Inactivation of porcine calcitonin by rat kidney microsome.

Biological activity of porcine calcitonin was most actively inactivated by the rat kidney homogenate than by other tissue homogenates. Among the various subcellular fractions of the rat kidney homogenate examined, microsome fraction was most active in the in vitro inactivation of porcine calcitonin. Inactivation of porcine calcitonin by the rat kidney microsome was dependent on pH and temperature. Inactivating activity of the rat kidney microsome was inhibited by 1 X 10(-3) M monoiodoacetate and 1 X10(-5) M p-chloromercuribenzoate. These results suggest that porcine calcitonin is probably inactivated by a SH-enzyme in the rat kidney microsomes. However, the participation of other enzymes cannot be ruled out, since the inactivating activity of the rat kidney microsome fraction is also inhibited by 1 X 10(-4) M diisopropylfuorophosphate.     

基因工程制备重组人降钙素类似物

构建融合表达人降钙素类似物的工程菌 ,然后纯化 GST- h CTa- Leu融合蛋白 ,凝血酶酶切后 ,再经离子交换层析、脱盐 ,获得纯度大于 90 %的 h CTa前体 .对 h CTa前体的 C末端进行修饰 ,得线形 h CTa- NH2 ,再通过复性 ,获得了具有与天然人降钙素活性相当的 h CTa.     

Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity

The calcitonin peptides [calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin] share many biological actions, including activity on bone cells. In the present study, CT (10(-11) to 10(-9) M) stimulated [(3)H]thymidine incorporation in primary cultures of human osteoblasts (hOB), as already demonstrated for CGRP and amylin. RT-PCR analysis showed that the calcitonin receptor and the calcitonin receptor-like receptor are both expressed in hOB. In these cells, CT (10(-10) M) and amylin (10(-9) M), in contrast to CGRP (10(-8) M), did not increase cAMP production. All three peptides stimulated protein kinase C (PKC) activity. To evaluate PKC involvement in hOB proliferation, cells were incubated with phorbol 12,13-dibutyrate, a stimulator of PKC activity; cell proliferation was increased in a dose-dependent manner (EC(50) = 3.4 x 10(-8) M). Staurosporine (10(-9) M), a PKC inhibitor, blocked phorbol 12,13-dibutyrate-induced PKC activity and cell proliferation. Inhibition of PKC by staurosporine also counteracted the stimulatory effect of CT, CGRP, and amylin on hOB proliferation. From these data, it is deduced that the activation of PKC is important for hOB proliferation and that it is involved in the anabolic effect of CT peptides on bone.     

Isolation and determination of the amino acid sequence of chicken calcitonin I from chicken ultimobranchial glands

A radioimmunoassay for chicken calcitonin in chicken ultimobranchial glands was established utilizing a rabbit antiserum against eel calcitonin. This assay method, which is about 100 times as sensitive as the usual bioassay for hypocalcemic activity, was used for monitoring chicken calcitonin during its purification. The immunoreactivity in chicken ultimobranchial extract was separated by SP-Sephadex C-25 chromatography into two fractions. Chicken calcitonin I, which was occurred in the major immunoreactive fraction, was further purified to homogeneity as shown by reverse phase HPLC. In the end, 39 nmol of chicken calcitonin I was obtained from 3,384 chickens following a 12,000-fold purification. The complete amino acid sequence of purified chicken calcitonin I was determined to be H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Ly s-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2 and confirmed by synthesis. The specific biological activity of chicken calcitonin I (4,500 MRCU/mg) was identical to that of eel calcitonin, which has the highest specific biological activity among the calcitonins so far isolated. Chicken calcitonin I resembled the calcitonins from the ultimobranchial glands both of salmon and eel in sequence, biological activity, and immunological property.     

Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells

Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor.     

Ectopic synthesis of high-Mr calcitonin by the BEN lung carcinoma cell line reflects aberrant proteolytic processing

Cloning and nucleotide sequence analysis of the human calcitonin mRNA from the BEN lung carcinoma cell line, a cell line known to secrete high-Mr forms of calcitonin, showed no difference in the coding region at the nucleotide level compared with calcitonin mRNA isolated from medullary thyroid carcinoma which secretes calcitonin monomer. Therefore, the secretion of high-Mr forms of calcitonin reflects the absence or limited activity of proteolytic processing enzymes within the secretory pathway of this cell line. In all other respects, as judged by RNA blotting and S1 mapping, calcitonin/alpha-CGRP expression was identical to that found in medullary thyroid carcinoma, including the differential use of an alternative splice donor site within intron 1. The BEN cell line also produces low levels of alpha-CGRP mRNA and secretes CGRP antigenic determinants. Analysis of plasma CGRP levels in 12 patients with anaplastic lung carcinoma showed elevated levels in 11 of these, suggesting that CGRP may be an important diagnostic marker for this disease.     

Regulation of cytochrome c oxidase activity by c-Src in osteoclasts

The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane-associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria.     

Effect of carbohydrate structure on biological activity of artificially N-glycosylated eel calcitonin

To reveal the function of the carbohydrate portion of glycopeptides and glycoproteins, we chemo-enzymatically synthesized artificially N-glycosylated derivatives of eel calcitonin and studied their three-dimensional structure and biological activity. The CD and NMR spectra in trifluoroethanol-H₂O solution showed that the glycosylation did not change the three-dimensional structure. All the derivatives retained the strong in vivo hypocalcemic activity of calcitonin. However, the relative activity was dependent on the structure of the attached carbohydrate. The single GlcNAc attachment best enhanced the activity, while larger carbohydrates decreased the activity. This relative activity order of compounds could be partly explained by their calcitonin-receptor binding affinity, though the affinity of the GlcNAc derivative did not exceed that of calcitonin. The enhanced hypocalcemic activity of the GlcNAc derivative was explained by its altered biodistribution. The GlcNAc attachment caused calcitonin to escape from the trap at the liver during the early circulation. Thus, the glycosylation was shown to modulate the biological activity of calcitonin depending on the carbohydrate structure without a change in the peptide backbone conformation.     

Glycosylation reduces the bioactivity of calcitonin

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.     

Regulation of cellular calcium metabolism and calcium transport by calcitonin.

Calcitonin was studied in isolated kidney cells and in isolated mitochondria. A concentration of 10 ng/ml of synthetic calcitonin increases the cellular accumulation of 45Ca and the total cell calcium. The mitochondrial pool is increased several-fold. Kinetic analysis of the data shows that although the total cellular exchangeable calcium pool is enlarged, calcium influx and efflux are significantly depressed by calcitonin. The absence of phosphate or the presence of inhibitors of mitochondrial calcium transport completely abolish the effects of the hormone. In isolated mitochondria, the hormone stimulates the active calcium uptake and depresses the extramitochondrial calcium activity. Calcitonin counteracts the effects of cyclic AMP which stimulates the release of calcium from mitochondria and increases the extramitochondrial calcium activity. These data indicate that cellular calcium homeostasis is controlled by the mitochondrial calcium turnover. They suggest that calcitomin regulates the cell calcium metabolism and inhibits the transcellular calcium transport by stimulating the rate of calcium uptake by mitochondria which depresses cytoplasmic calcium activity.     

Protein kinase A-independent activation of ERK and H,K-ATPase by cAMP in native kidney cells: role of Epac I

This study aimed at determining the signaling pathways underlying calcitonin- and isoproterenol-induced stimulation of H,K-ATPase in rat renal collecting duct. H,K-ATPase activity was determined in microdissected collecting ducts preincubated with or without either specific inhibitors or antibodies directed against intracellular signaling proteins. Transient cell membrane permeabilization with streptolysin-O allowed intracellular access of antibodies. The stimulation of H,K-ATPase by calcitonin and isoproterenol was mimicked by cAMP analogues and was abolished by adenylyl cyclase inhibition. Protein kinase A inhibition abolished isoproterenol but not calcitonin effect on H,K-ATPase. Calcitonin increased the phosphorylation of extracellular signal-regulated kinase (ERK) in a protein kinase A-independent manner, and the inhibition of the ERK phosphorylation prevented the stimulation of H,K-ATPase by calcitonin. Antibodies directed against either the cAMP-activated guanine-nucleotide exchange factor Epac I, the monomeric G protein Rap-1 or the kinase Raf-B, curtailed the stimulation of H,K-ATPase by calcitonin, whereas antibodies against the related monomeric G protein Ras or kinase Raf-1 had no effect. In conclusion, calcitonin stimulates H,K-ATPase through a cAMP/Epac I/Rap-1/Raf-B/ERK cascade.     

Characteristics of selective activation of cyclic AMP-dependent protein kinase isoenzymes by calcitonin and prostaglandin E2 in human breast cancer cells.

The characteristics of the cyclic AMP-dependent protein kinase isoenzyme response to calcitonin stimulation have been studied in two human breast cancer cell lines, T47D and MCF 7. Both cell lines possess calcitonin receptors, a calcitonin-responsive adenylate cyclase and the two isoenzymes of the cyclic AMP-dependent protein kinase, types I and II. The adenylate cyclase also responds to prostaglandin E2. Acute activation of the cyclic AMP-dependent protein kinase isoenzymes was determined by using a modification of a multiple small anion exchange column method [Livesey, Kemp, Re, Partridge & Martin (1982) J. Biol. Chem. 257, 14983-14987]. Control experiments showed that post-extraction activation did not influence the data. Calcitonin caused a rapid, selective activation of isoenzyme II in the T 47D cells with half-maximal response at 10(-10)M, and persisting for at least 24h. In MCF 7 cells calcitonin also caused a highly selective activation of isoenzyme II with half-maximal response at 5 X 10(-11) M, but the response was transient with a return to basal isoenzyme activity by 4-6 h. At this time further addition of calcitonin did not restimulate the cyclic AMP-dependent kinase activity. In neither cell line did calcitonin treatment result in activation of isoenzyme I. Prostaglandin E2, on the other hand, the only significant alternative agonist of adenylate cyclase in T 47D cells, activated isoenzymes I and II to an equal extent in these cells, illustrating that two hormones activating adenylate cyclase in the one cell type might exert different effects by their selective actions upon protein kinase isoenzymes.     

Biologically potent analogues of salmon calcitonin which do not contain an N-terminal disulfide-bridged ring structure

The disulfide bridge formed between the cysteine residues at positions 1 and 7 of salmon calcitonin (sCT) is not required for biological activity. The analogues [Ala1,7]sCT,[AcmCys1,7]sCT and [AmcCys1,Ala7]sCT (AcmC = S-acetamido-methylcysteine) are linear sequences which retain full hypocalcemic activity in the intact rat and ability to activate adenylate cyclase of rat renal membranes. The secondary structure of these peptides in aqueous solution in the presence or absence of lipid is not greatly perturbed by the opening of the disulfide ring. In contrast with salmon calcitonin, substitution of Cys by AcmCys in human calcitonin results in greatly reduced hypocalcemic activity but no loss in the ability of the peptide to activate renal adenylate cyclase. Thus in vitro activation of adenylate cyclase by human calcitonin analogues is not always correlated with in vivo hypocalcemic potency.     

Tumour calcitonin. Interaction with specific calcitonin receptors.

The human epidermoid bronchial carcinoma (BEN) cell line has been shown to have specific membrane binding sites for calcitonin and to secrete high-molecular-weight forms (ranging from 40000 to 10000) of immunoreactive calcitonin. Synthetic salmon and human calcitonins and a thyroid extract of porcine calcitonin have been shown to displace 125I-labelled salmon calcitonin from the receptors in a dose-related fashion. The binding to these receptors of calcitonins derived from the BEN cell line and a medullary thyroid carcinoma with molecular weights ranging from 28000 to 3500 (both separated by gel-filtration chromatography) has been investigated. Neither major peaks of BEN-cell-line calcitonin showed receptor binding activity. Only one form of medullary thyroid carcinoma calcitonin, that which co-eluted with synthetic calcitonin monomer on gel-filtration chromatography, caused any significant displacement of labelled hormone from the receptors.     

Failure to demonstrate the stimulative effect of calcitonin on cyclic AMP accumulation in avian bone in vitro

The effect of calcitonin on the metabolism of calcium and cyclic AMP in the avian bone was examined in vitro. In the chick embryonic cortical bone, calcitonin affected neither the level of cyclic AMP nor the parathyroid hormone-induced stimulation of calcium release from bone. Also, the level of cyclic AMP in the cortical or medullary bone of Japanese quails was not increased by calcitonin. The medullary bone did not respond to calcitonin either in freshly prepared tissue or in tissue cultured for 48 hr in the absence of calcitonin. The activity of renal adenylate cyclase of Japanese quails was also calcitonin-insensitive. The inability of calcitonin to increase the level of cyclic AMP and to antagonize the parathyroid hormone-induced stimulation of calcium release from bone may account for the lack of hypocalcemic effect of calcitonin in birds as reported by other investigators.     

Altered activation by calcitonin and inhibition by somatostatin of human embryonal carcinoma cell adenylate cyclase with retinoic acid-induced differentiation

Human teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early human development, we have characterized the alterations that occur in the hormonal responsiveness of human embryonal carcinoma cell adenylate cyclase with differentiation in response to 10 microM retinoic acid. Two cell lines CL12 and CL13, cloned from Tera 2 cells by Dr. C. F. Graham, have been used in these studies. Adenylate cyclase of CL12 and CL13 cells is stimulated in the presence of 10 microM GTP by epinephrine and calcitonin, with calcitonin being the most potent stimulator of cyclic AMP production. Exposure of these cells to retinoic acid leads to an arrest in growth and within 6 days to a differentiated cell population with a stable nonreversible phenotype. No changes in basal, GTP- and fluoride-stimulated adenylate cyclase activities are observed with retinoic acid treatment, but the cyclase of differentiated cells exhibits a greater stimulation by calcitonin (7.5-fold) and the appearance of a somatostatin inhibitory effect. Somatostatin specifically inhibits, by 25%, the hormonal stimulation of adenylate cyclase of cells treated for 5 days with retinoic acid. The increase in calcitonin stimulation of adenylate cyclase activity of the differentiated cells is related to an increase (congruent to 3-fold) in the number of hormonal receptors and not to a significant change in receptor binding affinity (Kd 4.6 X 10(8) M-1). These alterations in calcitonin and somatostatin responsiveness suggest a possible regulatory role for these hormones during embryonic development. Furthermore, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of human embryonal carcinoma cell differentiation.     

Complementation between LLC-PK1 mutants affected in polypeptide hormone-receptor function

The mutant LLC-PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP-dependent protein kinase (cAMP-PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I-labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half-maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC-PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC-PK1 cells exhibited normal hormone binding and activation of cAMP-PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the 'receptorless' mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC-PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.     

Calcitonin inhibits the phosphorylation of various proteins in rat brain synaptic membranes

The present report examines the effect of different calcitonins and analogs on the in vitro phosphorylation of brain synaptic membrane proteins. The findings demonstrate that calcitonin is a potent inhibitor of several brain synaptic proteins and that salmon and eel calcitonins are considerably more potent than thyrocalcitonin in eliciting this effect. Deletion of leucine from position 16 in salmon calcitonin sequence resulted in a drastic loss of inhibitory activity, indicating the importance for a hydrophobic residue at position 16 in the intact calcitonin molecule. The mechanism of calcitonin inhibition of protein phosphorylation was likely due to the blockade of stimulation of protein kinases by calmodulin.     

Modulation of Calcitonin Binding by Calcium: Differential Effects of Divalent Cations

Abstract

Binding of salmon calcitonin to bovine hypothalamic membranes is enhanced about 25% by calcium with a half-maximal effect at 15 mM calcium. In contrast, membranes prepared from a cell line expressing a recombinant human calcitonin receptor show no effect of calcium under similar conditions. The hypothalamic calcitonin receptor solubilized with CHAPS detergent retains an apparent Kd of 0.3 nM for salmon calcitonin; however, binding of calcitonin to the detergent-solubilized receptor complex can be inhibited by divalent cations in order of potency Mn>Ca Sr Mg NaCl with Mn and Ca having apparent Ki's of 5 mM and 20 mM respectively. Dixon and Scatchard plots of Mn and Ca inhibition of binding to the soluble receptor complex suggest a noncompetitive mechanism of inhibition. Calcium also inhibits calcitonin binding to a detergent-solubilized recombinant human calcitonin receptor. Inhibition of calcitonin binding is observed using two independent methods for determining soluble receptor-hormone complex and inhibition is reversed by EDTA.