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The glycosyl nature of the receptor for the peptide hormone calcitonin has been investigated in a human breast cancer cell line, T 47D. Studies have been carried out to assess the ability of various lectins and of the antibiotic tunicamycin to inhibit specific binding of calcitonin to the cells, to reduce cross-linking of photoactive calcitonin to a macromolecular receptor component and to influence calcitonin stimulation of cyclic AMP. Pre-incubation of cells with low concentrations of tunicamycin for 72 h resulted in a reduction of total specific binding by approx. 80% and a 40% reduction in calcitonin-stimulated adenylate cyclase; formation of the cross-linked receptor component was also inhibited. Wheat-germ lectin showed the most marked inhibition of total specific binding and cyclic AMP production. However, cross-linking of photoactive calcitonin to receptor component was totally inhibited by this lectin. Soya-bean lectin brought about very little reduction in total specific binding but had more profound effects on calcitonin-stimulated cyclic AMP production and cross-linking of photoactive calcitonin. Concanavalin A and lentil lectin showed some inhibition of all parameters. The data indicate that the calcitonin receptor in T 47D cells is associated with glycosyl moieties, the major contributors of which are N-acetyl-D-glucosamine residues, but N-acetyl-D-galactosamine and mannose residues are also associated.

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.

Parathyroid hormone related protein (PTHrP) has been demonstrated in the tumour cells of squamous cell carcinomas originating in a variety of organs, in undifferentiated small cell tumours of the bronchus, and in carcinoma of the kidney. The protein hormone is thought to produce the hypercalcaemia which may complicate some of these malignancies. By using an antibody raised in rabbits against the N-terminal portion of the molecule, the polypeptide may be demonstrated in cells of the prickle cell layer of normal skin and in the cells of hair follicles. Skin tumours showing squamous or hair follicle differentiation are shown to contain the protein antigen, while basal cell carcinomas and tumours with sweat gland differentiation do not. The hormone may be primordial in origin and the progenitor of parathyroid hormone.

A protein with biological activities similar to parathyroid hormone (PTH) has been purified from serum-free culture medium obtained from a human lung cancer cell line (BEN). A major protein band of 18 kDa was obtained on NaDodSO4/polyacrylamide gels, with faint bands at 35 kDa and 67 kDa. Biological activity was associated only with the 18-kDa band. Amino acid sequence analysis of the material purified by HPLC revealed that 8 of the 16 residues were identical with those of human PTH. Antibody raised to a corresponding synthetic peptide recognized the PTH-related material but showed less than 1% cross-reactivity with human PTH amino-terminal peptides. BEN cells contained PTH DNA, but not PTH messenger RNA, indicating involvement of another gene. The purified PTH-related protein had a specific biological activity approximately equal to 6 times greater than that of bovine PTH(1-34). The PTH-related protein may have a role in the syndrome of humoral hypercalcemia of malignancy.

Specific high-affinity receptors for 1,25-dihydroxycholecalciferol [1,25-(OH)(2)D(3)] have been described recently in broken-cell preparations of several cultured human breast cancer cell lines including the T47 D line. It was necessary to determine whether intact breast cancer cells in culture would bind 1,25-(OH)(2)D(3) specifically and whether the next step in the proposed scheme of action, i.e. nuclear translocation, occurred. The following results were obtained. (1) Specific uptake of 1,25-(OH)(2)D(3) by T47 D cells occurs in intact cells in culture. (2) The rate of uptake is proportional to medium 1,25-(OH)(2)D(3) concentration but is slow compared with that of other steroid hormones, e.g., oestradiol, under identical conditions. Even at 0.5nm-1,25-(OH)(2)D(3) in the medium, at least 4h are required to reach maximum compared with less than 1h for oestradiol binding. (3) Estimation of binding characteristics by Scatchard analysis indicates a single class of binding sites with K(d) of 68pm and 11800 binding sites/cell, which are similar results to those obtained with broken-cell preparations. (4) Inclusion of various vitamin D metabolites in the incubation medium decreased specific binding of 1,25-(OH)(2)D(3) by the intact cells in a manner identical with their effects in the broken-cell preparation and with potencies similar to their potency on Ca(2+) transport and bone resorption in vivo. Order of potency was 1,25-(OH)(2)D(3)>(24R)-1,24,25-trihydroxycholecalciferol >>25-hydroxycholecalciferol>(25R)-24,25-dihydroxycholecalciferol >>(25R)-25,26-dihydroxycholecalciferol. (5) In the 1,25-(OH)(2)D(3)-depleted state, 80% of the 1,25(OH)(2)D(3) receptor is found in the cytosol fraction of the cells even when the subcellular fractionation is performed under low-salt conditions. By contrast after incubation with [(3)H]1,25-(OH)(2)D(3), 59% of the specific 1,25-(OH)(2)D(3) binding is found in the partially purified nuclei fraction. These data indicate that nuclear translocation of the receptor-hormone complex takes place in the intact T47 D cell. The results also support the hypothesis that the 1,25-(OH)(2)D(3) receptor is functional in this cultured breast cancer cell line, which may provide a useful model for further study of the early biochemical events in 1,25-(OH)(2)D(3) action.

We have developed a mouse marrow culture system, in which multinucleated cells (MNCs) are formed within 6-8 days. These MNCs showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb calcified dentine. 1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3] stimulated the formation of TRACP-positive MNCs, and salmon calcitonin (CT) inhibited it. In this study, we examined whether the TRACP-positive MNCs formed from mouse marrow cells possess CT receptors, another typical characteristic of osteoclasts. Mouse marrow cells cultured for 8 days with 10 nM 1 alpha, 25(OH)2D3 and freshly isolated authentic mouse osteoclasts were incubated with [125I]-salmon CT in the presence or absence of excess amounts of unlabeled CT, stained for TRACP, and processed for autoradiography. The [125I]-CT exclusively bound to TRACP-positive mononuclear cells and MNCs formed in the 1 alpha, 25(OH)2D3-treated cultures and also to isolated mouse osteoclasts. Both [125I]-CT binding and TRACP activity were induced simultaneously on mononuclear cells on day 3 in the cultures treated with 1 alpha, 25(OH)2D3. CT markedly stimulated cAMP production in the 1 alpha,25(OH)2D3-treated cultures. The CT-dependent cAMP production increased in parallel with the increase in the number of TRACP-positive MNCs formed. Neither freshly isolated marrow cells nor cells cultured without 1 alpha, 25(OH)2D3 showed CT-induced cAMP accumulation. Furthermore, CT induced cytoplasmic contraction of both marrow-derived MNCs and isolated osteoclasts. These results clearly indicate that 1 alpha,25(OH)2D3 induces TRACP activity and CT receptors almost simultaneously in mouse marrow cultures, and the MNCs formed in vitro respond to CT as authentic osteoclasts do.

A specific receptor for 1,25-dihydroxyvitamin D has been demonstrated in a cultured human breast cancer cell line. This is the first such demonstration in any cancer cell. It may explain the high incidence of metastatic bone destruction and hypercalcaemia in this common malignancy, and the limited success of other steroid-receptor assays in predicting the response of breast cancer to therapy.

The effects of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP production were studied in osteoclast-rich cultures derived from medullary bone of laying hens and from the long bones of newborn rats. Cyclic AMP was assayed biochemically in replicate cultures, and furthermore, changes in cytoplasmic fluorescence were sought by indirect immunofluorescence with rabbit anti-cyclic AMP and FITC-labelled goat anti-rabbit IgG. Treatment of rat osteoclasts with calcitonin increased cyclic AMP formation as measured biochemically, and this was confirmed by the immunofluorescence method. No such increase took place in chick osteoclasts. Prostaglandin E2 increased cyclic AMP production in both rat and chick osteoclasts as determined by both methods. Since the immunofluorescence method failed to detect a response to parathyroid hormone either in chick or rat osteoclasts, its variable biochemical effects were concluded to be due to actions on contaminating osteoblasts in the cultures. Thus it has been possible with a combined biochemical and immunocytochemical approach to define the cyclic AMP responses to the calcium-regulating hormones in rat and chick osteoclasts. The failure of calcitonin to increase cyclic AMP in chick osteoclasts identifies a need to investigate the nature of calcitonin action on avian osteoclasts, which may contribute to understanding of its actions on mammalian cells.

Peptides containing residues 1-34 of parathyroid hormone-related protein (PTHrP) and of bovine parathyroid hormone (bPTH), and recombinant full-length PTHrP(1-141) were infused i.v. into anaesthetized thyroparathyroidectomized rats to compare their action and potency on the renal handling of calcium, phosphate and cyclic AMP (cAMP) in vivo. All three peptides decreased the excretion of calcium and increased the excretion of phosphate and cAMP in the urine, with PTHrP(1-34) and PTHrP(1-141) having virtually equipotent effects. Thus the essential requirements for the major physiological activity of PTHrP on the kidney are contained within the 34 amino-terminal amino acids. For all three peptides, the lowest infusion rate that increased phosphate and cAMP excretion was 0.01 nmol/kg per h, whereas the lowest infusion rate that decreased calcium excretion was 0.025 nmol/kg per h for the PTHrP peptides and 0.1 nmol/kg per h for bPTH(1-34). The response to the PTHrP peptides was maximal at an infusion rate of 0.1 nmol/kg per h for both calcium and phosphate. Since the kidney is either equally sensitive to PTHrP and bPTH(1-34), or more sensitive to PTHrP than to bPTH(1-34), the hypercalcaemia of humoral hypercalcaemia of malignancy may develop because uncontrolled secretion of PTHrP increases the renal reabsorption of calcium to such an extent that even a modest increase in the inflow of calcium into the blood raises plasma calcium concentration.

Induction of osteoclast-like multinucleated cells (MNCs) by various fragments of PTH-related protein (PTHrP) was examined in mouse marrow cultures. Osteoclast-like MNCs were defined as tartrate-resistant acid phosphatase (TRACP)-positive MNCs with calcitonin receptors. In all experimental protocols examined, PTHrP-(1-34) induced TRACP-positive MNCs at almost the same rate as PTH-(1-34). PTHrP-(1-29) was less potent than PTHrP-(1-34). PTHrP-(1-25) and PTHrP-(1-14) had no effect. PTHrP-(1-34) was more potent than PTH-(1-34) in increasing the accumulation of cAMP, but the former appeared to lose its activity more rapidly than the latter. Isobutylmethylxanthine increased the effect of PTHrP-(1-34) and PTH-(1-34) in inducing TRACP-positive MNCs. Furthermore, the calcium ionophore A23187 significantly increased the formation of TRACP-positive MNCs. The effect of PTH-(1-34) and PTHrP-(1-34) in inducing TRACP-positive MNCs was potentiated by adding A23187 but suppressed by adding verapamil simultaneously. The inhibition by verapamil was overcome by adding A23187. [Nle8,18,Tyr34]PTH-(3-34)amide inhibited the effect of not only PTH-(1-34) but also PTHrP-(1-34) in inducing both the accumulation of cAMP and the TRACP-positive MNC formation. These results show that PTHrP is a potent stimulator of osteoclast-like MNC formation to almost the same extent as PTH. It increases the number of osteoclast-like MNCs by a mechanism involving cAMP and calcium ions, and is most likely mediated through the same receptor. The controversial results of the bone-resorbing activity of PTH and PTHrP reported so far may be explained by the differences in the relative potencies of the respective hormones in increasing the intracellular cAMP and calcium ions and by the shorter half-life of PTHrP in culture medium.

Calcitonin receptors have been characterized for the first time in isolated osteoclasts. These receptors have been demonstrated by autoradiographic and biochemical methods, and the cells have also been shown to respond to calcitonin with a dose-dependent increase in cyclic AMP. The receptors in rat osteoclasts are specific and of high affinity (dissociation constant, Kd, 1 to 6 X 10(-10) M), and are present in greater numbers than in any cell previously studied (greater than 10(6) per cell). Chemical cross-linking of 125I-labeled salmon calcitonin to osteoclasts using disuccinimidyl suberate resulted in identification of a receptor component with a relative molecular weight of 80,000-90,000. By counting grains in autoradiographic experiments, we found that greater than 80% of specifically bound radioactivity was associated with multinucleate osteoclasts and the remainder was associated with mononuclear cells that are not osteoblasts, but that may be osteoclast precursors.

Peptides corresponding to the amino-terminal region of the parathyroid hormone-related protein (PTHrP) of humoral hypercalcemia of malignancy were synthesized. A 34-amino acid peptide, PTHrP(1-34), was two to four times more potent than bovine or human PTH(1-34) in bioassays promoting the formation of adenosine 3',5'-monophosphate (cAMP) and plasminogen activator activity in osteogenic sarcoma cells and adenylate cyclase activity in chick kidney membranes. Like parathyroid hormone itself, in which the activity resides in the first 34 residues, PTHrP peptides of less than 30 residues from the amino terminus showed substantially reduced activity. PTHrP(1-34) had only 6% of the potency of bovine PTH(1-34) in promoting bone resorption in vitro. PTHrP(1-34) strongly promoted the excretion of cAMP and phosphorus and reduced the excretion of calcium in the isolated, perfused rat kidney consistent with the symptoms seen in malignant hypercalcemia.

Recently we have shown that forskolin acts synergistically with calcitonin on stimulation of cyclic AMP (cAMP) production in isolated rat osteoclasts and now show that forskolin also augments later physiological responses to calcitonin. The sensitivity of calcitonin inhibition of cytoplasmic spreading in isolated rat osteoclasts was increased 3.5-fold by simultaneous treatment with 10(-7) M forskolin, which alone had no effect on the response. In weanling rats the threshold of the hypocalcemic response to salmon calcitonin was reduced fivefold by simultaneous treatment with 200 micrograms/kg forskolin i.v., which alone did not influence the plasma calcium. These results provide further evidence that calcitonin-induced inhibition of osteoclast function is mediated by elevation of cAMP levels and suggest that forskolin could be used to augment the therapeutic effects of calcitonin.

1. Incubation of parathyroid hormone with plasma membranes from rat kidney cortex resulted in rapid loss of all hormal activity. 2. Chick kidney membranes showed no ability to inactivate parathyroid hormone even with prolonged incubation. 3. Biologically active, labelled parathyroid hormone was degraded to fragments by rat kidney membranes, but not by chick kidney. 4. Hormone-responsive adenylate cyclase activity in a mixture of rat and chick kidney membranes was additive. 5. Parathyroid hormone bound specifically to chick kidney palsma membranes. 6. It is concluded that hormone in activation during incubation has little relevance to the effectiveness of parathyroid hormone in stimulating adenylate cyclase activity in kidney, and furthermore that failure of chick kidney to metabolize the hormone is not the explanation for the greater sensitivity of this species to the hormone.

Receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been described in several human breast cancer cell lines and more recently in human melanoma. The presence of 1,25-(OH)2D3 receptor (1,25-DR) in two cultured breast cancer cell lines was associated with receptors for calcitonin, another hormone thought to have effects on calcium handling. Therefore, it seemed important to examine a range of established human cancer cell lines for the presence of receptors for 1,25-(OH)2D3 and calcitonin. Thirty-three cancer cell lines were examined. 1,25-DR was found to be present in 23 lines, while calcitonin receptors were not detected in any of them. The 1,25-DR from several cell lines sedimented at about 3.5S in sucrose density gradients, had the appropriate specificity for vitamin D metabolites, had Kds of 0.8 to 2.2 x 10(-11) M, and had receptor concentrations of 12 to 99 fmol/mg protein. Ten malignant melanoma and nine colonic carcinoma lines constituted the largest groups of carcinoma cell lines, and seven and eight, respectively, of these were 1,25-DR positive. The high frequency of 1,25-DR positivity in the cultured colonic carcinoma cells is quite different from the low frequency of 1,25-DR in primary colonic carcinomas. It was also interesting that both of two cell lines derived from patients who had had both bone metastases and malignant hypercalcemia were 1,25-DR positive. These various cell lines may provide useful models for the examination of 1,25-(OH)2D3 action in vitro.

Two methods have been used to covalently cross-link [125I]-salmon calcitonin to its receptor on a human lung carcinoma cell line, BEN, and the human breast cancer cell lines T47D and MCF 7. The first method was to use a specific photoaffinity derivative of salmon calcitonin and the second employed the chemical cross-linker, disuccinimidyl suberate. In both cases a cross-linked component of approximate molecular weight 80-90,000 on BEN cells was identified by polyacrylamide gel electrophoresis. This is consistent with the size of the cross-linked component found on T47D breast cancer cells using the photoactive salmon calcitonin as described in previous work. Disuccinimidyl suberate was unable to cross-link [125I]-salmon calcitonin either on T47D or MCF cells. However, photoactive salmon calcitonin cross-linked to a component of approximately 80-90,000 Mr on the MCF 7 cells. Thus, whereas the photoactive salmon calcitonin could cross-link a similar receptor component in all cell lines, the ability of disuccinimidyl suberate to do so was apparently cell specific. These data confirm that the calcitonin receptor comprises a component of approximately 85,000 Mr in cell lines examined thus far.

Four human cancer cell lines (MCF 7, T 47D, ZR 75 and BEN), each of which possess calcitonin-responsive adenylate cyclase, have been investigated. Pre-treatment of cells with calcitonin resulted in two effects on their adenylate cyclase. First, the ability of adenylate cyclase to respond to further stimulation by calcitonin was reduced. Secondly, membranes from calcitonin-preincubated cells had increased activity of adenylate cyclase compared with control membranes, the activated state not only surviving washing and cell-disrupting steps but persisting for some hours after the removal of hormone from the incubation medium. This phenomenon has therefore been termed "persistent activation" and may be peculiar to the action of calcitonin. It is dose dependent and is seen with calcitonins of various origins (sCT, hCT, pCT and [Asu1-7] eCT). When pre-bound hormone was removed from the cell surface by acid washing, adenylate cyclase activities in preincubated membranes fell towards control activities and responsiveness of the enzyme to a second challenge with calcitonin returned. Inhibitors of protein synthesis prevented full recovery from desensitization but had no effect on the return towards control of residual enzyme activity in preincubated membranes. These results are consistent with continued receptor occupancy leading to a persistent activation of adenylate cyclase until internalization of the receptor has occurred.

Certain antigen-binding surface molecules and factors of T cells possess serological determinants related to immunoglobulin (Ig)-heavy-chain-variable regions (VH). We obtained sufficient quantities (greater than 100 micrograms) of homogenous VH-related T-cell molecules (VTM) for biochemical studies from normal murine thymocytes and by growing large quantities of monoclonal T-cell leukemia lines expressing the determinants. A solid phase immune adsorbent prepared from the IgG fraction of rabbit anti-IgT serum was used to isolate VTM from formic acid-solubilized T cells. The VTM from murine thymocytes and T-cell lines had Mr of 65,000-68,000. The VTM from distinct cell lines differ by isoelectric focusing and resolution of tryptic peptides indicating clonal restriction. VTM lack conventional light- or heavy-chain-constant region determinants but cross-react with antisera directed against defined VHa allotypes and JH peptides. The detection of a cross-reaction with a synthetic JH peptide is consistent with recently published data identifying JH-related sequences in putative T-cell receptor genes. The amino acid compositions of the VTM were distinct from those of mammalian Ig, major histocompatibility complex (MHC) antigens, and viral glycoproteins, but significant similarities occur with Ig V regions or heavy chains of primitive vertebrates. The results indicate that the VH-bearing T-cell products are not classical Ig, but bear limited VH-cross-reactive determinants.

Structural studies of T-cell immunoglobulin light chains have been carried out in order to ascertain whether they possess a unique structure or if they resemble standard kappa or lambda isotypes. T-cell immunoglobulin was isolated from 125I-labelled culture medium of monoclonal, continuously cultured T-lymphoma cells, and the purified 125I-labelled light chains were subjected to either cleavage by cyanogen bromide or digestion with trypsin. These peptides were then resolved and compared with those derived from 125I- and 131I-labelled murine kappa and lambda chains in mixed label experiments, by means of polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers and ion exchange chromatography. The profiles obtained suggested that T-lymphoma immunoglobulin light chains are immunoglobulin polypeptides and most closely resemble kappa chains. These results support available antigenic and mRNA hybridization data, which also suggest that T cells bear kappa-like light chains.

The epidermal growth factor receptor in cells of the UMR 106-06 clonal osteoblast line has been shown to be structurally similar to that previously characterized in other cell lines. A specific receptor component of approximately 165,000-185,000 Mr has been identified by polyacrylamide gel electrophoresis using the chemical crosslinker disuccinimidyl suberate to crosslink 125I-EGF to its receptor. Tunicamycin treatment of cells resulted in a dose-dependent loss of binding suggesting involvement of glycosyl moieties in EGF binding to its receptor. Competitive binding studies carried out using wheat germ lectin (WGL), concanavalin A (CON.A.), soybean lectin (SBL), and lentil lectin (ILL) to compete for binding of 125I-EGF revealed that CON A, WGL, and to a lesser extent LL could inhibit EGF binding; SBL was without effect. Treatment of the cells with neuraminidase which cleaves terminal sialic acid residues resulted in total loss of binding while alpha-glucosidase, beta-N-acetylglucosaminidase and alpha-mannosidase were without effect. These data indicate a specific interaction of EGF with terminal sialic acid residues of the EGF receptor. However, it would seem that the mannose residues which appeared to modify EGF binding were not available for the action of the above enzymes due to the presence of sialic acid.

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.

Osteoclasts freshly isolated from embryonic chicks have been examined for calcitonin receptors using radio-iodine-labeled salmon calcitonin. Calcitonin binding to chick osteoclasts could not be shown by either autoradiography or biochemical binding studies. Furthermore, calcitonin did not stimulate cyclic AMP production. By contrast, rat osteoclasts have abundant calcitonin receptors, and a sensitive cyclic AMP response to calcitonin has been shown previously. It is concluded that chick osteoclasts do not possess calcitonin receptors, a finding which could explain the lack of calcitonin responsiveness observed in other avian osteoclast culture systems.

Two human breast cancer cell lines (MCF 7 and T 47D) possess calcitonin-responsive adenylate cyclase systems. Suspended cells of both lines specifically bound 125I-labelled salmon calcitonin with mean dissociation constants of 1.7 nM (MCF 7) and 1.4 nM (T 47D); mean receptor numbers were 5300 and 24400 per cell respectively. Measurement of specific binding to MCF 7 cells was obscured by rapid and substantial degradation of the labelled hormone. Degradation of 125I-labelled salmon calcitonin: (i) was of high capacity; (ii) lacked the specificity displayed by 125I-labelled salmon calcitonin binding to the same cells; and (iii) was not related to binding since cell incubation supernatants retained full degrading activity. The degrading activity was inhibited by corticotropin (1-24)-tetracosapeptide, insulin and bacitracin. Inclusion of bacitracin in the incubation resulted in apparently fewer numbers of lower affinity receptors on MCF 7 cells, whereas these parameters were identical to T 47D cells incubated in the presence or absence of bacitracin. Eel [2-aminosuberic acid 1,7]-calcitonin was resistant to proteolysis in the presence of either cell line. Analysis of hormone-receptor interactions with calcitonin-responsive cells should take account of potent calcitonin-degrading activities in some cell lines.

Parathyroid hormone (PTH)-like bioactivity, assayed as adenylate cyclase response in UMR 106-01 osteogenic sarcoma cells, was present in extracts of sheep fetal and maternal parathyroid glands and placenta. Preincubation of extracts with PTH(1-34) antiserum inhibited approximately 40% of the bioactivity in fetal parathyroid extracts, 50% in maternal parathyroid extracts, but only 10% of the bioactivity in the placental extract. Partial purification of placental extracts by chromatography yielded fractions containing PTH-like bioactivity which were similar in behaviour to that of PTH-related protein (PTHrP) from a human lung cancer cell line (BEN). An antiserum against synthetic PTHrP(1-16) partially inhibited the bioactivity of the placental extract and synthetic PTHrP(1-34), but had no effect on the bioactivity of bovine PTH(1-34) or bovine PTH(1-84). The placental PTH-like bioactivity was higher in mid- than in late gestation. Fetal parathyroid glands contained the highest PTH-like bioactivity. Thyroparathyroidectomy of one fetal twin lamb in each of 16 ewes between 110 and 125 days of gestation resulted in decreases of the plasma calcium concentration and reversal of the placental calcium gradient that existed between the ewe and the intact fetus. Perfusion of the placenta of each twin in anaesthetized ewes was carried out sequentially with autologous fetal blood in the absence of the exsanguinated fetus. The plasma calcium concentration in the blood perfusing the placenta of each twin increased, but reached a plateau at a lower concentration in the perfusing blood of thyroparathyroidectomized fetuses than in that of the intact fetuses. Addition of extracts of fetal parathyroid glands or of partially purified PTHrP resulted in further increases in plasma calcium in the autologous blood perfusing the placentae of thyroparathyroidectomized fetuses, but addition of bovine PTH(1-84) or rat PTH(1-34) had no effect. The presence of this PTH-like protein in the fetal parathyroid gland and placenta may contribute to the relative hypercalcaemia of the fetal lamb. This protein, which is similar to PTHrP associated with humoral hypercalcaemia of malignancy, stimulates the placental calcium pump responsible for maintaining a relative fetal hypercalcaemia during gestation.

An analog of salmon calcitonin (sCT) has been synthesized, substituting Arg at positions 11 and 18 and Lys at position 14 to provide a free amino group for derivatization. The potency of [Arg11,18,Lys14]sCT was equivalent to that of sCT in activating adenylate cyclase in UMR 106-06 cells. The analog was derivatized with biotin, fluorescein, or 4-azidobenzoate without loss of activity. The derivatized analog was not degraded by lysine-specific endoprotease, whereas the underivatized [Arg11,18,Lys14]sCT was cleaved at Lys-14. The derivatized analogs were purified by HPLC and subsequently shown to possess full biological activity. The photoactive analog was used to photolabel 88,000 and 71,000 mol wt components of the calcitonin receptor in rat osteoclasts, but only an 88,000 mol wt component was photolabeled in the UMR 106-06 cells.