PubMed Health⌕ Search

Biomedical subjects

G C Nicholson

Publications and source records attributed to G C Nicholson.

72 records · Page 4Linked to original sources
PubMed1991

A carboxyl-terminal peptide from the parathyroid hormone-related protein inhibits bone resorption by osteoclasts.

PTH-related protein (PTHrP) interacts, via its amino-terminal 34 residues, with PTH receptors on osteoblasts to stimulate osteoclastic bone resorption indirectly. We now report that mature hPTHrP-(1-141) (EC50, approximately 10(-11) M) and a carboxyl-terminal fragment, PTHrP-(107-139) (EC50, approximately 10(-15) M), are potent inhibitors of resorption in an isolated rat osteoclast bone resorption assay, whereas hPTHrP-(1-108) and hPTHrP-(1-34) are inactive in this respect. PTHrP-(107-139) also inhibits resorption in a rat long bone organ culture system and reduces osteoclast spreading. PTHrP-(107-139) does not act on osteoclasts via a cAMP signal transduction mechanism, but its effects may be mediated by protein kinase-C. This previously unrecognized action of PTHrP, to inhibit osteoclastic bone resorption directly, indicates that PTHrP may be a precursor of multiple biologically active peptides with differing physiological functions.

Amino Acid Sequence↗
PubMed1991

A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP[107-111].

We have recently shown that a carboxyl-terminal sequence of parathyroid hormone-related protein, PTHrP[107-139], is a potent direct inhibitor of osteoclastic bone resorption. We now report that this bone resorption inhibitory activity, which we have called osteostatin, is entirely contained within the highly conserved pentapeptide PTHrP[107-111]. Our results indicate that processing at residue 106 and a free amino terminus is required for full activity of the peptide. The retroinverted peptide is considerably less potent than the parent peptide. The retention of full biological activity in such a short fragment was unexpected. This data provides the basis for the development of further analogs with potential therapeutic use in disorders associated with increased osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed1989

Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat, and mouse bone cells.

Mixed bone cell cultures obtained by sequential collagenase-trypsin digestion of newborn chick, rat, and mouse calvaria responded to calcitonin gene-related peptide (CGRP) with a dose-dependent increase in cyclic AMP formation. The amplitude of response to CGRP in each species was less than that to parathyroid hormone (PTH). The CGRP effect was not the result of an action as a weak calcitonin agonist, since in most instances a calcitonin effect was not observed. Only in early digests of mouse calvarial cells were consistent stimulatory effects of calcitonin on cyclic AMP noted, and these were always considerably less in amplitude than those to CGRP. It is concluded that chick, rat, and mouse bones contain cells in osteoblast-rich populations that respond specifically to CGRP with a rise in cyclic AMP.

Animals↗
PubMed1988

Solubilization of functional calcitonin receptors.

Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.

Azides↗
PubMed1988

Human placental calcitonin receptors.

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

Adenylyl Cyclases↗
PubMed1988

Forskolin augments the effects of calcitonin on cytoplasmic spreading of isolated rat osteoclasts and plasma calcium levels in the rat.

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.

Animals↗
PubMed1988

Biologically active, derivatizable salmon calcitonin analog: design, synthesis, and applications.

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.

Adenylyl Cyclases↗
PubMed1988

Induction of calcitonin receptors by 1 alpha, 25-dihydroxyvitamin D3 in osteoclast-like multinucleated cells formed from mouse bone marrow cells.

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.

Animals↗
PubMed1987

Chicken osteoclasts do not possess calcitonin receptors.

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.

Animals↗
PubMed1987

Calcitonin receptors of human osteoclastoma.

Osteoclast-rich cultures were prepared by disaggregation of osteoclastomas (giant cell tumour of bone) and settlement onto glass or plastic surfaces. Autoradiography using [125I]-salmon calcitonin ([125I]-sCT) revealed specific binding only to multinucleate giant cells (osteoclasts) and a minor population of mononuclear cells. [125I]-sCT competitive binding studies indicated a Kd of 5 x 10(-10) M and receptor number of approximately 1 million sites/osteoclast. sCT treatment resulted in a dose-dependent rise in cAMP (EC50 10(-10) M). Homogenates of an osteoclastoma also demonstrated specific binding of [125I]-sCT. Chemical cross-linking of a labelled synthetic sCT derivative. [125I]-[Arg11,18,Lys14]-sCT, using disuccinimidyl suberate, resulted in labelling of a receptor component of approximate Mr 85-90,000. The multinucleate giant cells (osteoclasts) of human osteoclastomas possess large number of CT receptors which exhibit the same binding kinetics and apparent Mr as those of other CT target cells.

Bone Neoplasms↗
PubMed1987

Control of cyclic adenosine 3',5'-monophosphate production in osteoclasts: calcitonin-induced persistent activation and homologous desensitization of adenylate cyclase.

Hormonal control of cAMP production in the osteoclast has not been investigated in detail because this bone-resorbing cell has been difficult to isolate. We have used osteoclasts freshly isolated by disaggregation from neonatal rat long bones and settled onto coverslips (100-150 cells per coverslip) to examine the effects of calcitonin and prostaglandin E2 on osteoclast cAMP levels and cytoplasmic spreading. Salmon, eel, and human calcitonin (CT), and various analogs, stimulated cAMP production in a dose-dependent manner with relative potencies as seen in other response systems. Forskolin (10(-7) M) increased the sensitivity and amplitude of the response. Pretreatment with pertussis toxin (200 ng/ml for 3 h) had no effect suggesting that CT does not act through Ni, the inhibitory guanine nucleotide regulatory unit of adenylate cyclase. CT treatment was associated with rapid and dose-dependent induction of a persistent activated state of adenylate cyclase and homologous desensitization, the former being a particular feature of CT action previously observed in nonosteoclastic cells. Quantitative histomorphometry demonstrated a sensitive, dose dependent, and prolonged (greater than 2 h) reduction in osteoclast plan area after exposure to salmon CT. Although prostaglandin E2 also stimulated cAMP production and resulted in cell contraction in osteoclasts this was not associated with persistent activation of adenylate cyclase nor with prolonged contraction. Persistent activation of adenylate cyclase may be an important mechanism in CT inhibition of osteoclast function.

Adenylate Cyclase Toxin↗
PubMed1986

Actions of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP formation in chicken and rat osteoclasts.

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.

Animals↗
PubMed1986

Abundant calcitonin receptors in isolated rat osteoclasts. Biochemical and autoradiographic characterization.

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.

Animals↗
PubMed1985

Parathyroid hormone radioimmunoassay: the clinical evaluation of assays using commercially available reagents.

This paper reports on the diagnostic usefulness of two commercial PTH assay kits and four "in-house" assays using commercially available reagents, studying the same samples from normal controls and different patient groups. The ability of such assays to discriminate proven primary hyperparathyroid (1 degree HPT) patients from normals varied significantly but without any apparent correlation with assay components. For all assays, performance declined markedly in 1 degree HPT patient groups with lower serum calcium levels. Patients with PTH secondary to chronic renal disease were well discriminated from normal by all assays. Although immunoassays are useful in many cases of 1 degree HPT, it is difficult to develop C-terminal or mid-region PTH assays that are uniformly diagnostically useful in the clinical situation where they are of greatest potential use i.e. in cases of mildly hypercalcaemic 1 degree HPT.

Antibody Specificity↗
PubMed1985

Estimation of 1,25 dihydroxyvitamin D by cytoreceptor and competitive protein binding assays without high pressure liquid chromatography.

Using two different cultured rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8) we have investigated the recently described cytoreceptor assay for 1,25-dihydroxyvitamin D (1,25-(OH)2D). The assay method is relatively simple and sensitive to 2.4 fmole per tube. Using either cell line, assay of serum samples, whose only preparation consisted of extraction and purification on a disposable diatomaceous earth column, produced variable values for serum 1,25-(OH)2D. Additional purification, using a disposable silicic acid minicolumn to remove other vitamin D metabolites resulted in consistent values and additional HPLC resulted in no further decrease in the values obtained. Our results show that a single two stage non-HPLC column can purify serum samples for assay in the cytoreceptor assay. The method is also applicable to the competitive protein binding assay employing calf thymus cytosol and the correlation between values obtained by both methods is highly significant. It is a sensitive, simple, and accurate method with technical advantages which allow greater sample throughput than other 1,25-(OH)2D assays.

Animals↗
PubMed1984

Autoimmune thyroid disease and giant cell arteritis: a review, case report and epidemiological study.

A 75-year-old man with the simultaneous onset of Graves' disease and giant cell arteritis is described. Although there have been few previous reports of simultaneous onset, literature review and a retrospective study of the records of this hospital suggest that the association of Graves' disease and the polymyalgia rheumatica-giant cell arteritis syndrome is not simple coincidence. The patient's course was complicated by cerebral infarction secondary to either arterial thrombo-embolism or arteritis.

Aged↗