Calcitonin Analogue Research Articles

Effect of Elcatonin on Experimental Gastric and Duodenal Ulcers

The antiulcer action of elcatonin, an analogue of natural eel calcitonin, was compared with that of cimetidine, secretin and solcoseryl. Elcatonin (3 to 10 u/kg, s.c.) inhibited the development of gastric ulcers induced by pylorus ligation, water-immersion stress, aspirin and reserpine and duodenal ulcers induced by cysteamine in rats. Moreover, once daily injections of elcatonin (1 to 10 u/kg/day, s.c.) promoted the healing of acetic acid-induced chronic gastric ulcers not only in rats but in dogs. The healing effect persisted after the cessation of administrations. Cimetidine (30 to 100 mg/kg, p.o.) inhibited the development of gastric ulcers induced by water-immersion stress, aspirin and reserpine and duodenal ulcers induced by cysteamine in rats. However, once daily administrations of cimetidine (30 to 100 mg/kg/day, p.o.) d>d not show significant effect on acetic acid-induced chronic gastric ulcers in rats. Secretin (30 to 100 u/kg, s.c.) inhibited the development of gastric ulcers induced by pylorus ligation, water-immersion stress, aspirin and reserpine in rats, but was not effective on cysteamine-induced duodenal ulcers and acetic acid-induced chronic gastric ulcers in rats. Solcoseryl (2 ml/kg, s.c.) inhibited only the development of water-immersion stress-induced gastric ulcers in rats. These results suggest that elcatonin is different from these reference drugs in its properties of action on experimental ulcers. Mechanisms of the antiulcer action of elcatonin which has a superior effect on experimental ulcers are discussed.

Enhanced Rectal Absorption of [Asu1,7]-eel Calcitonin in Rats Using Polyacrylic Acid Aqueous Gel Base

The effects of polyacrylic acid aqueous gel on the absorption of rectally administered [Asu1,7]-eel calcitonin, a calcitonin analogue, were investigated in rats. The [Asu1,7]-eel calcitonin (1 U/kg) was given into the rectal loop in gel bases at various pH (5.5-8.5) and polyacrylic acid concentrations (0.01-1.0% w/v). The maximum hypocalcemic effect was obtained in ∼30min after administration of the analogue in a 0.1% w/v polyacrylic acid gel base at pH 5.5. The plasma calcium level decreased by ∼18% from the initial level. Rectal administration in vehicles such as polyethylene glycol 1000, triglyceride fatty acid mixture base, or saline solution had little or no hypocalcemic effect at a dose of 5 U/kg. The results indicated that a polyacrylic acid aqueous gel base significantly improved the absorption of this analogue. Furthermore, rectal administration in a polyacrylic acid gel base (0.1% w/v; pH 5.5) required a dose 35 times greater than an intravenously administered dose to achieve an equivalent hypocalcemic effect.

Inhibitory effect of [Asu1 . 7]-eel calcitonin on glucagon-induced glycogenolysis in perfused rat liver.

In an attempt to elucidate the mechanism by which calcitonin modulates glucose metabolism, the effect of elcatonin ([Asu1 . 7]-eel calcitonin), a potent synthetic eel calcitonin analogue, on hepatic glycogenolysis was investigated by use of perfused liver from fed rats. Elcatonin, as infused into the portal vein at concentrations between 10 mU/ml and 200 mU/ml did not affect glucose output into the hepatic venous effluent. At concentrations higher than 100 mU/ml, it inhibited the glycogenolysis stimulated by submaximal concentrations of glucagon which was perfused concurrently. This aspect of elcatonin effect was reproduced by synthetic salmon calcitonin. Though elcatonin showed a marked inhibition of the glycogenolytic activity induced by glucagon at or less than 5.7 X 10(-11) M, the inhibitory effect became undetectable when higher concentrations of glucagon were employed. Elcatonin did not inhibit the glycogenolysis induced by dibutyryl cyclic AMP at near (0.5 microM) or less than half-maximally effective (0.2 microM) concentrations. In addition, it did not inhibit the glycogenolytic activity half-maximally stimulated by alpha-adrenergic agonist (phenylephrine, 0.4 microM) or vasopressin (0.2 mU/ml). Elcatonin inhibited the cyclic AMP production of the tissue induced by glucagon infusion. These data indicate that elcatonin modulates hepatic glycogenolysis by preventing the glucagon effect at a step before cyclic AMP production and with an apparently competitive kinetics. In view of the concept that Ca++ is involved in the glycogenolytic effect of alpha-adrenergic agonist and vasopressin, the fact that elcatonin did not influence the action of these agents suggests that Ca++ fluxes are not involved in the elcatonin effect on liver.

Growth inhibition of human breast cancer cells induced by calcitonin

The human breast cancer cell line (T47D) has specific, high affinity calcitonin receptors and calcitonin-responsive adenylate cyclase. Human, salmon and [Asu 1,7]eel calcitonin inhibited cell growth in a dose-related manner with almost equipotency. Analogues of human calcitonin demonstrated slight cell growth inhibition. We found extreme growth inhibition with daily treatment with dibutyryl cyclic AMP (10 −4 M). In contrast to calcitonin 1,25-(OH) 2D 3 had a biphasic effect on cell growth. Physiological doses (5 × 10 −10 M) of 1,25-(OH) 2D 3 stimulated growth of T47D, whereas treatment by supraphysiological amounts (2.5 × 10 −7 M) caused significant inhibition of growth. Calcitonin and 1,25-(OH) 2D 3 appeared to have additive effects.

Covalent cross-linking of a photoactive derivative of calcitonin to human breast cancer cell receptors.

A photoaffinity derivative of salmon calcitonin has been produced by transglutaminase-mediated incorporation of N-(beta-aminoethyl)-4-azido-2-nitroaniline into the hormone. The derivative, purified by high pressure liquid chromatography, retained the abilities to bind to cultured T47D breast cancer cells and to stimulate adenylate cyclase in these cells. In both these respects it was equipotent with synthetic salmon calcitonin. Photolysis of the 125I-labeled photoactive salmon calcitonin derivative bound to T47D cells was accompanied by specific labeling of only one component (Mr approximately 85,000) as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling was observed only upon photolysis and was inhibited by unlabeled synthetic salmon calcitonin but not by the inactive calcitonin analogue, 8-glycine human calcitonin. Reduction did not alter the apparent molecular weight of the calcitonin receptor complex. No macromolecular forms of calcitonin were produced by photolysis in the absence of T47D cells.

Des-Ser2 salmon calcitonin: a biologically potent synthetic analog.

We studied the biological activity of a synthetic analog of salmon calcitonin (SCT), from which the serine2 residue within the amino-terminal disulfide ring had been omitted (des-Ser2-SCT). This analog proved to be indistinguishable from SCT with respect to hypocalcemic activity in vivo, displacement of [125I]SCT from rat renal membranes, and accumulation of cAMP in incubated renal cortical slices, des-Ser2SCT was twice as potent as SCT with respect to adenylate cyclase activation in renal membranes. Since the deletion did not impair biological activity, it appears that the function of the intact ring structure does not critically depend on its encompassed peptide chain length.

Adenylate Cyclase Activation and Competitive Binding with Renal Tissue Using Synthetic Eel Calcitonin Analog and Its Fragments

The structure-function relationship of calcitonin (CT) was investigated using a synthetic eel CT (E-CT) analog and its fragments. Adenylate cyclase activation and competitive binding to rat renal receptors were used as parameters of function. [Asu1,7]E-CT analog, synthesized by replacing the S-S bond of Cys1-Cys7 in the natural hormone with ethylene linkage of 1-amino suberic acid (Asu), and E-CT fragment 11-32 had about 1/5th and 1/50th the potencies of synthetic E-CT, respectively, in both adenylate cyclase activation and competitive inhibition of 125I-labeled [Asu1,7]iodo-E-CT binding on rat renal plasma membranes. The minimal chain length required to activate adenylate cyclase in rat renal plasma membranes was between 12-18 amino acids, and the minimal chain length required to affect the CT receptor binding was between 6-12 amino acids, near the C-terminus. Fragments, including the C-terminus, show a disproportionate and potent inhibition of the binding compared with the potency required for adenylate cyclase activation. Thus, the amino acid sequence near the C-terminus probably plays an important role in the binding of CT to the receptor. (Endocrinology 108: 698, 1981.)

EFFECT OF [Asu1,7]-EEL CALCITONIN AND ITS FRAGMENTS ON GASTRIC ACID SECRETION IN RATS

Effect of [Asu1,7]-eel calcitonin ([Asu1,7]-E-CT), an analogue of natural eel calcitonin, and its fragments on gastric acid secretion was studied in pylorus-ligated rats. [Asu1,7]-E-CT markedly reduced the acid secretion with intravenous and intramuscular doses of 3 Medical Research Council units (MRC U)/kg or 0.6 μg/kg. This activity was comparable to that of salmon calcitonin and porcine calcitonin compared with equivalent hypocalcemie doses and was consistently evident during daily administrations for 10 days. Assay of six fragments of [Asu1,7]-E-CT showed that the C-terminal peptide consisting of 22 amino acids diminished the acid secretion but shorter peptides of the C-terminal fragment or the N-terminal fragments did not. These results suggest that the C-terminal peptide may play an important role in this action of [Asu1,7]-E-CT, and a peptide chain of over 18 amino acids is required.

Effect of (Asu1,7)-EEL calcitonin and its fragments on gastric acid secretion in rats.

Effect of [Asu 1,7 ]-eel calcitonin ([Asu 1,7 ]-E-CT), an analogue of natural eel calcitonin, and its fragments on gastric acid secretion was studied in pylorus-ligated rats. [Asu 1,7 ]-E-CT markedly reduced the acid secretion with intravenous and intramuscular doses of 3 Medical Research Council units (MRC U)/kg or 0.6 μg/kg. This activity was comparable to that of salmon calcitonin and porcine calcitonin compared with equivalent hypocalcemie doses and was consistently evident during daily administrations for 10 days. Assay of six fragments of [Asu 1,7 ]-E-CT showed that the C-terminal peptide consisting of 22 amino acids diminished the acid secretion but shorter peptides of the C-terminal fragment or the N-terminal fragments did not. These results suggest that the C-terminal peptide may play an important role in this action of [Asu 1,7 ]-E-CT, and a peptide chain of over 18 amino acids is required.

Calcitonin receptors in a cloned human breast cancer cell line (MCF 7)

A human breast cancer cell line, MCF 7, is shown to possess a specific calcitonin receptor and calcitonin responsive adenylate cyclase, and calcitonin treatment results in activation of cyclic AMP-dependent protein kinase. Studies with several analogues of calcitonin show that the receptor and adenylate cyclase response preserve the ability to discriminate among the structure-function relationships of the calcitonin molecule. The same cell line has been shown recently to possess a receptor for the steroid hormone, 1,25(OH) 2-vitamin D. Coexistence in MCF 7 cells of receptors for two calcium-regulating hormones may be related to the osteoclast-like properties of these cells.

Predicted secondary structure of calcitonin in relation to the biological activity

The secondary structure of the natural analogues of calcitonin have been predicted using the methods of Burgess et al., Chou and Fasman, and Lim. The predicted structures were similar in the N-terminal part of the chain, but were variable in other regions of the molecules. The higher hypocalcaemic potency of ultimobranchial calcitonins as compared to thyroidal calcitonins may be related to the higher potential for helical structure in the former.

Synthesis of [2-Serine, 8-VaIine]-Human Calcitonin

Abstract The [2-serine, 8-valine]-analog of human calcitonin (1) was synthesized by the liquid phase method. Analog 1 was built up from five fragments with minimized protection for the side functional groups. These fragments were linked step wise to the C-terminal fragments by the azide method. A gel filtration was effectively applied for the purification of the resulting pep tide in each fragment condensation. Analog 1 had about the same hypocalcemic activity as that of human calcitonin. The result showed that the high activity of eel or salmon calcitonin 1 or 2 is not ascribable only to the amino acid sequence of their N-terminal decapeptide.

Calcitonin and calcium ionophores: cyclic AMP responses in cells of a human lymphoid line.

Receptors for calcitonin, as assayed by the specific binding of 125I-labeled salmon calcitonin and stimulation of cyclic AMP formation, were found in 8866 cells derived from a human lymphoid line. The affinity of calcitonin from different species and of various analogues of human calcitonin for the binding sites and their ability to stimulate cyclic AMP formation were closely related to their hypocalcemic activity and presumably reflected biological properties of the hormones. Besides calcitonin, prostaglandin E1 and beta-adrenergic catecholamines stimulated cyclic AMP formation in these cells. The calcium ionophores, A23187 and Br-X-573A, did not influence the specific binding of 125I-labeled salmon calcitonin. A23187, however, suppressed basal and calcitonin-stimulated formation of cyclic AMP in the presence of at least 0.6 mM calcium in the incubation medium. Br-X-537A did not require extracellular calcium to suppress basal and calcitonin-stimulated formation of cyclic AMP, suggesting that the release of calcium from internal stores may regulate adenylyate cyclase activity in 8866 cells.

Radioimmunoassay of human calcitonin using labeled [Asu1, 7]-human calcitonin analogue.

Application of synthetic human calcitonin analogue ([Asul,7]-hCT) was attempted, since it is chemically more stable than native hCT. [Asu1, 7]-hCT was successfully labeled with 125I to a specific activity ranging from 410 to 540 microCi/microgram. Sensitivity, recovery and reproducibility of the assay system using 125I-labeled [Asu1,7]-hCT analogue, standard synthetic hCT and its antibodies, were 49 pg/ml, 99.8 +/- 4%, and 7.95% (C. V. within assay) or 21.9% (C. V. between assays), respectively. The dilution curve of the sera obtained from patients with medullary carcinoma paralled well with the standard curve. 125-I-labeled [Asu1, 7]-hCT analogue can be used for routine radioimmunoassay of hCT.

Structure-activity relationship of human calcitonin. III. Biological activity of synthetic analogues with shortened or terminally modified peptide chains (author's transl)

Assays of 8 synthetic analogues of human calcitonin in rats showed that their hypocalcaemic activity was drastically reduced by deletion of the C-terminal amide group, chain-shortening or opening of the disulphide ring, but unaffected or enhanced by modification of the N-terminal amino group.

Renal Receptors for Calcitonin: Coordinate Occurrence with Calcitonin-Activated Adenylate Cyclase

High affinity binding of 125I-labeled salmon calcitonin ([125I]SCT) and calcitonin-activated adenylate cyclase were detectable in renal plasma membranes from the rat. Addition of 5'-guanylyl-imidodiphosphate lowered the threshold for enzyme activation by peptide hormones. Renal plasma membranes from man, dog and cow contained little or no calcitonin-sensitive adenylate cyclase and showed no high affinity binding of [125I]SCT. High affinity binding sites were distributed during membrane fractionation in fractions where the specific activity of hormone-sensitive adenylate cyclase was greatest. Calcitonin binding and activation of the adenylate cyclase enzyme occurred at similar hormone concentrations. The relative potencies of calcitonin analogues were similar whether measured by competition for high affinity binding sites or by effect on adenylate cyclase. Low concentrations of Lubrol-PX, a nonionic detergent, did not affect catalytic function of the enzyme determined in the presence of sodium fluoride but caused parallel loss of high affinity [125I]SCT binding and hormonal sensitivity of the enzyme. This observation provided further evidence that interaction of calcitonin with specific receptors (identified with [125I]SCT binding) is essential for calcitonin activation of adenylate cyclase, but showed that catalytic activity of enzyme does not require functioning hormone receptors.

Analogues of human calcitonin. II. Influence of modifications in amino acid positions 1, 8 and 22 on hypocalcemic activity in the rat.

The calcitonin analogues [Val8] -HCT and [Tyr22] -HCT, each with a single modification, and [Val8, Tyr22] -HCT and [Bmp1, Val8] -HCT, with two replaced amino acids were compared with synthetic human calcitonin (HCT) in respect of their hypocalcemic effects in the rat. The introduction of either valine in place of methionine in position 8 or of tyrosine for phenylalanine in position 22 of the HCT molecule yielded analogues 4 to 5 times as potent and nearly twice as long-acting as HCT. The doubly substituted peptide [Val8, Tyr22] -HCT displayed properties closely similar to those of [Val8] -HCT and [Tyr22] -HCT. The analgoue [Bmp1, Val8] -HCT, with a deaminated cysteine residue at the N-terminus, was about 6 times more potent than HCT and slightly longer-acting than [Val8] -HCT.

Analogues of human calcitonin I. Influence of modifications in amino acid positions 29 and 31 on hypocalcaemic activity in the rat

Analogues of human calcitonin I. Influence of modifications in amino acid positions 29 and 31 on hypocalcaemic activity in the rat

Calcitonin Receptors on Cultured Human Lymphocytes

SUMMARY Established human lymphoid cell lines bound calcitonin with high affinity. Calcitonin binding was detectable on both B cell and T cell lines derived from patients with a spectrum of lymphoproliferative disorders. These cell lines had marked differences in binding capacity per cell even when adjusted for differences in surface area. The binding was linearly related to cell density and showed a complex temperature dependency. Steady state kinetic analysis suggested a single class of high affinity receptors with an apparent association constant of 4 x lOlo MP. Competitive binding studies with calcitonin analogs revealed an order of Ki values similar to those previously obtained using plasma membranes from known calcitonin target tissues. Human calcitonin, as found previously with renal or skeletal membranes from the rat, competed only weakly for the lymphocyte receptors confirming that the human analog has evolved into a weak or unstable form, or both.

Microelectrophoresis of cholinergic and aminergic drugs on paraventricular neurons

Microelectrophoresis of cholinergic and aminergic drugs on paraventricular neurons