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Calcitonin, the hypocalcemic hypophosphatemic hormone is produced in the thyroid by the C-cells. We recently detected the presence of calcitonin and its messenger in hepatic tissue in vivo and in vitro. The calcitonin precursor is composed of the N-terminal peptide, calcitonin and a carboxyterminal peptide. We previously reported that in normal human thyroid and in medullary thyroid carcinoma a second calcitonin messenger is expressed in low quantities. This mRNA differs in its 3' region from the first one. It also codes for the N-terminal peptide, calcitonin and a carboxyterminal peptide which differs by the last eight amino acids from the first. We report here that both calcitonin mRNAs are expressed in normal or tumoral liver. Direct estimation, by a specific immunoassay, of the levels of carboxyterminal peptide II, the specific peptide coded for by calcitonin mRNA II, confirmed that this peptide is synthesized in human liver.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH) inactivates prostaglandins. We recently reported an mRNA sequence coding for a predicted isomer (PGDH(rI)) of this enzyme. The TT cell line, derived from medullary thyroid carcinoma (MTC), expresses mRNAs for both isomers. We report here the expression by TT cells and MTC of a third 15-PGDH related mRNA (PGDH(rII)), 241 nt shorter than type-I 15-PGDH. RNase protection assays confirmed that TT cells expressed this mRNA (PGDH(rII)). Thus different splicing patterns could be involved in the post-transcriptional regulation of type-I 15-PGDH gene in MTC.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces the differentiation of monocytes into macrophage-like cells in vitro. To identify the genes expressed during this process, we performed differential display polymerase chain reaction on RNA extracted from cord blood monocytes (CBMs) treated with 1,25-(OH)2D3. Treated CBMs expressed type-I 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH), the key enzyme of prostaglandin E2 (PGE2) catabolism and a 15-PGDH-related mRNA (15-PGDHr). This newly described 15-PGDH-related mRNA was constitutively expressed in adult monocytes. 15-PGDH gene(s) transcription was accompanied by the appearance of the 15-PGDH activity in treated CBMs. In addition, the cyclooxygenase 2 mRNA level was decreased and PGE2 levels in the culture mediums were lowered (50%). Our results stress that 1,25-(OH)2D3, at least in neonatal monocytes, can exert, directly or indirectly, a dual control on key enzymes of PGE2 metabolism. In conclusion, we suggest that modifications in prostaglandin metabolism, induced by the expression of type-I 15-PGDH and the downregulation of cyclooxygenase 2, could be involved in monocytic differentiation.

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation.

Three isoforms of calcitonin (CT) exist in salmonids. Isohormones I and II are expressed in the pink salmon Oncorhynchus gorbuscha. We report here the existence in this species of a CT gene and of its transcripts, which encode for a fourth isohormone, the salmon CT (sCT) IV. This new CT gene was identified by PCR from genomic DNA and by sequencing the amplified DNA. The expression of this CT gene was established in ultimobranchial body and brain, by reverse transcription-PCR, hybridization and sequencing. The sCT IV gene, like the sCT I gene, is a complex transcription unit, containing exons encoding for a CT as a calcitonin gene-related peptide (CGRP) molecule. The predicted peptide, sCT IV, has a greater homology with the eel CT and the sCT II than with the sCT I. Alignment of the sCT IV with other fish and chicken CT showed amino acid modifications in similar positions as those found during evolution. The predicted salmon CGRP IV peptide is highly homologous to the known CGRP molecules in other species, confirming the high conservation of the molecule during evolution. This identification of a new salmon CT gene is interesting both for the therapeutic potential represented by the new molecules encoded by this gene and for phylogenetic studies.

We have characterized, by RT-PCR amplification using specific primers, the presence of glucagon-like peptide-1 (GLP-I) receptor mRNA in CA-77 cells, a C cell line derived from a rat medullary thyroid carcinoma. Down-regulation of the GLP-1 receptor mRNA was observed after exposure of CA-77 C cells with GLP-1 (7-37). Increased secretion of both calcitonin gene-related peptide (CGRP) and calcitonin (CT) occurred after treatment with GLP-1 (7-37) associated with elevated steady-state levels of CGRP and CT mRNA. GLP-1 (7-37) increased cAMP formation in CA-77 cells in a dose-dependent manner; exendin (9-39), a GLP-1 receptor antagonist, inhibited cAMP production. The GLP-1 peptide which is produced by intestinal cells could be involved in the control of CT secretion through an entero-thyroidal axis implying GLP-1 receptor and increased CT gene expression.

We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis.

Interleukin-1 receptor type I (IL-1RI) expression in cultured rabbit articular chondrocytes (RAC) was studied by reverse transcription-polymerase chain reaction (RT-PCR). A cDNA probe specific for the rabbit IL-1RI gene was constructed using primers derived from the sequence data of the human, murine and chick receptors. Transforming growth factor-beta 1 (TGF beta-1) was shown to transiently increase the level of expected 900-bp PCR product at 1 h of incubation and decrease the expression at 48 and 72 h with no effect at 24 h. In receptor binding assays using [125I]-IL-1 alpha, TGF beta decreased IL-1R bioactivity at all time points. These results suggest that TGF beta-induced down-regulation of IL-1 RI could be responsible for its ability to antagonize the effect of IL-1 and that TGF beta may have a role in the repair of articular cartilage.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, synthesizes large amounts of calcitonin (CT), its biological marker. However, in some cases with a poor prognosis, MTC is associated with low basal CT levels owing to a decrease in the thyroid CT content. Using a murine model of human MTC, we studied the relationships between CT biosynthesis, C cell proliferation, and the circulating CT level during MTC progression. Cell proliferation was revealed by autoradiography of radioactive thymidine incorporation in dividing nuclei, after CT or CT mRNA detection by immunocytochemistry (ICC) or in situ hybridization (ISH). All rat thyroids showed a severe hyperplasia of C cells containing significant amounts of CT and CT mRNA, and a very low mitotic index. Tumours were found in 68% of the thyroids. In the strongly immunoreactive small nodules (ICC+), many labelled nuclei were observed. Subsequently some nodular cells, still containing detectable CT mRNA (ISH+), were not detected by immunocytochemistry (ICC-) owing to a dramatic decrease in secretory granules. Their mitotic index increased, and a rise of the basal CT plasma level was noted. These ISH+, ICC- tumour MTC cells represent a modified aggressive tumour C cell population exhibiting an increased ability to proliferate and were detected by the rise in the basal circulating CT level.

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.

We recently reported that human liver and primary cultures of hepatocytes express calcitonin. We therefore studied the expression of calcitonin gene related peptide (CGRP), the alternative splicing product of the calcitonin gene, in hepatocytes and liver. We used polymerase chain reaction amplification with specific primers to detect the presence of CGRP I and II messengers and a specific radioimmunoassay to measure the peptide. We report here that CGRP is synthesized by primary cultures of hepatocytes and in liver. As liver also possesses specific receptors for CGRP in non-parenchymal cells, a paracrine system could be involved in liver metabolism.

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.

We amplified, using the polymerase chain reaction and calcitonin receptor (CTR) specific primers, RNA extracted from medullary thyroid carcinoma (MTC) and the derived TT cell line. Both secrete large amounts of calcitonin. Electrophoresis of amplification products revealed, in both cases, an ethidium bromide-stained band that hybridized to a CTR probe. Sequencing the band amplified from TT cells revealed an open reading frame identical to the sequence of H-CTR but lacking 16 amino acids in the first intracellular loop. This demonstrates the existence of an mRNA coding for a subtype of H-CTR which is expressed in TT cells and MTC.

Calcitonin (CT), a hypocalcemic and hypophosphatemic hormone, is produced by the C-cells of the thyroid gland. It is the main tumoral marker of medullary thyroid carcinoma (MTC). Hypersecretion of CT is also associated with other types of tumors. Thus, heterogeneity of circulating CT can play an important role in the accurate determination of hormone levels in blood samples obtained from MTC patients. Further studies will be necessary to establish the predictive value of the several peptides coded by the calcitonin gene family. All of them specifically reflect the ways and the pattern of alternative splicing of the primary transcript of the Calc I gene. Such relations implicate further investigations concerning the relationship between calcitonin circulating levels, biosynthetic activity of C-cells and the expression of gene encoding for this hormone, in normal and neoplastic conditions.

Immunoreactive calcitonin (CT) is present in liver. This could represent hormone synthesized by liver cells, degraded or bound to specific receptors reported in this organ. We report here that the calcitonin gene is expressed in liver. We proved this by demonstrating, by PCR amplification using specific primers, the presence of calcitonin messenger in human liver and in primary cultures of human hepatocytes and detected by radioimmunoassay CT in hepatic tissues and cells. The synthesis of hormone by liver that also possesses specific receptors for CT favors the presence of an autocrine or paracrine system involving calcitonin in this organ.

We report the isolation of calcitonin gene-related peptide (CGRP) mRNAs and expression of these RNAs in different tissues in the pink salmon, Oncorhynchus gorbuscha. Hybridization of poly(A+) RNAs indicated a mature CGRP RNA of 1.1 kb. The CGRP-like immunoreactivity occurring in tissues and plasma had the same relative molecular weight as the synthetic molecule. Variations in CGRP plasma levels were observed during migration, spawning, and postspawning states. These data suggest that CGRP may play an important role during the reproductive cycle of salmon.

We studied the hormonal secretion of a human mixed follicular and medullary carcinoma. Thyroglobulin (Tg) secretion, especially by large cells and sometimes by small ones, was visualized with immunoenzymatic staining. Calcitonin (CT) was produced by small spindle-shaped cells. Moreover, immunofluorescence double staining performed on the resected thyroid tissue showed the secretion of both Tg and CT in a small number of cells. The cells lost their hormonal secretion after 2 months of culture. Hormonal secretion was modulated by different additives in the medium. Tg secretion was induced when TSH was added to the culture medium; the maximal effect was produced with the addition of 1 mU TSH/ml and 1 microM cortisol, which potentiated the effect of TSH on Tg production. A durable Tg secretion was obtained by embedding the cells in Engelbretch-Hohn-Swarn (EHS) tumour matrix. The CT production was reinduced by the addition of 4 mM Ca2+, 1 microM glucagon and 1 microM cortisol to the culture medium. These findings show that different cells are found in a mixed follicular and medullary carcinoma, some of which can secrete both CT and Tg. They can remain differentiated for a long period after being embedded in EHS tumour matrix with Ca2+ and hormonal components.

We report here an efficient and rapid method for the specific detection of calcitonin in tumor C-cells of medullary thyroid carcinoma (MTC). This occasionally aggressive tumor arises from the endocrine thyroid C-cells. Its principal marker is calcitonin, the predominant C-cell secretion, which is detected in patients and in our animal model by radioimmunoassay of the plasma, as well as by immunohistochemistry of thyroid tissues. Although calcitonin is easily detectable in normal C-cells, its content is greatly reduced in tumor cells owing to the disappearance of the secretory granules that store the mature peptide. This finding suggests cell dedifferentiation correlated with an increasing aggressivity of the tumor. We therefore developed a rapid detection of calcitonin mRNA by in situ hybridization on routine paraffin sections, using a synthetic oligodeoxyribonucleotide probe labeled with digoxigenin-dUTP. The reaction was detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase, and the enzyme catalyzed the appearance of a dark blue color. The signal was exclusively restricted to the normal, hyperplastic, and tumor C-cells. It was specific, as increasing concentrations of the unlabeled oligonucleotide led to progressive disappearance of the reaction. Its sensitivity was slightly diminished as compared with corresponding frozen sections, but the intensity of the signal was quite acceptable. High levels of calcitonin mRNA were found in all normal and hyperplastic C-cells. They were increased in most of the tumor MTC cells, which did not correlate with the amount of intracellular peptide stores but explained the abnormally high basal levels of circulating calcitonin of the tumor-bearing rats. ISH is therefore of greater value than ICC for an early anatomopathological detection of this tumor. Our data show that the tumor cells are not "dedifferentiated." They only lack the granular compartment storing the mature peptide before exocytosis, but CT biosynthesis and the rest of the secretory process seem to be complete. Our results suggest that factors expressed in malignant C-cells affect basic cell mechanisms involved in the storage of the mature calcitonin, rather than the expression of the CALC gene.

Ageing can affect both the secretion of a hormone and the number of its specific receptors. An autoradiographic method was used to quantify renal binding sites for calcitonin (CT) in Wistar rats aged 1, 3, 6, 12 and 18 months. In 1-month-old rats, high densities of calcitonin binding sites were observed in the outer cortex and in the outer medulla. However, an increasing number of rats presenting very low calcitonin binding site density in the outer medulla (that we called 'deficient') appeared during ageing. Ageing also involved a gradual decrease in calcitonin receptor densities in the kidney outer medulla in the non-'deficient' rats. The basal calcitonin concentrations in plasma did not vary with age. The increase in plasma calcitonin in response to a calcium injection increased with age, but this increase was not cor- related with the decrease in binding site density. In 18-month-old rats suffering from C cell hyperplasia or carcinoma, both basal and stimulated levels of calcitonin were increased (basal: x 3; stimulated: x 5), but no major modification in calcitonin binding site densities was observed. Thus in the Wistar rat, receptor density is apparently age-regulated and a relative increase in endogenous CT level is without effect on receptor density.

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.

The calcitonin/calcitonin gene-related peptide gene expresses two different mRNAs by tissue-specific alternative processing. The calcitonin mRNA is produced in thyroid C cells by splicing of the first three exons to the fourth polyadenylated exon. It encodes a protein precursor containing an amino-terminal peptide, calcitonin, and a carboxyl-terminal peptide (CCP I). Calcitonin gene-related peptide (CGRP) mRNA is produced in neuronal cells by splicing of the three common exons to the fifth exon and the polyadenylated sixth exon, leading to the production of a CGRP precursor. Our studies concerning the expression of the calcitonin/CGRP gene in human medullary thyroid carcinoma revealed the presence of a new RNA transcript. Amplification by polymerase chain reaction and direct sequencing showed that the novel transcript is composed of exons 1, 2, and 3, part of exon 4, exon 5, and a polyadenylated exon 6. This transcript contains an open reading frame coding for the known amino-terminal and calcitonin peptides, as well as for a novel calcitonin carboxyl-terminal peptide, CCP II. This third alternative pathway utilizes an internal donor site within the exon coding for calcitonin. The presence of CCP II was demonstrated in plasma and thyroidal tissues of medullary thyroid carcinoma patients, implying that this novel mRNA is actively translated in medullary thyroid carcinoma.

We localized specific binding sites for human calcitonin gene related peptide (hCGRP) in different organs of the trout using labelled human CGRP. Maximal binding was observed in gill and spleen membranes. The binding of 125I-hCGRP was time and temperature dependent. Scatchard analysis of binding data for the spleen and the gills disclosed two binding sites. The constants for the site of high affinity and low capacity (KAM-1 and Bmax (fmol/mg of proteins] were 2.9 x 10(9) for the spleen and 70 and 3.5 x 10(9) for the gill. Salmon calcitonin (sCT) inhibited the binding of 125I-hCGRP to spleen membranes with the same order of potency as hCGRP. In contrast sCT was less effective than hCGRP in suppressing the specific binding of 125I-hCGRP to gill membranes.

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.

mRNAs were isolated from 2 patients suffering from a familial form of a rare variant of medullary carcinoma of the thyroid (MTC), called mixed follicular and medullary carcinoma. The presence of calcitonin (CT) and thyroglobulin (Tg) mRNAs was checked by northern and in situ hybridization and compared with immunohistochemical results. In each case, mRNAs hybridizing to probes specific for CT and Tg were detected. Both proteins were quantified by radioimmunoassay determination in tissue extracts. Patient 1 had 20 ng Tg and 68 ng CT per micrograms total protein, and patient 2 had 0.4 ng Tg and 1.7 ng CT per micrograms total protein. Northern analysis showed that mixed carcinoma expressed several species of both CT mRNAs and Tg mRNAs. The main Tg transcripts present in neoplastic cells (8.5 and 4.8 kb for patient 1 and patient 2) were identical to or smaller than those of normal thyroid tissue (8.5 kb). The tumor CT mRNA (1 kb) was identical to that of normal tissue. In situ hybridization confirmed the presence of CT and Tg mRNA in the great majority of tumor cells. Furthermore, the presence of small amounts of organified iodine was evidenced by analytical ion microscopy in 35% of these cells. This raises an important question regarding the histogenesis of this tumor.

Plasma calcitonin levels increase during reproduction in female salmon. Whether these changes in circulating levels of the hormone are due to increased secretion and or increased biosynthesis is not established. We measured total and poly A+ RNAs and calcitonin content of ultimobranchial bodies of male and female pink salmon during the different stages of the reproductive cycle. Calcitonin mRNAs were analysed by hybridization of northern and dot blots with a specific probe for salmon calcitonin. Dot blot autoradiography indicated that female animals had higher levels of CT mRNA than males and these values were correlated with plasma calcitonin levels. In conclusion, sexual hormones still to be identified, are probably implicated in the expression of the calcitonin gene in females during the reproductive cycle.

Two genes code for calcitonin gene-related peptides (CGRPs). One expresses by tissue-specific alternate splicing calcitonin and CGRP I mRNAs, the other CGRP II mRNA. Calcitonin is the marker of sporadic or hereditary human medullary thyroid carcinoma (MTC). CGRP II expression is not well established in normal or tumoral thyroid. After amplification by polymerase chain reaction, CGRP I and II mRNAs were detected in six cases of MTC associated with other endocrine neoplasia (MEN IIa) and in two cases of isolated MTC. CGRP I was detected in all non-C cell tumoral thyroids (6 samples), CGRP II was barely detectable in three out of six cases. CGRP II could be a specific tumoral marker of MTC.

The physiological significance of calcitonin gene-related peptide (CGRP) was investigated by assessing the CGRP stimulated adenylate cyclase activity in various tissues of trout. The highest enzyme concentration was found in gill and stomach membranes. The maximal activity (190% of the basal value) was observed for a concentration of 53.3 nM CGRP I or II. In the presence of 58 nM sCT, the maximal enzyme activity represented 120% of the basal value. No additive effect was observed; this suggests that both CGRP and sCT activities are mediated through the same receptor. The present data are in favour of a role for this neuropeptide operating in branchial cell functions such as calcium transfer from the external to the internal milieu.

The effect of calcitonin gene-related peptide (CGRP) on prolactin (PRL) secretion was studied in female rats. Prepubertal female rats were submitted to the stressful stimuli of injection, blood puncture and thermal stress. Lactating rats were exposed to suckling stimulus. The effects of CGRP on PRL release were compared to those of calcitonin (CT). CGRP i.p. or s.c. prevents the increase in circulating PRL induced by stress. This effect was observed two hrs and even 24 hrs after CGRP administration. It was elicited at all doses tested, 2.5, 1.25 and 0.6 micrograms per animal. The time course of the actions of CGRP and CT are different. It is proposed that CGRP and CT act on different receptors.

Calcitonin (CT) levels in the ultimobranchial body and in plasma were radioimmunoassayed in rainbow trout, Salmo gairdneri, as a function of the annual cycle. In male and female, there was an important increase in the CT levels in the ultimobranchial body and in plasma. These variations in CT levels in both male and female suggest that sexual maturity influences the synthesis and the secretion of calcitonin in fishes. A positive correlation was observed between plasma and ultimobranchial CT levels and the gonadosomatic index in both sexes, suggesting that CT has a role in the processes involved in gonadal development.

C-cells tumours are frequent (50%) in old WAG/Rij rats. In comparison to the original Wistar strain, three month old WAG/Rij rats are characterized by higher calcitonin synthesis and secretion, in addition to a genetically transmitted loss of calcitonin binding sites in the outer renal medulla. In order to determine if the increase of calcitonin gene expression is also of genetic origin, we quantified calcitonin and its specific messenger in the thyroid glands of second generation (Wistar x WAG/Rij) hybrids. These parameters ranged from low Wistar like to higher WAG/Rij like values. The amount of calcitonin messenger in the thyroid was highly correlated to the release of the hormone in plasma elicited by a calcium challenge and inversely correlated with the number of its binding sites in the kidney. Our results suggest that an enhanced expression of the calcitonin gene is genetically transmitted, probably as a consequence of the mutation involved in the loss of renal calcitonin binding sites. It may represent the first event leading to malignancy.

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.

The binding of salmon calcitonin was investigated in subcellular fractions obtained from normal porcine lung. Only the membrane fraction (density, 1.14 g/cm3) showed specific binding for calcitonin. Specific binding of 125I-labeled salmon calcitonin was competitively inhibited by concentrations of unlabeled homologous hormone in the range 0.01-1 nM. Half-maximal inhibition of binding was observed with 0.12 nM salmon calcitonin. Scatchard analysis of the data suggested the presence of one class of binding sites with a mean affinity constant of 0.9 X 10(10) M-1 and a mean receptor number of 40 X 10(8)/mg of protein. The binding of salmon calcitonin was highly specific; half-maximal inhibition of binding was observed with 63.8 nM bovine calcitonin, the hormone corticotropin having no effect in this system.

The primary step of calcitonin biosynthesis was studied in a normal organ: chicken ultimobranchial gland, a tissue particularly rich in calcitonin secretory cells. Poly(A)-rich RNA was extracted and purified from ultimobranchial organs and translated in a reticulocyte lysate in the presence of labelled methionine. Polyacrylamide gel electrophoresis of specific immunoprecipitates revealed a major band of Mr 14 500 and a band of Mr 13 300. Thus, in chicken the precursor of calcitonin is a Mr 14 500 polypeptide. The minor component of Mr 13 300 could represent limited processing by the reticulocyte lysate.

The ultimobranchial gland (UBG) of birds is particularly rich in calcitonin, the hypocalcaemic hypophosphataemic hormone, that is secreted by the C-cells of the mammalian thyroid. The principal cells of the UBG have a striking resemblance with the mammalian C-cells, i.e., they possess small intracytoplasmic dense-core secretory granules, 150-300 nm in diameter. The gland also contains a second, morphologically distinct, endocrine cell type with larger granules, 500-800 nm in diameter. A sensitive immunocytochemical reaction was developed with the use of antibodies against salmon calcitonin. By means of this technique the presence of calcitonin-immunoreactive molecules was demonstrated in both secretory cell types of the UB gland of the chicken. This gland can thus be considered as a homogeneous calcitonin-producing tissue. Whether the secretory products are identical is discussed and differences in the secretory pathways are suggested.

Hypercalcaemia always results in serious clinical sequalae and, if not treated, carries a most unfavourable prognosis. The clinician will gain major diagnostic help from an evaluation of the calcitonin and parathyroid hormone blood levels. With regard to parathyroid hormone we have developed, for the first time, a radioimmunoassay which is specific for the estimation of biologically active hormone in the circulation. We are dealing here with an unusual radioimmunological situation as the immunochemical sites are generally quite distinct from those associated with hormonal activity. We are presenting in this first paper the normal values and also the variations that occur in different types of hypercalcaemia. The comparison of these results with those obtained by the usual methods of estimation for parathyroid hormone assay lacking in biological activity shows the value of this new technique.

Previous studies have shown that certain patients suffering from poorly differentiated follicular carcinoma (PDFC) of the thyroid had high levels of calcitonin (CT) and carcinoembryonic antigen (CEA) in the plasma. In this work, both CT and CEA were localized in tissue sections obtained at operation from patients suffering from PDFC. The results confirm the hypothesis that certain cases of PDFC are in fact CT-secreting tumors and represent another type of thyroid neoplasma. Patients suffering from PDFC should be screened using both CT and CEA assays.

DNA complementary to mRNA extracted from the thyroid glands of patients suffering from medullary carcinoma of the thyroid (MCT), a calcitonin-producing tumour, was inserted in the Pst site of pBR 322 by G-C tailing. The recombinant plasmids were used to transform Escherichia coli DP 50. Ampicillin-resistant clones were screened using a 32P-labelled cDNA to mRNA extracted from a case of MCT particularly rich in calcitonin (CT) mRNA. Positive clones were subsequently rescreened using a 32P poly(T) probe. Eighty clones were thus purified, and the inserts obtained by digestion with PstI were subjected to positive hybridization selection with subsequent translation in vitro. An insert stimulating synthesis of the protein and containing restriction sites compatible with the previously published complete sequence of calcitonin mRNA from rat was sequenced. This cDNA insert contained the entire coding region of 426 bp, 70 bp at the 5'-end, and 295 bp upstream from the poly(A) tail. The complete amino acid sequence of human preprocalcitonin could thus be deduced.

The bindings of calcitonin was investigated in trout bone, kidney and gill and rat bone and kidney. Specific binding of calcitonin was observed in all tissues tested except fish kidney membranes. The affinity constants for the sites of high affinity-low capacity (in trout bone and rat kidney) or for the unique site (in trout gill and rat bone) were of the same order of magnitude (2.0-9.0 x 10(9) M-1), the number of binding sites per mg of protein being higher in rat bone homogenates than in other tissues. These studies strongly support the theory that the gill in fishes is likely to perform some of the functions of the kidney in mammals.

The detection of high circulating levels of calcitonin is a most valuable procedure to diagnose advanced cases of medullary carcinoma of the thyroid. However, the diagnosis of a primitive tumor in the early stages of development (as in cases of the familial form of the disease) or of a metastasis following ablation of the tumor is more difficult. In the latter cases, the levels of circulating calcitonin may be within normal limits, and for diagnosis one must then resort to tests to stimulate the secretion of calcitonin. We are reporting from our personal experiences the advantages, disadvantages and inconveniences of the three most well-known tests. All patients responded positively to administration of calcium or pentagastrin. The alcohol test, however, produced inconsistent results.

The effect of calcitonin (CT) and parathyroid hormone (PTH) on carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1.) activity was tested in human erythrocyte hemolysates and with purified carbonic anhydrases I and II. The most important effect was on carbonic anhydrase II: CT showed a 2-fold increase and PTH showed a 50% decrease of carbonic anhydrase activity. This effect was observed at low hormonal concentrations and suggests the importance of CT in regulating carbonic anhydrase activity in the two important sites of CO2 exchange, erythrocyte and lung.

In order to determine the effect of beta-blocking agents on secretions of parathyroid hormone and calcitonin, 9 patients with renal failure were given single doses of propranolol (a blocker of the beta 1 and beta 2 receptors) or an equivalent amount of metoprolol (a beta 1 selective agent). Propranolol causes a decrease of plasma parathyroid hormone (p less than 0.02) as well as of calcitonin (p less than 0.05) whereas metoprolol has no effect on the plasma levels of these hormones. These findings suggest that parathyroid tissue and thyroid C cells have receptors that are exclusively of the beta 2 type which are modulating the secretion of parathyroid hormone and calcitonin.

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.