Porcine Calcitonin Research Articles

A novel effect of salmon calcitonin on in vitro Ca-uptake by rat brain hypothalamus: the regional and hormonal specificities.

It was found that salmon calcitonin-I (sCT) inhibited in vitro 45Ca2+-uptake by rat brain hypothalamus blocks in a dose-dependent manner. The minimum effective concentration was estimated to be 10 nM or less. The effect appeared to be specific to the hypothalamus and was not observed with the pons plus medulla oblongata or the cerebral cortex. Two C-terminal fragments of the fish hormone, sCT (10-32) and sCT (22-32), and porcine calcitonin failed to inhibit the ion-uptake though tested in concentrations abolishing 125I-sCT binding to these brain tissues, indicating that the whole structure of sCT is essential for the inhibitory effect but not for the binding. Another finding to be noted was a possible dependency of this effect on the integrity of the cell membrane structure. A crude synaptosomal fraction subsequently prepared from sCT-exposed hypothalamus blocks exhibited a decreased uptake of 45Ca2+, while a corresponding fraction from unexposed tissue did not respond to the hormone. These characteristics of this novel in vitro effect of sCT suggest its possible relevancy to the anorectic effect which also appears to be specific to the fish hormone.

A NOVEL EFFECT OF SALMON CALCITONIN ON IN VITRO Ca-UPTAKE BY RAT BRAIN HYPOTHALAMUS: THE REGIONAL AND HORMONAL SPECIFICITIES

It was found that salmon calcitonin-l (sCT) inhibited in vitro 45Ca2+-uptake by rat brain hypothalamus blocks in a dose-dependent manner. The minimum effective concentration was estimated to be 10 nM or less. The effect appeared to be specific to the hypothalamus and was not observed with the pons plus medulla oblongata or the cerebral cortex. Two C-terminal fragments of the fish hormone, sCT (10-32) and sCT (22-32), and porcine calcitonin failed to inhibit the ion-uptake though tested in concentrations abolishing 125I-sCT binding to these brain tissues, indicating that the whole structure of sCT is essential for the inhibitory effect but not for the binding. Another finding to be noted was a possible dependency of this effect on the integrity of the cell membrane structure. A crude synaptosomal fraction subsequently prepared from sCT-exposed hypothalamus blocks exhibited a decreased uptake of 45Ca2+, while a corresponding fraction from unexposed tissue did not respond to the hormone. These characteristics of this novel in vitro effect of sCT suggest its possible relevancy to the anorectic effect which also appears to be specific to the fish hormone.

Rat enterocyte Na + transport in vitro action of parathyroid hormone and calcitonin

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na + transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na + efflux rate constant ( o K Na ) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of o K Na induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on o K Na was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na + pump. When incubating the isolated epithelial cells in an EGTA-containing Ca 2+-free medium, bovine PTH lost its capacity to inhibit ( o K Na ). Thus, the presence of extracellular Ca 2+ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on o K Na , bovine PTH induced no change in net Na + uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na + transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive o K Na of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca 2+ in the incubation medium to be operative.

Paget's disease of bone: experiences with 100 patients treated with salmon calcitonin.

ONe hundred patients with Paget's disease of bone were treated with salmon calcitonin. Seventy per cent of patients who presented with pain reported improvement of symptoms. Side effects which occurred in 53 patients were more severe in women, necessitating the withdrawal of therapy in 30% of women as against 7.5% of men. Five of the patients with intolerable side effects were subsequently able to tolerate porcine calcitonin, while six were unable to tolerate salmon, porcine or human calcitonin. No audiological improvement occurred. Aortic valve lesions were detected in 11 patients. After a mean treatment period of 7.8 months, serum alkaline phosphatase level was reduced by 39.3%. Total 24-hour urinary hydroxyproline was reduced by 46.1% after 11.2 months of treatment.

Primary structure of the major human pituitary pro-opiomelanocortin NH2-terminal glycopeptide. Evidence for an aldosterone-stimulating activity.

The isolation and complete purification of a human glycopeptide representing the major immunoreactive form of the pituitary NH2-terminal segment of pro-opiomelanocortin is presented. The complete sequence of this peptide was determined following CNBr fragmentation and it is shown to be 76 amino acids long. It bears an O-glycosylation site at Thr 45 and an N-glycosidic linkage at Asn 65. Compared to the reported genomic DNA sequence (Chang, A. C. Y., Cochet, M., and Cohen, S. N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4890-4894), one variation exists, namely Arg 22 replacing Gly 22. Two disulfide bridges linking Cys 2 to 8 and Cys 20 to 24 have been determined. Based on the sequence and disulfide bridge localization, a large degree of homology exists between the NH2-terminal sequence of the human peptide and all calcitonins, especially porcine calcitonin. The human NH2-terminal peptide is shown to stimulate the release of aldosterone from isolated cells of a human adrenal tumor, in at least equipotency to adrenocorticotropic hormone and the porcine NH2-terminal analogue, which is 100 times more potent than angiotensin II. Finally, this reported sequence completes that of the human DNA which was lacking the first 19 amino acids due to the presence of a 2-kilobase intron.

Characterization and regional distribution of calcitonin binding sites in the rat brain

Binding sites for calcitonin (CT), as assayed by the displacable binding of [125-I] iodo salmon CT ([125-I]sCT), were found on a membrane fraction prepared from rat brain. The half times of association varied between 23 and 7 min as a function of the temperatures used in the incubation medium, ranging from 6° to 37°C. Salmon CT in amounts as low as 10 −10 M inhibited the binding of [125-I]sCT to the membranes, whereas the virtually biologically inactive free acid of human CT and human CT sulfone did not affect the binding. The specific binding of [125-I]sCT to the membranes was directed to structural and/or conformational features in the COOH-terminal half of salmon CT. 133 to 8,900 times higher amounts of porcine CT and human CT and analogues thereof were required to achieve an inhibition of binding equal to that produced by salmon CT. Sixty-seven percent of specific binding of labeled hormone was not dissociable, even after 6 h of incubation with an excess of unlabeled hormone. [125-I]sCT extracted from the membranes was not degraded, as judged by gel permeation chromatography, and retained binding activity. Specific binding was highest in the hypothalamus, followed by the brainstem. It was intermediate in the midbrain-thalamus and the striatum, lower in the cortex and negligible in the hippocampus, and cerebellum and the spinal cord.

Release of somatostatin-like immunoreactivity from the perfused canine thyroid. Selective stimulatory effect of calcium ions.

It is well accepted that the C cells of the thyroid contain somatostatin, but the role in local endocrine function has not yet been firmly established in this organ, and it has not been proved that thyroidal somatostatin is released into the circulation. We have measured the contents of somatostatin-like immunoreactivity in the effluent of canine thyroid glands perfused without recirculation with a synthetic buffer medium. During basal conditions a definite release was consistently found in the order of 10 pg/ml corresponding to 12 pg/min. The somatostatin-like immunoreactivity was studied in dilution experiments and by gel-filtration chromatography, and found to have properties identical to those of synthetic cyclic somatostatin, which was also recovered quantitatively when added to sampling tubes. Various compounds were infused in concentrations that are highly active in pancreas perfusion experiments. 14-min infusion of arginine, 5 and 11.5 mmol/liter; isoproterenol, 10 and 23.7 nmol/liter and 68.7 mumol/liter; acetylcholine, 5 mumol/liter, carbamylcholine, 10 and 100 mumol/liter; glucagon, 1 and 30 nmol/liter; and porcine calcitonin, 1 and 100 ng/ml did not affect the basal release of somatostatin-like immunoreactivity significantly. Neither did an increase from the control level of 4 mmol/liter glucose of 10 or 20 mmol/liter, nor an increase in the control level of 4.4 mmol/liter K+ to 7.5 or 14.4 mmol/liter. Each of these compounds were tested in three or four dogs. The effect of an increase in Ca++ from the control level of 1.5 mmol/liter to 2.25, 3.0, and 4.5 mmol/liter was tested in random order in five thyroid lobes. All three doses elicited an immediate increase in effluent somatostatin-like immunoreactivity. In most experiments the response was biphasic with an early spike, followed by a stable level that was maintained during prolonged Ca++ infusion. The secretory response was not diminished through a series of repeated short pulses of calcium infusion. The response to 3.0 mmol/liter Ca++ (control period 8.4 +/- 1.5, test period 337 +/- 110 pg/ml, mean +/- SE) and 4.5 mmol/liter Ca++ (control period 9.5 +/- 1.4, test period 386 +/- 125) were significantly higher than 2.25 mmol/liter Ca++ (control period 7.2 +/- 1.0 test period 140 +/- 39), while there was no significant difference between responses to the two high doses. Infusion of salmon calcitonin, 10 ng/ml and 1 microgram/ml; or porcine calcitonin, 1 microgram/ml during calcium stimulation (2.25 mmol/liter of Ca++) did not induce alterations in the release of somatostatin-like immunoreactivity. The results demonstrate that thyroidal somatostatin is mobilizable, and it appears to be selectively sensitive to calcium stimulation, indicating a possible role in calcitonin release control.

Calcitonin receptors in the central nervous system of the rat.

Specific binding sites for calcitonin (CT) were sought in preparations of rat brain in vitro. Whole rat brains or discrete regions of the brain were homogenized, and the 10,000 × g fraction was tested for binding of 125I-labeled salmon CT ([125I]sCT). The highest total and specific binding occurred in the hypothalamus, and the least occurred in the cortex and cerebellum, relative to whole brain. Intermediate levels of binding of the radioligand were found in the pons-midbrain and medulla. More detailed characterization of the hypothalamusradioligand interaction revealed that the binding was saturable and linear with increasing concentrations of tissue protein from 25–800 μg/ml. Furthermore, it was both time and temperature dependent, reaching equilibrium by 1 h at 4, 23, and 37 C. After 1 h of incubation at 4 C, half-maximal inhibition of binding of the radioligand occurred at a sCT concentration of 1 × 10-9 M. Both porcine and human CT were less effective than sCT in inhibiting the binding of [125I]sCT t...

SPECIFIC BINDING OF 125I-SALMON CALCITONIN TO RAT BRAIN: REGIONAL VARIATION AND CALCITONIN SPECIFICITY

AbstractRat brain particulate fraction was found to contain binding sites for 125I-Salmon Calcitonin-I (125I-SCT). Maximum binding occurred in the physiological pH range of 7.25–7.5. The binding reaction proceeded in a temperature-dependent manner. Binding sites were broadly distributed among the various rat brain regions and considerable regional differences existed in the affinity and density as detected by Scatchard analysis. The highest affinity was recorded in the case of the hypothalamus and the lowest in the case of the cerebellum. The KD (nM) and Bmax (pmole/mg protein) estimated for the binding to four regions were as follows: hypothalamus: 1.4 and 0.19, midbrain, hippocampus plus striatum: 1.5 and 0.08, pons plus medulla oblongata: 3.0 and 0.15 and cerebellum: 8.3 and 0.20. Using a particulate fraction of rat brain void of cerebellum and cortices, a binding assay for calcitonins was developed. Binding of 125I-SCT was inhibited by unlabeled salmon, [Asu1,7]-eel* and porcine calcitonins in a dose-dependent manner and the IC50s were 2.0, 8.0 and 30 nM, respectively. The IC50s were comparable to those estimated using a kidney particulate fraction. Human calcitonin, β-endorphin and substance P were weak inhibitors of the binding. Other peptides, drugs and putative neurotransmitters tested (totally 23 substances) failed to inhibit the binding at concentrations of 1.0 μM. The physiological significance of brain binding sites for calcitonin, with the possiblity that the brain may possess endogenous ligands for these sites are discussed.

Evoked potential studies of porcine calcitonin in rabbits

Effects of porcine calcitonin on evoked potentials of rabbits to painful stimuli were examined. The development of the long latency cortical potential evoked by electrical stimulation of both the tooth pulp and sciatic nerve in gallamine-immobilized rabbits was inhibited by i.c.v. calcitonin of 17 U/kg, but not by the intravenous injection of 50 U/kg. Pretreatment with naloxone (0.5 mg/kg i.v.) showed no influence upon the inhibitory effect of calcitonin, though it completely antagonized the effect of morphine. The results suggest the analgesic effect of i.c.v. porcine calcitonin can be attributed to blocking of pain pathways in the central nervous system in a manner distinct from the narcotic analgesic.

Specific binding of 125I-salmon calcitonin to rat brain: regional variation and calcitonin specificity.

Rat brain particulate fraction was found to contain binding sites for 125I-Salmon Calcitonin-I (125I-SCT). Maximum binding occurred in the physiological pH range of 7.25-7.5. The binding reaction proceeded in a temperature-dependent manner. Binding sites were broadly distributed among the various rat brain regions and considerable regional differences existed in the affinity and density as detected by Scatchard analysis. The highest affinity was recorded in the case of the hypothalamus and the lowest in the case of the cerebellum. The KD (nM) and Bmax (pmole/mg protein) estimated for the binding to four regions were as follows: hypothalamus: 1.4 and 0.19, midbrain, hippocampus plus striatum: 1.5 and 0.08, pons plus medulla oblongata: 3.0 and 0.15 and cerebellum: 8.3 and 0.20. Using a particulate fraction of rat brain void of cerebellum and cortices, a binding assay for calcitonins was developed. Binding of 125I-SCT was inhibited by unlabeled salmon, [Asu1,7]-eel and porcine calcitonins in a dose-dependent manner and the IC50s were 2.0, 8.0 and 30 nM, respectively. The IC50s were comparable to those estimated using a kidney particulate fraction. Human calcitonin, beta-endorphin and substance P were weak inhibitors of the binding. Other peptides, drugs and putative neurotransmitters tested (totally 23 substances) failed to inhibit the binding at concentrations of 1.0 microM. The physiological significance of brain binding sites for calcitonin, with the possibility that the brain may possess endogenous ligands for these sites are discussed.

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.

The effect of porcine calcitonin on intestinal calcium and phosphate fluxes in the young piglet

The effects of a physiological (3.8 ng.mL-1) and a pharmacological (120 ng.mL-1) concentration of porcine calcitonin (CT) on transmucosal calcium and phosphate flux rates were determined in intestinal mucosa from young piglets (4--40 days) mounted in Ussing flux chambers. The physiological concentration of the hormone inhibited net absorptive calcium flux rates in the proximal jejunum and distal ileum but not in the duodenum. No effect of either concentration of CT on phosphate flux rates was observed. The inhibitory effect of the physiological concentration may indicate a role for CT in the maintenance of calcium homeostasis in the young suckling mammal.

Effects of ketoprofen on the calciuric and uricosuric activities of calcitonin in man.

The action of ketoprofen on the calciuric and uricosuric effects of porcine calcitonin in man is reported. The drug is capable of inhibiting both effects. This action is considered like an effect on CT receptors in the kidney or a competitive action on prostaglandin synthesis.

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.

Immunohistological studies of artiodactyl and teleost pituitaries with antisera to calcitonin

The pituitary glands of pigs, cows, sheep (artiodactyls), and trout and goldfish (teleosts) were studied by immunohistology for the presence of calcitonin with antisera specific for the respective thyroidal and ultimobranchial calcitonins of these species. The specificity of the antisera was demonstrated by the lack of reactivity with other pituitary hormones and by the lack of cross-reactivity among the three groups of biochemically distinct thyroidal or ultimobranchial calcitonins: (a) rat (and human), (b) artiodactyl, and (c) teleost. The antiserum to salmon (teleost) calcitonin demonstrated immunohistologically positive cells in the teleost pituitaries, but not in the pig, sheep, or cow pituitaries. The antiserum to porcine calcitonin demonstrated immunohistologically positive cells in the artiodactyl but not in the teleost (or rat) pituitaries. The human calcitonin antiserum, cross-reacting with rat thyroidal calcitonin and demonstrating immunohistologically positive cells in the rat pituitary gland, did not demonstrate immunohistologically positive cells in the artiodactyl or teleost pituitaries. These studies demonstrate the presence of immunoreactive pituitary calcitonin in teleosts and artiodactyls and the species and immunochemical specificity of this phenomenon.

Evaluation of a Radioimmunoassay Technique Measuring Calcitonin in The Bovine, Ovine and Porcine Species

A heterologous RIA system was set up for measuring bovine, ovine and porcine calcitonin (CT). The system consisted of porcine GT used as standard and for the preparation of an iodinated tracer. The antiserum used was raised against ovine GT. For each analysis was used 25–200 µl blood plasma. Practical detection limit was 0.25 µg of CT per litre of blood plasma. The parallelism between the dose response curves for the p-GT standard and for the assay of increasing amounts of bovine, ovine and porcine blood plasma showed the suitability of the present assay system to study the GT secretion in these species. Furthermore, the reliability of the method was verified by a clearly recognized CT response to calcium infusion.

Presence by radioimmunoassay of a calcitonin-like substance in porcine pituitary glands.

We studied acidic acetone extracts of whole porcine pituitary glands for the presence of immunoreactive calcitonin (CT) using a porcine CT (pCT) RIA which did not react with other known pituitary hormones. Four preparations of porcine pituitary extract contained immunoreactive CT. Three of these displayed inhibition of binding parallel to that of authentic pCT in the pCT RIA and contained a single peak of immunoreactivity similar to pCT when studied by two different gel filtration chromatography systems. One preparation of porcine pituitary extract showed nonparallelism in RIA dose-dilution experiments and multiple immunoreactive species both similar to and larger than pCT on gel filtration in 6 M guanidine HCl. The effect of the reduction of disulfide bonds, followed by carboxymethylation of sulfhydryl groups, on immunoreactivity and apparent molecular size was similar for the CT-like substance in porcine pituitary extract and for authentic pCT. Preliminary immunohistological studies showed cytoplasmic staining in cells of the porcine adenohypophysis. These results demonstrate that the porcine pituitary gland contains a substance which has some of the immunochemical and biochemical properties of thyroidal pCT.

Effects of calcitonin in a case of congenital hypophosphatasia: 42

A female infant with congenital limb deformities developed craniotabes and x-ray revealed rachitic metaphyses. Initial biochemistry showed serum calcium 11.6 mg%, phosphate 7.8 mg%, alkaline phosphatase 6 King Armstrong units. Subsequent alkaline phosphatase values were persistently low (range 0.9-2.7 K.A.Units) and a diagnosis of the severe form of congenital hypophosphatasia was made supported by levels of alkaline phosphatase in cultured cells and tissues. Family studies showed a pattern consistent with autosomal recessive inheritance. At the age of three months she was irritable, feeding poorly and failing to thrive. Porcine calcitonin 50 M.R.C. Units/day was given intramuscularly for three weeks. Within two weeks hardening of the skull bones was noted with disappearance of craniotabes which recurred, however, six weeks later. X-rays over the following months showed increased mineralisation and periosteal new bone. Metabolic balance studies for calcium, phosphorus and magnesium before, during and after calcitonin therapy showed an alteration from an average positive calcium balance of 15 mg/day to 103 mg/day with treatment. No consistent change in phosphate or magnesium balance was evident. At the time of calcitonin treatment evidence of craniosynostosis developed. The child died of chest infection at 14 months of age. In this case of severe congenital hypophosphatasia there is evidence of clinical, radiological and biochemical response to calcitonin. Treatment may have precipitated craniosynostosis and failed to alter the grave prognosis. It is possible that smaller doses of calcitonin over a longer period of time would be safer and more effective.