peptides in bone remodelling. It was anticipated that deletions of the CT/alpha-CGRP and CTR genes would lead to bone loss, since CT has been shown to inhibit bone resorption in vitro and in vivo and has been used to treat patients with excessive bone resorption. Surprisingly, it was found that CT/alpha-CGRP-/- and CTR+/- mice have increased bone mass due to increased bone formation. Mice with deletion of the amylin gene, however, exhibited bone loss due to enhanced bone resorption. Selective deletion of the alpha-CGRP gene also leads to bone loss, but due to decreased bone formation. Thus, our understanding of the role of the CT family of peptides has been changed dramatically and much more data have to be gained before we fully understand the roles these peptides have in bone biology.     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

Amyloid formation by salmon calcitonin

It is demonstrated using three independent methods that salmon calcitonin can form amyloid fibrils in vitro. Large aggregates are shown to exhibit a blue-green birefringence in cross polarised light after staining with congo red. Individual fibrils were observed using electron microscopy. These fibrils are approx. 50–60 Å in diameter and up to 20 000 Å in length and are similar in appearance to those observed in Alzheimer's disease. Finally, X-ray diffraction studies of the large aggregates reveal the cross-β conformation characteristic of the monomers in the fibre.     

Interrelation of prolactin and calcitonin

Intraperitoneal prolactin injection (3.5 U/200 g bw, daily, for 5 days) caused a marked rise in blood calcitonin concentration in female Wistar rats. It is common knowledge that exogenous calcitonin administration results in an obvious drop of blood prolactin level. Hence, the interrelation between anterior pituitary lactotropic function and blood calcitonin level may be regarded as a negative feedback, since prolactin activates calcitonin production and secretion, with the latter, in its turn, inhibiting adenohypophysial lactotropic function.     

Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes

Calcitonin, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin belong to a unique group of peptide hormones important for homeostasis in diverse tissues. Calcitonin is essential for calcium balance, whereas CGRP and ADM are important for neurotransmission and cardiovascular and respiratory regulation. Based on phylogenetic analysis, we identified intermedin as a novel member of the calcitonin/CGRP peptide family. Analysis of intermedin expression indicated that intermedin is expressed primarily in the pituitary and gastrointestinal tract. Intermedin increased cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competed with labeled CGRP for binding to its receptors in these cells. In addition, treatment of 293T cells expressing recombinant calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMPs) showed that a CRLR/RAMP receptor complex is required for intermedin signaling. In contrast to CGRP and ADM, which exhibited a preferential stimulation of CRLR when co-expressed with RAMP1 and RAMP2 or RAMP3, respectively, intermedin represents a nonselective agonist for the RAMP coreceptors. In vivo studies demonstrated that intermedin treatment led to blood pressure reduction in both normal and spontaneously hypertensive rats via interactions with the CRLR/RAMP receptor complexes. Furthermore, in vivo treatment in mice with intermedin led to suppression of gastric emptying activity and food intake. Thus, identification of intermedin as a novel member of the calcitonin/CGRP peptide family capable of signaling through CRLR/RAMP receptor complexes provides an additional player in the regulation of peripheral tissues by CRLR and will allow development of new therapeutic agents for pathologies associated with diverse vascular and gastrointestinal disorders.     

Utilization of synthetic peptides for the study of calcitonin and biosynthetic precursors for calcitonin

By using synthetic peptides and a library of moncclonal anti-peptide antibodies, we have developed a panel of techniques that allow the dissection of circulating immunoreactive calcitonin in the serum. C Cells of the thyroid were found to release both mature calcitonin and biosynthetic intermediates in the circulation. Finally, these products were found to circulate as heterogenous molecular species. A methodology for the standardization of the measurement of calcitonin is proposed in the form of a two-site immunoradiometric assay specific for mature calcitonin.     

Human placental calcitonin receptors.

Receptors for the hypocalcaemic hormone, calcitonin (CT), have been identified in a membrane fraction prepared from term human placentae. Binding of 125I-labelled salmon CT (125I-sCT) to the membranes was time- and temperature-dependent, saturable (Bmax. 58 +/- 11 fmol/mg of protein), of high affinity (Kd 80 +/- 21 pM) and poorly reversible. Species-specific CTs and CT analogues competed for 125I-sCT binding with potencies proportional to their known biological potencies. Various unrelated peptide hormones did not compete, indicating that receptor binding was specific for CT. Photoaffinity labelling using a derivatized biologically active sCT analogue, [Arg11,18,3-nitrophenylazide-Lys14]sCT, identified a receptor component of Mr approximately 85,000, comparable with findings in osteoclasts and other target cells. The presence of CT receptors in the human placenta supports other evidence that CT may have a role in the regulation of placental function.