Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.     

Amylin receptors mediate the anorectic action of salmon calcitonin (sCT)

The teleost salmon calcitonin (sCT), but not mammalian CT, shows similar biologic actions in the skeletal muscle as amylin and calcitonin gene-related peptide (CGRP). The peptides have also been shown to reduce food intake in rams. Because sCT, but not amylin, binds irreversibly to amylin binding sites, the aim of the present study was to compare the anorectic potency of both peptides. To determine whether sCT reduces food intake through interaction with amylin binding sites, we also tested whether appropriate antagonists (CORP 8-37, AC 187) attenuate the anorectic effect of sCT. Finally, we wanted to know whether rat calcitonin (rCT) and sCT reduce food intake to the same extent. Peptides were injected intraperitoneally at dark onset in 24 h food-deprived rats. At doses of 5 or 0.5 microg/kg, the anorectic effect of sCT was more potent and lasted much longer (e.g. 5 microg/kg: sCT > 10 h; amylin approx. 2 h) than that of amylin. Both CORP 8-37 and AC 187 (10 microg/kg) markedly reduced the anorectic action of sCT (0.5 microg/kg). In contrast to sCT, rCT (0.5 microg/kg) had no effect on food intake. It is concluded that sCT s anorectic effect is partly mediated by amylin receptors. Irreversible binding of sCT to amylin receptors may lead to a stronger and prolonged effect in comparison to amylin due to a sustained activation of the binding sites. Similar to other actions of CTs, the anorectic potency of sCT in rats was higher than that of mammalian (rat) CT. This agrees with binding profiles of amylin, sCT, and rCT at amylin binding sites as observed in in vitro studies.     

Calcitonin inhibition of physiological and stimulated prolactin secretion in rats

The administration of salmon Calcitonin (sCT) intravenously (2.5 or 10 μg/kg) or into the lateral cerebral ventricles (2.5 or 25 ng/rat, i.c.v.) of unanaestized male rats induced clearcut decreases in plasma prolactin(PRL) levels. The i.c.v. injection of one of these doses of sCT (25 ng/rat) into rats with median eminence lesions was completely ineffective, while it induced a dramatic decrease in plasma PRL levels of sham-operated rats. Morphine- and heat stress-stimulated PRL levels were also abolished by sCT injection (250 ng/rat i.c.v.). The sCT-induced decrease in PRL levels was completely overcome by haloperidol, a dopamine-receptor blocker. We conclude that sCT may affect PRL secretion via an hypothalamic system, probably involving dopaminergic neurons. The present results indicate that CT, like many others peptides, may affect PRL secretion, directly or indirectly, even though further research is necessary to determine whether this effect has pharmacological or physiological importance.     

Anti-hypercalcemic effect of orally administered recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing salmon calcitonin on hypercalcemic rats

Oral delivery of salmon calcitonin (sCT) to rats via a recombinant Saccharomyces cerevisiae was assessed. A synthetic sCT gene was cloned and expressed in S. cerevisiae yAGA2-sCT. Recombinant salmon calcitonin (rsCT) expression was detected by flow cytometry. The resorption activity of osteoclasts was inhibited by 3 × 10⁻⁶ M rsCT. Oral administration of 5 g lyophilized yAGA2-sCT/kg to hypercalcemic rats decreased serum calcium from 2.8 ± 0.02–2.7 ± 0.02 mM.     

The effects of calcitonin on plasma calcium levels and bone metabolism in the fresh water teleost Channa punctatus

Administration of salmon calcitonin (sCT) caused significant reduction in total and ultrafiltrable plasma calcium content in the plasma of a fresh water female teleost Channa punctatus. A time-bound analysis on the effect of sCT showed a highly significant short duration reduction in total and ultrafiltrable plasma calcium content in fish kept in normal tap water and low-calcium water and a moderate hypocalcemia in fish kept in high-calcium water. Sexually immature adult fish showed a greater response than the sexually mature ones. Using tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) activities in plasma and hydroxyproline (HYP) excretion in urine, the effect of sCT on the inhibition of bone calcium resorption were examined. In both sexually mature and immature adult fish, kept in normal tap water, sCT significantly suppressed TRACP and ALP activities in plasma and excretion of HYP in urine within 2-6 h with a maximum at 4 h after injection. Salmon CT treatment to sexually immature adult fish caused significant increase in skeletal bone calcium concentration. Taken together, all this information indicates that CT in a fresh water female teleost is an effective regulator of plasma calcium levels, and its action, at least in part, operates through inhibition of bone calcium resorption.     

Application of the yeast‐surface‐display system for orally administered salmon calcitonin and safety assessment

High manufacturing costs and oral delivery are the constraints in clinical application of calcitonin. We selected surface‐displayed Saccharomyces cerevisiae as a low‐cost and safe carrier for oral delivery of salmon calcitonin (sCT). The sCT DNA fragment, optimized according to the codon preference of S. cerevisiae, was synthesized and cloned into the plasmid M‐pYD1 to yield recombinant yAGA2‐sCT, which was induced to express sCT by galactose for 0, 12, and 24 h. sCT expression was detected on the cell surface by indirect immunofluorescence and peaked at 12 h. About 65% recombinants expressed sCT on flow cytometry. The in vivo and in vitro activity of recombinant sCT was determined by detecting bioactivity of antiosteoclastic absorption on bone wafers and orally administering yAGA2‐sCT to Wistar rats, respectively. For safety assessment of yAGA2‐sCT, we observed abnormalities, morbidity, and mortality and determined body weight, serum chemistry parameters, hematological parameters, and organ weight. In vitro bioactivity of the recombinant sCT was similar to that of commercial sCT, Miacalcic; oral administration of 5 g/kg yAGA2‐sCT induced a long‐term hypocalcemic effect in Wistar rats and no adverse effects. This study demonstrates that yAGA2‐sCT anchoring sCT protein on a S. cerevisiae surface has potential for low‐cost and safe oral delivery of sCT. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of amylin-related peptides on food intake, meal patterns, and gastric emptying in rats

We previously demonstrated that amylin inhibits food intake and gastric emptying in rats with half-maximal effective doses (ED(50)s) of 8 and 3 pmol x kg(-1) x min(-1) and maximal inhibitions of 78 and 60%, respectively. In this study of identical design, rats received intravenous infusions of salmon calcitonin (sCT), rat calcitonin (rCT), rat calcitonin gene-related peptide (rCGRP), and rat adrenomedullin (rADM) for 3 h at dark onset, and food intake was measured for 17 h or for 15 min and gastric emptying of saline was measured during the final 5 min. sCT, rCGRP, and rADM inhibited food intake with estimated ED(50)s of 0.5, 26, and 35 pmol x kg(-1) x min(-1) and maximal inhibitions of 88, 90, and 49%, respectively. rCT was not effective at doses up to 100 pmol x kg(-1) x min(-1). sCT, rCGRP, rADM, and rCT inhibited gastric emptying with ED(50)s of 1, 130, 160, and 730 pmol x kg(-1) x min(-1) and maximal inhibitions of 60, 66, 60, and 33%, respectively. These results suggest that amylin and sCT may act by a common mechanism to decrease food intake, which includes inhibition of gastric emptying.     

人降钙素类似物的合成及活性研究

通过对鲑鱼降钙素和人降钙素结构的比较,设计并合成了人降钙素类似物ｍｈＣＴ－２。利用空气氧化获得分子内二硫键,经分离纯化,产物达ＨＰＬＣ及毛细管电泳均一,蛋白质序列分析和质谱分析符合理论值。在大鼠降血钙生物活性测定中,利用量反应平行线法进行ｍｈＣＴ－２的生物效价判定达２０００ＩＵ／ｍｇ,比ｈＣＴ的效价高一个数量级。     

Acute effect of large doses of calcitonin on calcium, magnesium and inorganic phosphate levels in rat serum in correlation to differences in thryroid function.

The acute effect of pharmacological doses of porcine calcitonin were studied on the calcium, inorganic phosphorus and magnesium level in correlation to thyroid function in experimental rats. In every case, and with all three doses, the serum calcium and inorganic phosphorus level fell significantly within 60 minutes after calcitonin administration. The drop in both minerals was most pronounced in the group with experimental hyperthyroidism, in which all different doses had the same effect. In experimental hypothyroidism, the decrease was correlated to the dose. In the case of the serum magnesium level, a statistically significant drop was observed in animals with hyperthyroidism after the two largest doses of calcitonin, while only a mild increase occurred in animals with hypothyroidism after the smallest dose. None of the doses of calcitonin affected the magnesium level in animals with normal thyroid function.     

Effect of the secretin family of peptides on gastric emptying and small intestinal transit in rats

We have compared the effects of the secretin family of peptides and their synthetic fragments on gastric emptying (GE) and small intestinal transit (SIT) using an unanesthetized rat model which simultaneously measures the GE and SIT of both solids and liquids. The meal consisting of 5% polyethylene glycol w/v, 5% Indian ink v/v and 20 non-digestible plastic beads was given intragastrically 10 minutes after the intraperitoneal injection of 0.5 ml of saline or peptides (2 and 5 micrograms/kg). Plasma secretin and the immunospecificity of secretin fragments were determined. In control rats, the t1/2 for the GE of both solids and liquids were 56 +/- 3.8 and 19 +/- 2.3 minutes, respectively. Liquids emptied faster than the solids and liquids travelled ahead of the solids in the intestine. Secretin (5 micrograms/kg) inhibited GE of both solids and liquids by 33-37%. Secretin delayed the SIT of the meal by approximately 35%. Fragments of secretin and of VIP had no effect on GE and SIT of both solids and liquids. The whole molecule of secretin was required to inhibit GE and to delay SIT of solids and liquids. Glucagon, PHI and growth hormone releasing factor (GHRF1-44) inhibited GE and SIT of both solids and liquids. For all peptides tested, the inhibition of SIT was proportional to the inhibition of GE suggesting that the prolongation of SIT was secondary to delayed GE. These observations indicate that the peptides of the secretin family inhibit GE and prolong SIT. Thus, the structural requirement required for the secretin family of peptides to effect their motor actions on the stomach is similar to that required for pancreatic enzyme secretion.     

The influence of NMDA receptor agonist and antagonist on morphine state-dependent memory of passive avoidance in mice

The measurement of step-down latency in passive avoidance has been used to study memory in laboratory animals. The pre-training injection of 5 mg/kg morphine impaired memory, which was restored when 24 h later the same dose of the drug was administered. To explore the possible involvement of NMDA modulators on morphine-induced memory impairment, we have investigated the effects of intracerebroventricular (i.c.v.) administration of NMDA and the competitive NMDA antagonist, DL-AP5, on morphine-induced memory impairment or recall, on the test day. Morphine (5 mg/kg, s.c.) was administered 30 min before training to induce impairment of memory and 24 h later, 30 min before test to improve it. Pre-test administration of NMDA (0.00001, 0.0001 and 0.001 microg/mouse, i.c.v.) did not alter the retention latency compared to the saline-treated animals. But restored the memory impairment induced by pre-training morphine (5 mg/kg, s.c.). Pre-test administration of DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) by itself decreased the retention latencies. The same doses of DL-AP5 increased pre-training morphine-induced memory impairment. Co-administration of NMDA (0.0001 and 0.001 microg/mouse, i.c.v.) and morphine (5 mg/kg, s.c.) on the test day increased morphine memory improvement. Conversely, DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) inhibited morphine-induced memory recall. It is concluded that NMDA receptors may be involved, at least in part, in morphine state-dependent learning in mice.     

Central injection of nicotine increases hepatic and splenic interleukin 6 (IL-6) mRNA expression and plasma IL-6 levels in mice: involvement of the peripheral sympathetic nervous system.

Accumulating evidence suggests that plasma levels of interleukin 6 (IL-6), a major cytokine stimulating the synthesis of acute-phase proteins, are intimately regulated by the central nervous system. Nicotine, one of the major drugs abused by humans, has been shown to affect immunological functions. In the present study, effects of intracerebroventricular (i.c.v.) injection of nicotine on plasma IL-6 levels were investigated in mice. Nicotine administered i.c.v. dose-dependently increased plasma IL-6 levels; the lowest effective dose was 0.3 ng/mouse and the maximal effect was attained with the dose of 105 ng/mouse. The nicotine (105 ng/mouse, i.c.v.)-induced plasma IL-6 levels peaked at 3 h and approached basal levels 6 h after injection. Mecamylamine, a nicotinic receptor antagonist, blocked nicotine-induced plasma IL-6 levels. Depletion of peripheral norepinephrine with 6-hydroxydopamine [100 mg/kg, intraperitoneal (i. p.)] inhibited the nicotine-induced plasma IL-6 levels by 57%, whereas central norepinephrine depletion with 6-hydroxydopamine (50 microgram/mouse, i.c.v.) had no effect. Pretreatment with prazosin (alpha1-adrenergic antagonist; 1 mg/kg, i.p.), yohimbine (alpha2-adrenergic antagonist; 1 mg/kg, i.p.), and ICI-118,551 (beta2-adrenergic antagonist; 2 mg/kg, i.p.), but not with betaxolol (beta1-adrenergic antagonist; 2 mg/kg, i.p.), inhibited nicotine-induced plasma IL-6 levels. Among the peripheral organs, including the pituitary, adrenals, heart, lung, liver, spleen, and lymph nodes, nicotine (105 ng/mouse, i.c.v.) increased IL-6 mRNA expression only in the liver and spleen, which was inhibited by peripheral norepinephrine depletion. These results suggest that stimulation of central nicotinic receptors induces plasma IL-6 levels and IL-6 mRNA expression in the liver and spleen via the peripheral sympathetic nervous system, alpha1-, alpha2-, and beta2-adrenoreceptors being involved.     

Intravenous human rabies immunoglobulin for post-exposure prophylaxis: serum rabies neutralizing antibody concentrations and side-effects

The beneficial effect of passive immunization for post-exposure rabies prophylaxis is associated with the appearance of serum neutralizing antibody (SNA) earlier than occurs with vaccine alone. We compared the SNA response and the side-effects in 30 previously unimmunized healthy volunteers given a commercially available human rabies immunoglobulin (HRIG) intramuscularly (i.m.) or an experimental HRIG prepared by DEAE Sephadex column chromatography, intravenously (i.v.) with or without human diploid-cell culture rabies vaccine (HDCS). The subjects were divided into five equal groups: HDCS alone, HDCS + i.m. HRIG 20 IU/kg (currently recommended), i.v. HRIG alone 15 IU/kg, HDCS + i.v. HRIG 15 IU/kg or HDCS + HRIG 5 IU/kg i.v. plus 10 IU/kg i.m. to simulate local bite wound infiltration. HDCS, 1.0 ml, was injected subcutaneously (s.c.) on days 0, 3, 7, 14 and 28. Only local discomfort at injection sites was observed without differences between groups. SNA was demonstrated in all HRIG recipients at day 1, but the concentrations were higher in those receiving it intravenously. No difference in the SNA response to vaccine was observed between the i.v. and i.m. HRIG groups given the same vaccine lot. It would appear that i.v. HRIG 15 IU/kg can be substituted for i.m. HRIG 20 IU/kg for post-exposure prophylaxis. Since the current regimen is almost 100% protective, there is no way of proving that i.v. HRIG 15 IU/kg is more efficacious. The immediate SNA level and economy are the chief advantages of i.v. HRIG 15 IU/kg.     

Preparation and Characterization of Salmon Calcitonin–biotin Conjugates

This study was performed to prepare and characterize the biotinylated Salmon calcitonin (sCT) for oral delivery and evaluate the hypocalcemic effect of biotinylated-sCTs in rats. Biotinylated sCTs was characterized by using high performance liquid chromatography (HPLC) and MALDITOF-MS. The effect of biotinylation on permeability across Caco-2 cell monolayers was examined. Their hypocalcemic effect was determined in rats. Mono- and di-bio-sCTs were separated by reverse phase HPLC. The molecular weights of mono-bio-sCT and di-bio-sCT were determined to be 3,660.5 and 3,900.2 Da, respectively. The permeability of biotinylated-sCTs across Caco-2 cell monolayers was observed with a significant enhancement compared with sCT. Intrajejunal (ij) administration of mono-bio-sCT and di-bio-sCT resulted in sustained reduction in serum calcium levels, with a maximum reduction (% max(d)) of 21.6% and 30% after 4 h and 6 h of application, respectively. The biotin conjugation of sCT may be a promising strategy for increasing the oral bioavailability of sCT and achieving sustained calcium-lowering effects.     

Influence of verapamil on central and peripheral effects of prostacyclin on circulatory system in rats

The influence of verapamil on cardiovascular effects of prostacyclin (PGI2) in rats was examined. PGI2 administered into the lateral brain ventricle (i.c.v.) or intravenously (i.v.) in a dose of 2.7 x 10(-8)mol evoked hypotension and tachycardia. Pretreatment with verapamil in a dose of 2.0 x 10(-5)mol/kg given intraperitoneally (i.p.) diminished hypotensive effect of PGI2 i.c.v. as well as inhibiting the influence of PGI2 i.c.v. and i.v. upon the heart rate. Bolus injection of PGI2 in a dose of 2.7 x 10(-10), 2.7 x 10(-9) or 2.7 x 10(-8)mol evoked biphasic inotropic and chronotropic effects on isolated rat heart. Short-term increase of the contractile force together with bradycardia and afterwards long-lasting decrease of contractility with sustained, slight tachycardia were observed. Verapamil in a concentration of 1.0 x 10(-6)M blocked biphasic inotropic effect and bradycardia after PGI2 administration. Because some central and peripheral cardiovascular effects of PGI2 were inhibited by verapamil, it is concluded that PGI2 may participate in transmembrane calcium ions movements.     

Simple liquid chromatography-electrospray ionization mass spectrometry method for the routine determination of salmon calcitonin in serum

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the quantification of salmon calcitonin (sCT) in serum. Serum samples from rats and dogs were deproteinized and freeze-dried. The residue was then reconstituted with 57% acetonitrile in water containing 0.1% trifluoroacetic acid and 0.005% benzalkonium chloride. A 20-microl aliquot of the reconstituted solution was injected onto a polymer based RP-C18 column, The outlet was connected to an ion-trap mass spectrometer equipped with an ESI source, and spectra were recorded in a positive-ion, selected-ion monitoring mode. The limit of quantification of the method was 10 ng/ml. Biexponential curves were observed for the temporal serum concentration of sCT following intravenous administration of sCT to rats (100 microg/kg) and dogs (250 microg/kg), resulting in reasonable pharmacokinetic parameters. The present method appears applicable to routine analysis of serum sCT in pharmacokinetic studies with good selectivity, accuracy and precision.     

Inhibition of whole body Ca2+ uptake in fresh water teleosts, Channa punctatus and Cyprinus carpio in response to salmon calcitonin

In many fish, ultimobranchial-derived calcitonin (CT) has been shown to be a potent hypocalcemic regulator. But an equal number of studies failed to show any correlation between CT and plasma calcium levels. Again, in fish, where CT has been shown to function as a hypocalcemic hormone, the way this is brought about is not well known. While the regulatory role of many hormones e.g., stanniocalcin, pituitary-derived prolactin and cortisol on gill calcium (Ca2+) transport (GCAT) has been well established, very few studies have been done to examine the effects of CT on GCAT in fish. In the present study we examined the effects of synthetic salmon calcitonin (sCT) in vivo on GCAT in two distinctly different species of fresh water teleost, Channa punctatus (partially air breathing) and Cyprinus carpio (fully gill breathing). Whole body calcium uptake, a measure of GCAT, was lower in the partial air breathing fish. We found that salmon CT had significant inhibitory effect on GCAT in both the fish species, kept either in normal tap water or low-calcium water. Fish, kept in high-calcium water, showed little response. In parallel studies we also observed that inhibition of GCAT was correlated with simultaneous changes in plasma calcium levels in response to exogenous administration of sCT. The present findings therefore suggest that CT in fresh water teleosts regulate its hypocalcemic action through inhibition of GCAT.     

Biological actions of human and rat calcitonin and calcitonin gene-related peptide

We assessed the central and peripheral biological actions of human and rat calcitonin and calcitonin gene-related peptide (CGRP). After intravenous administration, human and rat calcitonin, but neither human nor rat CGRP significantly decreased plasma calcium and phosphorus concentrations in awake, freely moving rats. After intracerebroventricular as well as after intravenous administration, human and rat calcitonin and human and rat CGRP significantly inhibited gastric acid secretion in conscious rats. Intracerebroventricular administration of rat calcitonin did not alter plasma calcium and phosphorus concentrations. Linear, partially protected CGRP and calcitonin did not exhibit any biological effects. These studies indicate that calcitonin, but not CGRP, affects calcium and phosphorus homeostasis while both peptides decrease gastric acid secretion similarly. Furthermore, these studies support the hypothesis that the calcium and phosphorus lowering effects of calcitonin are peripheral while the gastric inhibiting actions of the calcitonin and CGRP are mediated by the central nervous system.     

Effect of 6-azauridine on calcium metabolism in rats.

After 12 days of intramuscular administration of 6-azauridine (500 mg/kg b.w.) rats displayed a significant decrease in plasma calcium, inorganic phosphorus, and total hydroxyproline levels and alkaline and acid phosphatase activity. The biological method employed revealed no changes in calcitonin activity. 6-Azauridine reduced the citric acid concentration in the kidneys, liver, heart and bones. Alkaline phosphatase activity in the kidneys, heart and liver was unaffected. The results indicate that 6-azauridine inhibits calcium resorption from the bones and interferes with collagen synthesis. It cannot be ruled out that the described changes are elicited by the antimetabolic effect of this cytostatic drug.     

Intracerebroventricular administration of calcitonin enhances glucose-stimulated release of insulin

Intracerebroventricular (i.c.v.) administration of salmon calcitonin (500 ng) augmented glucose-stimulated release of insulin in rats. Vagotomy increased this enhancement effect of i.c.v. calcitonin significantly, whereas peripheral atropine treatment did not change it. Adrenal catecholamines did not participate in the centrally mediated insulinotropic effect of calcitonin since acute adrenalectomy did not modify the enhancement effect of i.c.v. calcitonin. Destruction of the sympathetic ganglia by neonatal treatment with 6-hydroxydopamine abolished the enhancement effect of i.c.v. calcitonin, which suggests that the sympathetic nervous system participates in the central action of calcitonin to enhance glucose-stimulated release of insulin.