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Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT) and CT gene-related peptide (CGRP), the two splice peptide products of the CALC I gene. Normal and hyperplastic C cells are intrafollicular, in contact with the basement membrane (BM) that is maintained around the differentiated tumors. To investigate the relationships between MTC evolution and BM constituents, we examined the modifications induced by laminin-1 and -2 (merosin), two isoforms colocalized in the follicular BM, on three MTC cell lines: murine rMTC 6-23 and CA-77 cells, and human TT cells. Laminin exerted a mitogenic activity on rMTC 6-23 and on TT cells, causing a concurrent decrease in both CT and CGRP mRNA levels and production of the peptides. Conversely, laminin reduced the proliferation rate and enhanced CGRP synthesis and secretion in CA-77 cells. This antiproliferative response, which coincides with an increase in differentiation markers, is comparable to that reported in normal cells and also in the neoplastic Caco-2 cell line. This suggests that laminin could exert opposite effects depending on the stage of tumor evolution.

Using a sensitive calcitonin (CT) immunoassay and a newly developed bioassay capable of detecting 0.025 MRC mU CT, we have studied acute and chronic plasma CT fluctuations in male and female rats. Immunoassay of serial plasma samples revealed progressive increases in plasma CT concentrations during aging; female rats have higher CT concentrations than age-matched males. Acute periodic CT fluctuations were discovered by immunoassay of plasmas obtained at 3-h intervals; the greatest values occurred just before and during feeding. Fed rats have higher CT than starved rats. We have used immunoadsorbent chromatography to concentrate specifically CT moieties from large volumes of plasma for concurrent immunoassay and bioassay measurements of circulating CT. These concurrent measurements of immunoextracted plasma CT demonstrate that for normal rats, our immunoassay measurements correspond to bioassay measurements. In 1-yr-old rats on a regulated feeding schedule, the biological (hypocalcemic) activity of CT recovered from 15 ml peripheral plasma ranged from less than 0.15 mU in starved males to 0.78 mU in feeding females. We conclude that biologically active CT circulates in normal rats and that the blood concentration of biologically active CT progressively increases during somatosexual maturation, being highest in old females, and increases acutely just before and during feeding.

We have measured plasma calcitonin in 135 untreated eucalemic men with lung cancer and a control/smoker population. Calcitonin levels were determined by radioimmunoassay and validated by immunoextraction. Plasma immunoreactive calcitonin moieties were purified by immunoadsorbent chromatography, treated with mercaptoethanol and urea, and characterized by gel filtration. Artifacts in human calcitonin radioimmunoassays of cancer-patient plasmas were detected by parallel plasma incubations in a salmon calcitonin radioimmunoassay system which does not detect human calcitonin and by immunoprecipitation of tracer at the end of radioimmunoassay incubations. Heating fresh plasmas to 65 degrees C for 1.5 hours reduced radioimmunoassay artifacts without loss of calcitonin moieties. Such characterization of hypercalcitoninemia in each of the histopathological types of lung cancer has raised some important questions about the interpretation of plasma calcitonin radioimmunoassay measurements in lung cancer. Based on inhibition of tracer-antibody binding, plasma calcitonin seemed to be elevated in 18% (14/80) of basal plasma samples obtained from patients with epidermoid or with anaplastic lung cancer. Unequivocal hypercalcitoninemia (heat stable, causing no inhibition of antibody-tracer binding in the salmon calcitonin radioimmunoassays, and immunoextractable with human calcitonin antibodies) was not found in any of the apparently hypercalcitoninemic plasmas from persons with epidermoid or anaplastic lung cancer. By contrast, unequivocal hypercalcitoninemia was found in 27% (15/55) of plasmas from patients with small cell carcinoma or adenocarcinoma. Most of the immunoreactive calcitonin recovered from small cell and adenocarcinoma lung cancer plasmas with unequivocally elevated calcitonin is much larger than calcitonin monomer.

A rat medullary thyroid carcinoma cell line, CA-77, has been established as a model system for investigating calcitonin biosynthesis and secretion. Growth of this cell line in serum-free defined medium provided suitable conditions for studying steroid hormone effects on the production of calcitonin and related peptides. After exposure for 5 days to a variety of steroids, only dexamethasone and corticosterone increased cellular content of calcitonin and a second secretory peptide (CCAP) derived from the same mRNA translation product as calcitonin. Glucocorticoids had no effect on cellular somatostatin, another secretory product of these cells. Increasing doses of dexamethasone progressively elevated cellular calcitonin and CCAP, with a maximal effect at 10(-8) M; 10(-9) M and lower doses were ineffective. On a molar basis, corticosterone was approximately 50-fold less potent than the synthetic glucocorticoid. An increase in cellular calcitonin content was observed only after 48 h of glucocorticoid treatment; a maximum increase (13-fold) occurred after 7 days. Glucocorticoids also increased basal calcitonin secretion. Similar effects were observed for cellular and secreted CCAP. Withdrawal of dexamethasone after 4 days of treatment lowered cellular calcitonin toward the level of control cultures. Dexamethasone pretreatment potentiated the acute secretory response to calcium for both calcitonin and CCAP, while no such enhancement was noted for calcium stimulation of somatostatin secretion. We conclude that the glucocorticoids specifically stimulate the production and secretion of calcitonin and CCAP, two secretory peptides derived from preprocalcitonin.

Crude and highly purified preparations of enteric peptide hormones were shown to stimulate trout calcitonin (tCT) secretion in vitro. Since the maximal stimulatory effects of crude pancreozymin/secretin preparations were seen to be greater than the secretory effects obtained with the individual purified enteric peptides, the current study has focused on the secretory effects of several combinations of enteric peptides. The additive and synergistic secretory effects of various specific peptide combinations are demonstrated. Marked (85-fold) stimulation tCT secretion occurs in response to combinations of synthetic secretin (5 X 10(-5)M) with pentagastrin (10(-6)M) or the carboxylterminal octapeptide of pancreozymin (10(-6)M). These findings have significance with regard to the potentiation of hormone action, and are preliminary evidence for the presence of separate receptors for various enteric peptide secretagogues on C-cells.

Calcitonin gene-related peptide (CGRP) and calcitonin (CT) are secreted by medullary thyroid carcinoma (MTC). Relationships between extracellular calcium ([Ca2+]e), cytosolic free calcium ([Ca2+]i) (as measured with fura-2), and secretion of immunoreactive CGRP and CT have been investigated in rat and human MTC cell lines. Rat MTC 6-23 cells responded to a rise in [Ca2+]e from 0.5 to 3.0 mM with a transient increase of [Ca2+]i, and the secretion of CGRP and CT was raised from 19 +/- 2 (mean +/- SE) to 122 +/- 28 pg rat CGRP/mg protein . min and from 33 +/- 8 to 155 +/- 42 pg rat CT/mg protein . min (P less than 0.01). In the human MTC (TT) cell line, a rise of [Ca2+]e from 0.5 to 3.0 mM did not affect [Ca2+]i, and the secretion of CGRP and CT remained unchanged at 7.0 +/- 1.1 ng CGRP/mg protein . min and 1.0 +/- 0.1 ng CT/mg protein . min. However, when the plasma membrane was bypassed by electropermeabilization, the release of CGRP and CT was stimulated by calcium with an ED50 of 0.5 microM and 0.3 microM, respectively. With ionomycin, the secretion of CGRP and CT was also stimulated up to 17-fold in [Ca2+]i-dependent manner. The results indicate a role of [Ca2+]i in the secretion of CGRP and CT and provide evidence for a defect in Ca2+ signal transduction in the human MTC cell line.

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.

Messenger RNAs for calcitonin (CT) and calcitonin gene related peptide (CGRP) have been detected in a human medullary thyroid carcinoma cell line. DNA sequences of their cloned cDNAs, and genomic restriction mapping, indicate that both mRNAs probably originate from a single gene; the separate mRNAs are derived by alternative processing. The calcitonin gene is expressed in 10 of 10 examined culture lines of human lung cancer; most of these lines express a higher ratio of CGRP to CT specific mRNA than does the medullary thyroid carcinoma cell line.

Recent studies of rat and human calcitonin (CT) gene expression have uncovered a diversity of secretory peptides. Here we report the results of testing two such non-CT secretory peptides for CT-like action in live rats and in cultured fetal rat long bone. One peptide, the carboxyl-terminal CT-adjacent peptide that is cosynthesized with CT, has no hypocalcemic effect and no inhibitory action on bone resorption in vitro. The other peptide, CT gene-related peptide, lowers blood calcium and inhibits bone resorption. In vitro experiments are consistent with the idea that CT gene-related peptide is acting at CT receptors in bone.

Evidence is presented which supports a model proposing that differential RNA splicing events may be used in expression of genes of the endocrine system to generate alternative polypeptide hormones. We previously reported that variation in the expression of the gene encoding the small polypeptide hormone calcitonin is associated with the production of a new calcitonin-like or pseudo-calcitonin (psi Cal) mRNA. A plasmid containing psi Cal cDNA sequences has been constructed, and calcitonin genomic clones have been isolated. Hybridization analysis reveals that calcitonin and psi Cal sequences are chromosomally linked and are present in the same nuclear RNA transcripts. Both calcitonin and psi Cal mRNAs are functional and encode different polypeptide products. These data are compatible with the proposed model that alternative RNA splicing of the transcript(s) of the calcitonin gene ultimately results in the production of different polypeptide products.

Messenger RNA from calcitonin-secreting rat medullary thyroid carcinoma lines directs the synthesis of a putative precursor of calcitonin when translated in wheat embryo lysate. The major translation product directed by mRNA isolated from a rat tumor with a calcitonin content representing greater than 2% of the total protein mass has an apparent molecular weight of 17,500 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitations of the cell-free translation products demonstrate that the 17,500-dalton protein has immunologic determinants which are specifically recognized by antisera to calcitonin. In addition, the relative prominence of this protein is significantly diminished when mRNA from lines with low calcitonin content is used to direct cell-free protein synthesis. This evidence suggests that the initial translation product of calcitonin mRNA is a 17,500-dalton species which must undergo subsequent processing to generate the secreted polypeptide.

A strategy is presented for identification of putative neurohormones that were predicted by sequence analysis of recombinant cDNa molecules. The nucleotide sequence of a cloned calcitonin cDNA insert predicts that the proteolytic excision of calcitonin from a 136-amino acid precursor should generate two additional peptides derived from regions flanking the calcitonin sequence on the NH2 and carboxyl termini. The predicted 16-amino acid COOH-terminal cleavage peptide (NH2-Asp-Met-Ala-Lys-Asp-Leu-Glu-Thr-Asn-His-His-Pro-Thr-Phe-Gly-Asn-COOH) was chemically synthesized using solid phase procedures. Antibodies against this synthetic hexadecapeptide detect immunologically cross-reactive material in tissue extracts from normal thyroid glands and calcitonin-producing medullary thyroid carcinomas. Each of the four calcitonin mRNA-directed cell-free translation products contain the immunological determinants for both calcitonin and the COOH-terminal cleavage peptide.

The rat calcitonin/alpha-CGRP and beta-CGRP genes are members of a neuroendocrine gene family that are coexpressed in a distinct cell-specific pattern. The alpha- and beta-CGRP gene sequences predict highly related peptides that differ by only a single amino acid. Using rat medullary thyroid carcinoma cells as an in vitro system for testing hormone responsiveness, we have shown that the calcitonin/alpha-CGRP and beta-CGRP genes are differentially regulated. The synthetic glucocorticoid, dexamethasone, caused a 2- to 5-fold increase in calcitonin and alpha-CGRP mRNAs due to an increase in the nuclear transcription rate. In contrast, the levels of beta-CGRP mRNA did not significantly change following dexamethasone treatment. Differential regulation within neuroendocrine gene families might be a common mechanism for generating functionally similar peptides in response to a variety of hormonal signals.

The selective control of gene expression results in diversified morphology and physiological function. Understanding the expression of differentiated function in molecular terms requires detailed characterization of the regulation of mRNA synthesis and catabolism. Although considerable emphasis has been placed on transcriptional control, the discovery of HnRNA and 'split genes' gives rise to the possibility of post-transcriptional regulation at the level of processing of nuclear precursors. A rat calcitonin-producing medullary thyroid carcinoma (MTC) line was used as a model for definition of certain aspects in regulation of gene expression. Serial transplantation of several rat MTC lines containing and secreting large amounts of calcitonin generated tumours in which calcitonin biosynthesis was decreased more than 10-fold. We report here that the conversion from a 'high' to a 'low' calcitonin producing state is associated with specific modifications of the calcitonin mRNA synthetic pathway and a consequence of these changes seems to be the production of a new cytoplasmic mRNA.

The ratios of calcitonin (CT) to calcitonin gene-related peptide (CGRP) mRNA, both generated by alternative RNA processing from the same primary RNA transcript, are shown by Northern blotting of cytoplasmic RNA to vary as a function of growth in a human medullary thyroid carcinoma cell line (TT). Upon initial seeding, CT mRNA levels are relatively high, and CGRP mRNA levels are relatively low. During the early logarithmic growth phase, CGRP mRNA levels rise severalfold, while CT mRNA levels change only slightly. As the cells approach confluence, both CT and CGRP mRNA levels rise. Subsequently, CGRP mRNA levels fall substantially in postconfluent cells, while CT mRNA levels remain high. By actinomycin D blocking of nascent transcription, we have shown that these growth-related, reversible changes in the ratio of CT to CGRP mRNA are not due to changes in mRNA stability. Our data rather suggest that TT cells reversibly alter alternative RNA-processing patterns dependent upon growth conditions in vitro, such that CT mRNA is lowest and CGRP mRNA is highest during rapid growth. The mechanisms underlying this RNA-processing alteration may play a role in certain patients with aggressive forms of medullary thyroid carcinoma, in whom a decrease or loss of CT levels heralds a poor prognosis.

Calcitonin contains an amino acid sequence that provides a potential site for glycosylation of the peptide at the asparagine at position 3. Preliminary evidence has suggested that there are glycosylated forms of calcitonin and its precursor, procalcitonin. The CA-77 rat medullary thyroid carcinoma cell line, recently developed to study calcitonin biosynthesis, was used to demonstrate the synthesis of glycosylated forms of this hormone by intact cells. Cultures were incubated in medium containing either [3H]mannose or [35S]methionine. Two species incorporating both labels were specifically immunoprecipitated when cell extracts were treated with calcitonin antibodies. Gel filtration chromatography in 6 M guanidine hydrochloride indicated that one peptide had a molecular weight of 5500, approximately 2000 daltons larger than calcitonin, while the second peptide had a molecular weight of 14 400, the approximate size of procalcitonin. Treatment of the [3H]mannose-labeled cell extract with endo-beta-N-acetylglucosaminidase H before immunoprecipitation removed the labeled sugar from the calcitonin species. Microsequence analysis of the radiolabeled immunoreactive 5500-dalton calcitonin species showed methionine at cycle 8 and mannose at cycle 3, suggesting that this peptide is calcitonin containing an N-linked oligosaccharide at Asn-3. These results suggest that in this cell line a minor but significant biosynthetic pathway exists for the production of glycosylated calcitonin from glycosylated procalcitonin.

We have recently reported immunochemical methods for the measurement and purification of rat calcitonin (CT). To identify a convenient source of CT-producing cells for studies of rat CT biosynthesis, we have propagated 16 transplantable series of medullary thyroid carcinoma (MTC) from 2 types of WAG/Rij rat MTC from The Netherlands. Tumor CT concentrations and growth and plasma CT accumulation were studied in successive transplant generations of each MTC series. Although most MTC series maintained characteristic predictable tumor CT levels, some MTC series acquired nearly 90% lower CT levels. While related MTC series tended to maintain similar predictable growth rates, several spontaneous and propagatable increases (up to 5-fold) in tumor growth rates were noted. These increases occurred with and without concomitant decreases in tumor CT. Tumor CT production ranged over 20-fold among these 16 MTC series. Serial plasma CT analyses can be used to estimate tumor growth and CT production in live MTC rats. Using serial plasma CT analyses, we can select for serial transplantation tumors with specific growth and CT characteristics. This should facilitate propagation of MTC series needed for particular studies of CT production and cancer.

Studies were carried out on specific pathogen-free rats to evaluate the effects of aging and dietary manipulation on serum and thyroid calcitonin (CT) levels. Male Fischer 344 rats were randomized at 6 weeks of age to six dietary groups and subsequently maintained on the following dietary regimens. Group 1 rats were fed ad libitum throughout life; group 2 rats were fed 60% of the ad libitum food uptake, but received the same amounts of calcium, phosphorus, and vitamin D; group 3 rats were fed as the group 2 animals until 6 months of age and from then on were fed ad libitum; group 4 rats were fed ad libitum until 6 months of age and then switched to 60% food restriction; group 5 rats were fed ad libitum on food isocaloric with that of group 1 rats, but containing only 60% of the protein. Group 6 rats were killed at 6 weeks of age to serve as baseline controls. Ten rats were killed in each of the remaining five groups 15 h postprandial at 6-month intervals. The following observations were made. Serum CT increased with age similarly in the ad libitum fed group 1 and 5 rats. Food restriction markedly inhibited the increase in serum CT, and the effect was more profound in animals whose food intake was restricted after 6 months of age (group 4) than in animals on lifelong food restriction (group 2). In rats switched from food restriction to ad libitum feeding (group 3) at 6 months of age, serum CT increased with age to levels identical with those of lifelong ad libitum fed group 1 animals. Thyroid CT showed a similar pattern of age-dependent and dietary modulated changes. In contrast, aging and dietary modulation had no appreciable effect on serum calcium levels, except at 27 months of age when the serum calcium level of group 1 animals increased dramatically from the level for 24-month-old animals. There was a weak positive correlation between serum calcium and serum CT (r = 0.627; P = 0.02) and a highly significant positive correlation between serum CT and thyroid CT (r = 0.917; P = 0.001). These findings indicate that elective and therapeutic restriction of food intake might also attenulate CT levels in humans, with potentially adverse implications for skeletal homeostasis.

The biological significance of peptide hormone glycosylation is uncertain. To examine the effect of Asn-linked glycosylation on calcitonin's bioactivity we purified glycosylated calcitonin from a transplantable rat medullary thyroid carcinoma. Glycosylated calcitonin constituted 2.3% of the total extracted immunoreactive calcitonin. The structure of this peptide differed from nonglycosylated calcitonin only by the oligosaccharide modification of asparagine 3. Affinity of glycosylated calcitonin for lentil lectin indicated that the oligosaccharide was a complex processed form. In a standard in vivo bioassay glycosylated calcitonin had a markedly reduced hypocalcemic activity compared to nonglycosylated calcitonin, an effect most likely due to the presence of the oligosaccharide.

Calcitonin (CT) deficiency has been suggested as an etiologic factor in postmenopausal osteoporosis (PM-OP). Basal immunoreactive calcitonin (iCT) was measured with a sensitive radioimmunoassay (RIA) in 62 PM-OP women with compression fractures (CF) and in 28 normal age-matched women. Mean iCT values in the two groups were not significantly different (43.5 and 45.1 pg/ml, p greater than 0.10). In the 62 PM-OP females, no significant correlation was noted between basal plasma iCT levels and (1) age; (2) severity of disease as assessed by number of CF; (3) serum calcium, phosphorus, alkaline phosphatase, and immunoreactive parathyroid hormone; and (4) total bone mass as assessed by neutron activation analysis determinations of total body calcium (TBC). In 20 PM-OP patients treated for 24 mo with 100 Medical Research Council (MRC) units daily of synthetic salmon CT, no correlation was observed between basal plasma iCT and response of bone mass (TBC) to therapy. These data suggest that basal CT is not decreased in women with PM-OP, and that the level of circulating CT does not influence therapeutic changes in bone mass during CT therapy. CT is probably not a major etiologic or pathogenetic factor in PM-OP.

Improved methods for isolation, characterization, and quantitation of immunochemically heterogeneous forms of calcitonin (CT) in tissue and plasma must be developed before the biological origins and clinical importance of CT moieties can be elucidated. We are now proposing reversed-phase high-performance liquid chromatography (RP-HPLC) as one possible means of achieving high recovery, high resolution of CT moieties. In this paper we report RP-HPLC analyses of trace amounts of radiolabeled and unlabeled synthetic human and rat CT. We have systematically evaluated our application of RP-HPLC by employing several elution modes, including isocratic and gradient elution, as well as several elution reagents. We determined that high recovery and high resolution were best achieved with alkyl ion-pairing reagents, such as tetrabutylammonium phosphate, pH 7.5, or sodium sulfonyl hexane, pH 3.5. The most sensitive UV detection of trace amounts of CT was achieved with tetrabutylammonium phosphate buffer (TBAP). We recommend for RP-HPLC of CT a C18-bonded silica column and elution with a 20-min linear gradient of methanol-water (20:80 to 80:20, v/v) containing 0.005 M TBAP. Combined with appropriate extraction procedures, such as silica adsorption or immuno-adsorbant chromatography, this paired-ion RP-HPLC method can be an important aid in achieving more accurate and extensive information about CT moieties in biological samples. This method will also allow the rapid, optical detection and quantitation of CT moieties recovered from tissues, and perhaps from plasmas.

Serum immunoreactive calcitonin concentration (iCT) was determined in nine subjects with idiopathic hypercalciuria (IH), prior to and during oral phosphate supplementation (500 mg qid) to test the hypothesis that a renal phosphate leak was the primary defect resulting in IH in these patients. Prior to the oral phosphate supplementation, serum iCT was significantly elevated in the IH group, when compared to 26 normal individuals (75 +/- 7 vs 45 +/- 4 pg/ml, mean +/- SE, P less than .001). During oral phosphate treatment, however, serum iCT decreased to levels not significantly different from normals (39 +/- 3 and 50 +/- 5 pg/ml after 4 and 8 weeks, respectively). When data prior to and during phosphate supplementation were pooled, there was a significant correlation (r = .70, N = 26, P less than .001) between serum iCT and serum calcium. These observations suggest that the increased serum iCT in these subjects was a response to slight elevations in serum calcium, which are the result of normal physiological mechanisms to correct the renal phosphate leak.

A previous report from this laboratory (Birnbaum R. S., O'Neil, J. A., Muszynski, M., Aron, D. C., and Roos, B. A. (1982) J. Biol. Chem. 257, 241-244) provided immunochemical and biochemical evidence for the existence of a secretory peptide derived from the noncalcitonin region of rat preprocalcitonin. By a variety of criteria, we demonstrated that this naturally occurring peptide was similar, if not identical, to a synthetic peptide which consisted of the NH2-terminal 16 residues of the calcitonin mRNA translation product. We have now purified this peptide from rat medullary thyroid carcinoma and sequenced it. A rat tumor of the 1-2-4 tumor series was extracted in 0.1 N HCl yielding 900 micrograms of immunoreactive peptide. The peptide was purified to homogeneity by: 1) trichloroacetic acid precipitation of contaminating protein; 2) gel filtration; and finally, 3) reverse phase high pressure liquid chromatography. Overall yield was approximately 24%. Amino acid analysis and sequencing of the peptide yielded a composition and sequence identical with that of the synthetic peptide.

Calcitonin was measured in the plasma of 13 patients with medullary throid carcinoma and one patient with a calcitonin-producing islet cell carcinoma of the pancreas. The hormone was measured by the simultaneous application of two calcitonin antisera which had differing specificity. Calcitonin measurements were also made of gel filtration (Bio Gel P30) fractions of plasma samples. Although the two antisera were of comparable sensitivity for the measurement of human calcitonin standards, they reacted differently with the calcitonin in the plasma samples. One antiserum (LD-1) gave consistently higher estimations of hormone concentration in the plasma of patients with medullary thyroid carcinoma than the other (LD-26). By contrast, the concentration of calcitonin from the islet cell carcinoma of the pancreas was higher in the LD-26 assay. Multiple gel filtration peaks of immunoreactive calcitonin activity were present in both thyroidal and nonthyroidal calcitonin. Furthermore, the two different antisera identified differing immunochemical peaks of calcitonin activity for agiven plasma sample. These findings demonstrate the presence of immunochemical heterogeneity in plasma calcitonin and suggest the presence of immunological differences between thyroidal and nonthyroidal calcitonin.

We have developed a sensitive immunoassay for murine calcitonin which can measure the basal concentration of the hormone in peripheral plasma of rats as well as the calcitonin, extracted from rat thyroid tissue. Human calcitonin was used for tracer, standard, and antibody production. Assay methods were devised to minimize artifacts which have been shown to spuriously influence immunoassay performance. The basal plasma concentration of calcitonin in rats was 6 to 75 pg. per milliliter. Infusions of calcium, pentagastrin, and PGE2 produced a 2- to 7-fold increase in plasma calcitonin. Elevated concentrations of calcitonin were demonstrated in tumor specimens obtained from the second and fourth generations of a transplanted rat medullary thyroid carcinoma. Our results demonstrate that calcitonin circulates in the peripheral plasma of rats in the basal state and that hormone concentrations change during functional tests of secretion. This assay will permit studies of calcitonin secretion in physiological and pathophysiological states in the rat.

To test whether mobilisation of immunoreactive calcitonin in response to calcium challenge is reduced in postmenopausal osteoporosis, seventeen postmenopausal osteoporotic women with compression fractures and ten normal age-matched women were given intravenous infusions of 3 mg/kg elemental calcium over a 10 min period. Blood samples were obtained 5 min before and at 0, 10, 20, and 60 min after start of infusion for the measurement of serum calcium and plasma immunoreactive calcitonin. Serum calcium increased significantly from baseline in both normal and osteoporotic groups; immunoreactive calcitonin increased significantly in the controls 10 min and 20 min after the start of infusion, but in the women with osteoporosis calcitonin levels did not change significantly at any time. 20 min after the start of infusion the change in immunoreactive calcitonin from baseline was significantly less in osteoporotic women than in the controls. These data are consistent with a decreased immunoreactive calcitonin response to calcium infusion in postmenopausal osteoporotic women, and suggest that calcitonin deficiency may be involved in the development of postmenopausal osteoporosis.

We have developed methods for the long-term maintenance of monolayer cultures of both human and trout calcitonin-secreting (C-) cells (Roos et al., 1974a, 1975). Secreted calcitonin (CT) can be measured by specific and sensitive radioimmunoassays for human calcitonin (HCT) and trout calcitonin (TCT) (Deftos, 1971; Deftos et al., 1974; Roos et al., 1974b). The combined application of these cell culture and radioimmunoassay methods to the in vitro study of CT secretion has revealed fundamental differences in the regulation by divalent ions of CT secretion in mammalian and nonmammalian C-cells. Our results also indicate a specific role for secretin as a CT secretagogue in trout. In addition to establishing the individual secretory effects of calcium and the enteric polypeptide hormones on the C-cells of both species, we have investigated the combined effects of these agents in both human and trout C-cell cultures. The noncompetitive augmentation of the secretory effects resulting from the actions of specific combinations of these secretagogues in each of these C-cell populations suggests that CT secretion is regulated by co-ordinate interaction of specific ionic and peptide secretagogues. An elucidation of the cellular mechanisms involved in such multifactorial control of C-cell function should result from the continued investigation of CT secretion in these C-cell cultures.

We have recently reported the establishment of 16 series of calcitonin-producing transplantable rat medullary thyroid carcinoma. In the present study, these tumor series have been evaluated for the presence of somatostatin-like immunoreactivity. Each of the series contained detectable levels of both peptides. Immunoreactive somatostatin varied from less than 1 ng/mg of protein to almost 500 ng/mg of protein. The range of immunoreactive calcitonin was 0.3 to 30 micrograms/mg of protein. Although somatostatin-like immunoreactivity was always less than that of calcitonin, the levels in certain series were as high as those found in neural or endocrine tissues used for in vitro studies of somatostatin elaboration. No significant correlation was found between tissue levels of these two peptides. Two tumor lines were generated by initiation of tumor growth with cells from primary monolayer cultures. Levels of both immunoreactive calcitonin and somatostatin significantly differed from those of the parent lines, which were maintained by serial passage of tissue fragments only. Plasma somatostatin-like immunoreactivity assessed in two tumor series with high (149 ng/mg of protein) and low (1.5 ng/mg of protein) tissue levels was 3100 and 50 pg/ml, respectively. Gel filtration chromatography of tissue and blood extracts showed a predominant peak (greater than 90%) of immunoreactive somatostatin eluting at the position of the native hormone. Three other peaks were resolved in the tissue extract with estimated molecular weights of 14,000, 8,700, and 5,000. The high level of somatostatin-like immunoreactivity and the presence of multiple large forms suggest that certain tumor lines will prove valuable for studies of somatostatin biosynthesis and secretion.

Extracts of transplantable rat medullary thyroid carcinomas were fractionated by concanavalin A-agarose chromatography and SDS-polyacrylamide gel electrophoresis. While tumor extracts contained mostly (greater than 90%) native calcitonin, 4 distinct larger calcitonin species (Mr = 27,000, 17,000, 13,700, and 9,600) were also observed. Glycoprotein fractions from lectin-affinity chromatography of the tumor extracts contained 0.3-1.5% of the total immunoreactive calcitonin. Gel electrophoresis of the pooled glycoprotein fractions demonstrated that the predominant forms of immunoreactive calcitonin were larger than calcitonin monomer. One high molecular weight species (Mr = 9,600), isolated in sufficient quantity from the polyacrylamide gels for reapplication to concanavalin A-agarose, was specifically eluted from the lectin, indicating that it was a glycoprotein. These results are the first evidence that intact C-cells contain at lest one high molecular weight glycoprotein form of immunoreactive calcitonin and suggest that addition and removal of carbohydrate side chains occur during processing of calcitonin precursors to the native hormone.

cDNA analyses predict that the human calcitonin gene encodes the two precursor proteins of calcitonin and calcitonin gene-related peptide (CGRP). The mRNAs for the putative precursors of these peptides are derived from alternative processing of the primary transcript of this gene. The 75 amino-terminal residues of each preprohormone are predicted to be identical. We have developed the TT human medullary thyroid carcinoma cell line as a model to study human calcitonin gene expression. Mature calcitonin and CGRP are major secretory products of this cell line. Extracts of TT cells radiolabeled with amino acids for 1 h contained only one peak of either immunoprecipitable calcitonin or CGRP on reverse-phase high-pressure liquid chromatography. Each of these species had an Mr = 12,800 as estimated by gel filtration chromatography. The results of partial amino-terminal microsequencing of these two precursors were identical. Residues 2, 11, and 14 of each precursor were proline, and residues 7, 17, 24, 25, 26, and 29 were leucine. The unique alignment of the positions of these amino acids with the cDNA-predicted sequence for the common region of preprocalcitonin and preproCGRP suggests that the site of signal sequence cleavage occurs after residue 25 of both preprophormones. These studies represent the first identification of procalcitonin (116 amino acids) from a human cell line and of proCGRP (103 amino acids) from any tissue.

We have studied the effects of several porcine enteric polypeptide hormones on trout calcitonin (tCT) secretion in long-lived monolayer cultures of calcitonin-secreting (C-) cells derived from trout ultimobranchial glands. Gastrin, pancreozymin (CCK-PZ), glucagon, and secretin have dose-related stimulatory effects on tCT secretion; the distinct effects of secretin on tCT secretion are in contrast to its lack of CT secretagogue activity in some mammals. Synthetic peptide hormones and/or their structural analogs have variable secretory effects which correspond in general to the potency of the analogs for the well-recognized biological actions of these various peptide hormones in mammals. Although enteric peptide hormones are present in fish, their physiological role in the regulation of CT secretion has not been studied. These in vitro studies indicate a possible role for these hormones in the control of tCT secretion and support the concept that there are differences in the regulators of C-cell function in higher and lower vertebrates. In vitro studies of fish CT secretion are needed to establish the physiological significance of these in vitro studies of fish C-cell function.

The parafollicular-cell (C-cell) hormone calcitonin (CT) can preserve or even augment skeletal mass by inhibiting osteoclast-mediated bone resorption. The possibility of an additional anabolic skeletal influence has also been raised: C cells might, via CT or other secretory products, affect osteoblast-mediated bone formation. The 57-residue amino-terminal procalcitonin cleavage peptide, N-proCT, has recently been identified in human and rat C cells, where it is made and secreted in equimolar amounts with CT. The coelaboration of N-proCT and CT and N-proCT's sequence conservation during evolution prompted us to investigate the potential skeletal bioactivity of N-proCT. We found that synthetic human N-proCT, at nanomolar concentrations, stimulated proliferation of normal and neoplastic human osteoblasts. At maximally effective doses, human N-proCT caused more than a 100% increase above the control rate of DNA synthesis, an effect comparable to the maximal growth effect of insulin, a potent mitogen for osteoblasts. Human N-proCT exerted a similar maximal mitogenic effect in chicken osteoblast cultures but at 1000-fold greater concentrations than in human bone-cell cultures. The bone-cell action of N-proCT was potentiated with insulin with a greater than 200% increase in DNA synthesis at high insulin concentrations. In sharp contrast to these findings for N-proCT, the other bioactive C-cell peptides, CT and somatostatin, showed no mitogenic effects in human or chicken osteoblast cultures. Our results indicate that the action of N-proCT on cultured bone cells is separate from and potentiated by insulin, a known growth factor. Unlike insulin and related growth factors such as insulin-like growth factor I, N-proCT is not mitogenic in skin fibroblast cultures. We propose that N-proCT is a C-cell hormone that promotes bone formation via stimulatory actions on osteoblasts and preosteoblasts.

The calcium regulatory role of calcitonin (CT) in murine physiology was evaluated with a combination of immunological techniques, bioassays, and gland ablation. Passive immunization of rats with neutralizing CT antibodies caused an immediate but transient increase in plasma calcium with a time course similar to that observed in thyroparathyroidectomized animals. In animals fasted during the day and fed at night (1700-0700 h), acutely decreasing endogenous CT (< 100 pg/ml) by thyroparathyroidectomy or by passive immunization with CT antibodies resulted in a greater increase in plasma calcium in the preprandial (1600 h) than in the postprandial inverse correlation between immunoreactive CT and plasma calcium, a known secretagogue for CT. In these animals on a restricted feeding schedule, a sharp preprandial (1600 h) rise in immunoreactive and bioactive CT and a circadian rhythm in plasma calcium were also observed. The onset of the increase in circulating CT preceded the onset of feeding and coincided with or shortly preceded the daily nadir of plasma calcium. These findngs establish that CT has a primary calcium regulatory role in murine physiology. More specifically, they demonstrate that 1) the postthyroparathyroidectomy increase in plasma calcium is due specifically to the loss of CT, 2) passive immunization is a feasible technique for evaluating the actions of endogenous CT, 3) the role of CT in calcium regulation cannot be restricted to that of preventing prandial and postprandial increase in blood calcium, and 4) factors other than calcium must be involved in the control of CT secretion in the nonfed state.

The sequence of rat procalcitonin reveals that calcitonin is located within the precursor's midregion, flanked by two potential polybasic cleavage sites that separate it from amino- and carboxyl-terminal domains. Cleavage at the polybasic sites during precursor processing to generate the 32-residue calcitonin should also generate 57- and 16-residue peptides from the amino- and carboxyl-terminal flanking regions. The carboxyl-terminal flanking hexadecapeptide and its coordinate secretion from C cells with calcitonin have been previously reported. In the present study we have focused on the predicted 57-residue amino-terminal procalcitonin cleavage peptide (N-proCT). We raised antisera to synthetic peptides homologous to the carboxyl- and amino-terminal regions of the putative 57-amino-acid N-proCT and screened calcitonin-rich neoplastic and nonneoplastic C-cells for these two immunoreactivities. A single species of 7.4 kilodaltons detected in C cells by gel filtration and reversed-phase HPLC analyses accounts for most of the carboxyl- and amino-terminal immunoreactivities and possesses the biochemical and biological features predicted for N-proCT. When C cell hyperplasia is induced by a high fat diet, thyroidal levels of calcitonin and N-proCT increase in parallel. In neoplastic C cell cultures, N-proCT and calcitonin concentrations are nearly equimolar in both cellular extracts and basal medium; dexamethasone increases both the cellular and secreted concentration of these peptides. Basal and dexamethasone-treated cultures show calcium-dependent, parallel secretion of N-proCT and calcitonin. Thus, the 57-residue N-proCT predicted from analysis of the procalcitonin sequence is a secretory peptide that appears to be present in equimolar amounts and coordinately regulated with calcitonin in vivo and in vitro.

A chimeric plasmic containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. The cloned calcitonin DNA was used to characterize the expression of calcitonin mRNA. Cytoplasmic mRNAs from calcitonin-producing rat medullary thyroid carcinoma lines and from normal rat thyroid glands contain a single species, 1050 nucleotides long, whch hybridizes to the cloned calcitonin cDNA. The concentration of calcitonin mRNA sequences is greater in those tumors that produce larger amounts of immunoreactive calcitonin. RNAs from other endocrine tissues, including anterior and neurointermediate lobes of rat pituitary, contain no detectable calcitonin mRNA.

Medullary thyroid carcinoma is a tumor of the calcitonin-secreting thyroid C-cell, with a variable malignant potential. Virulent tumors are characterized by decreased calcitonin production, suggesting the emergence of a less differentiated medullary thyroid carcinoma cell. In order to further delineate relationships between tumor progression and status of differentiation of medullary thyroid carcinoma cells, we have sought to chemically manipulate the TT cell line, an established culture of human medullary thyroid carcinoma, derived from a patient with aggressive disease. We found that the phorbol esters, 12-O-tetradecanoyl phorbol 13-acetate and phorbol 12,13-dibutyrate 1) altered the morphology of the TT cells towards that of high-calcitonin-containing cells; 2) enhanced calcitonin secretion 7-fold; 3) increased calcitonin production at the transcriptional level by 2-fold; 4) inhibited cellular proliferation; and 5) decreased, by 80%, the levels of the c-myc gene mRNA. These data suggest that phorbol esters induce, in human medullary thyroid carcinoma cells in culture, a programmed pattern of events resulting in differentiation of these cells.

The 32-amino acid form of the peptide hormone calcitonin is the product of a series of post-translational processing steps of a 13,400-dalton precursor, procalcitonin. We have now identified the steps involved in proteolytic paring of the precursor to the mature secretory form. Cultures of the CA-77 cell line were radiolabeled and the various forms of calcitonin were isolated by specific immunoprecipitation followed by fractionation on gel filtration and reversed-phase high performance liquid chromatography. Pulse-chase kinetics showed that procalcitonin was cleaved to a 6,500-dalton biosynthetic intermediate which was subsequently processed to the size of mature calcitonin (3,400 daltons). Partial microsequencing of the [35S] methionine-labeled intermediate indicated that the sequence consisted of the COOH-terminal 52 residues of procalcitonin. Partial microsequencing of the [35S]methionine- or [3H]proline-labeled 3,400-dalton species revealed that it was indistinguishable from naturally occurring, amidated calcitonin. These data define the major pathway for calcitonin biosynthesis in this neoplastic cell line and presumably in normal cells.

Monolayer cultures of human calcitonin-secreting cells (C-cells) have been derived from medullary thyroid carcinomas. The cultures were prepared from cell suspensions obtained by enzymatic digestion of surgical specimens of the tumor. Human calcitonin (hCT) secretion by these cells was studied using a specific radioimmunoassay for the hormone. The cultures could be used for reproducible secretion studies up to 40 days after their initiation; they demonstrated a linear rate of hormone secretion in the basal state for at least 4 h. Calcium, pentagastrin, and prostaglandin E2 (PGE2) each produced a marked increase (up to 7-fold) in hormone secretion. Magnesium had no apparent secretory effect; and compared to PGE2, PGF2alpha had only a small secretory effect. In addition to responding to specific secretagogues shown to regulate calcitonin secretion in vivo, the secretory effects of each of the secretagogues could be raipdly reversed. Therefore, these cultures of human C-cells exhibit secretory responses which are quantitatively and qualitatively similar to C-cells in vivo. Accordingly, such cultures provide a useful model to study the regulation of calcitonin secretion in human C-cells.