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Studies the distribution of the alleles of the calcitonin receptor gene (CRG) in post-menopausal Caucasian women suffering or not from osteoporotic fractures. Increase of bone mineral density at the femoral neck; Heterozygous advantage effect of homozygous leucine subjects; Establishment of the genotype of the CRG. more...

Abstract: Calcitonin inhibits bone resorption by acting on osteoclasts via a specific receptor. The calcitonin receptor (CTR) is also found in many other normal and malignant tissues and cell lines. It has been cloned and sequenced in several species including humans. It belongs to a subclass of seven-transmembrane G protein-coupled receptors. Four human CTR (H-CTR) isoforms generated by alternatively spliced mRNA have previously been described. Two H-CTR encoding DNAs containing an unidentified 50-bp insert are now reported from T47D cells. The 50-bp insert corresponds to a DNA region located between exon 9 and exon 10, and appears to originate from an alternative splicing process. The two H-CTR cDNAs encode 274 and 290 aa long isoforms. Both are deleted from the putative fourth transmembrane domain to C-tail. They differ by the presence (H-CTR5) or absence (H-CTR6) of a previously known 16-aa insert in the putative first intracellular loop. Cell- and tissue-distribution analysis using RT-PCR demonstrates that the shorter one, HCTR6, is more prevalent. The mRNA of both isoforms was detected in giant cell tumor, whereas only H-CTR6 mRNA was detected in TT cells and kidney tissue. Neither H-CTR5 nor H-CTR6 could be detected in peripheral blood mononuclear cells cultured in the presence of RANKL, in MCF7 cells, and in cortical brain and ovarian tissues. When H-CTR6 was transiently expressed in HEK293 cells, CT failed to induce production of cAMP or to bind to the receptor. These suggest either an intrinsic loss of ligand binding function, or an altered intracellular trafficking. Our findings therefore indicate the existence of two novel splice variants of the H-CTR and confirm that multiple splicing patterns could be involved in the post-transcriptional regulation of the gene. [Copyright &y& Elsevier] more...

RNAs of ultimobranchial bodies (U.B.) from the pink salmon, Oncorhynchus gorbuscha, were studied using the polymerase chain reaction (PCR) with specific oligodeoxyribonucleotides (oligos) of the salmon calcitonin (sCT) mRNA selected in exon 2 or 3 and a poly(T) oligo. We observed two amplified DNA fragments, differing by 200 bp which hybridized with a specific exon 4 probe. Sequence analysis indicated that they both encoded exon 4, but differed in the length of their 3' non-coding regions by use of a putative polyadenylation signal situated 200 bp upstream from the established polyadenylation site. These two polyadenylation signals very likely were regulated differently, as the larger expressed transcript was predominant. To date, such use of an alternative polyadenylation signal in a CT mRNA has not been described in other vertebrates, and only the chicken CT mRNA possesses a second classical polyadenylation signal which is not known to be used. This characteristic of sCT biosynthesis appears to be typical in lower vertebrates and is of phylogenic interest. Moreover, it engenders a hypothesis of a relationship between the high concentration of the peptide observed in females of this species and their capacity to produce sCT by different biosynthetic pathways. more...

Calcitonin is an important physiological regulator of salmon gills. Although the calcitonin receptor was found in salmon gills, the critical question concerning the source of the hormone remained unanswered. In this communication, evidence is presented for expression of calcitonin mRNA and its encoded peptide in gills of the pink salmon, Oncorhynchus gorbuscha. The expression of calcitonin gene transcripts was demonstrated by reverse transcription-polymerase chain reaction, Southern hybridization, and sequencing. The sequencing identified a sequence corresponding to that of exon 4 of the salmon calcitonin gene. Expression of the encoded calcitonin gene in gills was detected by radioimmunoassay in gill extracts. This synthesis of calcitonin in gills, which also possess specific receptors to the peptide, suggests function of an autocrine or paracrine process producing calcitonin in this tissue. These observations confirm and extend previous reports on the physiological role of calcitonin in fish gills. more...

Alternative splicing of the primary transcript of the CALC I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (exons 1-4), whereas CGRP mRNA (exons 1, 2, 3, 5, and 6) is mainly produced in neuronal cells. The CT mRNA encodes for a protein precursor containing an amino terminal peptide, CT, and a carboxyl terminal peptide (CCP I). CGRP precursor is composed of the same amino terminal peptide and CGRP. Recently we reported the presence of a third mature transcript of the CALC I gene in human medullary thyroid carcinoma (MTC) tissues. This transcript encodes for a precursor containing the amino terminal peptide CT and a novel carboxyl terminal peptide, CCP II. This finding was further confirmed in the TT-cell line derived from a human MTC. We produced monoclonal antibodies against CCP II and developed a rapid and specific immunofluorescence method for this peptide. We demonstrated CCP II-specific immunoreactivity in TT-cells and in MTC tissues. CCP II labeling was relatively homogeneous in contrast to CT and CGRP, which presented striking heterogeneity for intensity of labeling. Therefore, CCP II mRNA is translated in tumor cells in an apparently constitutive way. more...

We show that an autocrine system for calcitonin gene-related peptide (CGRP) exists in F9 teratocarcinoma cells. Synthesis of CGRP by F9 cells was demonstrated by measuring the peptide concentration in cells and medium and by determining specific mRNA in cells. During six days of culture, CGRP secretion did not vary significantly in the medium, while intracellular CGRP and CGRP mRNA levels increased. F9 cells contained a CGRP-sensitive adenylate cyclase system and CGRP increases the accumulation of cAMP in the culture medium. Interestingly affinity purified antibodies against CGRP specifically inhibited growth of F9 cells by 50%. CGRP therefore stimulates F9 cell growth by an autocrine process, suggesting that CGRP may be a growth factor during early embryogenesis. more...

Immunoreactive calcitonin-gene-related peptide (ir-CGRP) was detected in the crustacean Nephrops norvegicus. High levels of ir-CGRP were present in the foregut and hepatopancreas (3 +/- 0.7 and 4.6 +/- 1.0 micrograms eq per 100 mg of fresh organ, respectively). Molecular sieving of acidic extracts of anterior gut of Nephrops norvegicus showed a high molecular weight immunoreactive peptide in the range 15,000 to 25,000 Da. Immunoreactivity related to salmon calcitonin was present in the high molecular weight fraction. more...

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species. more...

We have characterized the binding parameters of renal receptors (Scatchard analysis revealed the presence of two binding sites: site I, Ka1 = 1.29 X 10(9) M-1, number of binding sites = 9.9 X 10(6)/micrograms protein; site II, Ka2 = 0.93 X 10(8) M-1, number of binding sites = 4.27 X 10(8)/micrograms protein) and studied the effect of solubilization. The high-affinity sites are preserved during affinity chromatography and the process results in a 6080-fold purification of those sites. The lower-affinity sites are also preserved but the overall purification factor is about 40% lower than that obtained using molecular sieving. The purification of the renal calcitonin receptor by molecular sieving (Sephacryl S-200) is accompanied by total loss of the high-affinity site; however, the low-affinity site is enriched over 1642-fold. Binding parameters were obtained for the purified fractions. Synthetic salmon calcitonin was also bound to renal membranes using the bifunctional reagent disuccinimidyl suberate and photo-affinity cross-linking using hydroxysuccinimidyl azidobenzonate reagent. Cross-linked receptor eluted in the same volume as solubilized membranes specifically binding salmon calcitonin (S-200 chromatography). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified fractions showed several protein bands with apparent molecular masses ranging from 18 000 Da to 100 000 Da in the presence or absence of a reducing agent (2-mercaptoethanol). Autoradiography of polyacrylamide gels of cross-linked calcitonin receptor showed only three protein bands specifically binding salmon calcitonin. Their molecular masses were 70 000 Da, 40 000 Da and 33 000 Da respectively. The 40 000-Da molecule represents a major band (47% total binding species). This suggests that these three proteins are the principal components of the calcitonin receptor and that S-S bonds are not involved in the assembly of the receptor subunits. more...

The presence of calcitonin in fetal and postnatal mouse "C" cell was demonstrated by radioimmunoassays of fetal and adult thyroid extracts and by immunohistochemistry following the use of Bouin's fixative from 18 days postcoitum onwards. However, after fixation with formaldehyde no calcitonin could be detected in fetal or early postnatal samples, although it was still easily detectable in adult tissues treated with this fixative, raising the possibility that there may be age-dependent differences in the chemistry or physiological state of intracellular calcitonin. Experiments conducted with several other fixatives and the results of the radioimmunoassays suggest that the loss of calcitonin reactivity is not due to differences in the anti-genic sites of the molecule at the two stages in development. Neither can the positive detection of fetal material treated with Bouin's fixative be due to any action of this liquid. However, the inhibiting action of the aldehydes could be related to the preservation of the granule membranes in the fetal "C" cells: our ultrastructural studies show that the granules can be morphologically classified in dense-core and homogeneous matrix. Such granule membranes are abundant in the young and adult "C" cell and would be reactive, while the small dense-core granules are prevalent in the fetus and would be unreactive, confirming the hypothesis of a difference of structure of the granule membrane and not of calcitonin between fetal and late postnatal "C" cells. more...

Plasma levels of calcitonin (CT), parathyroid hormone (PTH), and estradiol (E2) were measured in the rat during the estrous cycle. Changes in the circulating levels of CT were observed, in particular a significant fall during estrus. No significant changes in PTH levels were recorded using a N terminal assay. E2 levels were not correlated with CT or PTH levels. more...