Role of Electrostatic Interactions in Calcitonin Prefibrillar Oligomer-Induced Amyloid Neurotoxicity and Protective Effect of Neuraminidase.

Salmon calcitonin is a good model for studying amyloid behavior and neurotoxicity. Its slow aggregation rate allows the purification of low molecular weight prefibrillar oligomers, which are the most toxic species. It has been proposed that these species may cause amyloid pore formation in neuronal membranes through contact with negatively charged sialic acid residues of the ganglioside GM1. In particular, it has been proposed that an electrostatic interaction may be responsible for the initial contact between prefibrillar oligomers and GM1 contained in lipid rafts. Based on this evidence, the aim of our work was to investigate whether the neurotoxic action induced by calcitonin prefibrillar oligomers could be counteracted by treatment with neuraminidase, an enzyme that removes sialic acid residues from gangliosides. Therefore, we studied cell viability in HT22 cell lines and evaluated the effects on synaptic transmission and long-term potentiation by in vitro extracellular recordings in mouse hippocampal slices. Our results showed that treatment with neuraminidase alters the surface charges of lipid rafts, preventing interaction between the calcitonin prefibrillar oligomers and GM1, and suggesting that the enzyme, depending on the concentration used, may have a partial or total protective action in terms of cell survival and modulation of synaptic transmission.

Calcitonin native prefibrillar oligomers but not monomers induce membrane damage that triggers NMDA-mediated Ca2+-influx, LTP impairment and neurotoxicity.

Amyloid protein misfolding results in a self-assembling aggregation process, characterized by the formation of typical aggregates. The attention is focused on pre-fibrillar oligomers (PFOs), formed in the early stages and supposed to be neurotoxic. PFOs structure may change due to their instability and different experimental protocols. Consequently, it is difficult to ascertain which aggregation species are actually neurotoxic. We used salmon Calcitonin (sCT) as an amyloid model whose slow aggregation rate allowed to prepare stable samples without photochemical cross-linking. Intracellular Ca2+ rise plays a fundamental role in amyloid protein-induced neurodegerations. Two paradigms have been explored: (i) the "membrane permeabilization" due to the formation of amyloid pores or other types of membrane damage; (ii) "receptor-mediated" modulation of Ca2+ channels. In the present paper, we tested the effects of native sCT PFOs- with respect to Monomer-enriched solutions in neurons characterized by an increasing degree of differentiation, in terms of -Ca2+-influx, cellular viability, -Long-Term Potentiation impairment, Post-Synaptic Densities and synaptophysin expression. Results indicated that PFOs-, but not Monomer-enriched solutions, induced abnormal -Ca2+-influx, which could only in part be ascribed to NMDAR activation. Thus, we propose an innovative neurotoxicity mechanism for amyloid proteins where "membrane permeabilization" and "receptor-mediated" paradigms coexist.

The Interaction between Amyloid Prefibrillar Oligomers of Salmon Calcitonin and a Lipid-Raft Model: Molecular Mechanisms Leading to Membrane Damage, Ca2+-Influx and Neurotoxicity.

To investigate the interaction between amyloid assemblies and "lipid-rafts", we performed functional and structural experiments on salmon calcitonin (sCT) solutions rich in prefibrillar oligomers, proto- and mature-fibers interacting with liposomes made of monosialoganglioside-GM1 (4%), DPPC (48%) and cholesterol (48%). To focus on the role played by electrostatic forces and considering that sCT is positive and GM1 is negative at physiologic pH, we compared results with those relative to GM1-free liposomes while, to assess membrane fluidity effects, with those relative to cholesterol-free liposomes. We investigated functional effects by evaluating Ca2+-influx in liposomes and viability of HT22-DIFF neurons. Only neurotoxic solutions rich in unstructured prefibrillar oligomers were able to induce Ca2+-influx in the "lipid-rafts" model, suggesting that the two phenomena were correlated. Thus, we investigated protein conformation and membrane modifications occurring during the interaction: circular dichroism showed that "lipid-rafts" fostered the formation of ß-structures and energy filtered-transmission electron microscopy that prefibrillar oligomers formed pores, similar to Aß did. We speculate that electrostatic forces between the positive prefibrillar oligomers and the negative GM1 drive the initial binding while the hydrophobic profile and flexibility of prefibrillar oligomers, together with the membrane fluidity, are responsible for the subsequent pore formation leading to Ca2+-influx and neurotoxicity.

Procalcitonin Impairs Endothelial Cell Function and Viability.

BACKGROUND: Procalcitonin is used as a diagnostic tool for the identification and risk stratification of septic patients. Procalcitonin plasma concentrations tightly correlate with the severity of the ongoing inflammatory reaction and can rise up to 10,000-fold. Impairment of endothelial cell function plays an important role in the pathogenesis of hypotension and disturbed organ perfusion during sepsis. We investigated the possible effects of procalcitonin itself on endothelial cell function and viability. METHODS: Human endothelial cells were exposed to 0.01 to 100 ng/mL procalcitonin and investigated for endothelial permeability using transwells, migration in a scratch wound assay and new capillary formation on extracellular matrix in vitro. Tumor necrosis factor-α and vascular endothelial growth factor served as positive controls. Procalcitonin's impact on the response of endothelial cells toward ischemia was investigated in vivo in the murine model of unilateral femoral artery ligation. Procalcitonin-exposed endothelial cells were subjected to immunoblot for the investigation of vascular endothelial-cadherin expression and angiogenic signaling pathways. Flow cytometry was used for the detection of inflammatory activation and viability, and genomic analysis was performed. Data are presented as difference in means and 95% confidence intervals; statistical analyses were performed using analysis of variance/Bonferroni, and P values are reported as adjusted for multiple comparisons (Padjust). RESULTS: Tumor necrosis factor-α and 0.1 ng/mL procalcitonin induced endothelial barrier disruption after incubation of endothelial monolayers for 6 hours (-2.53 [-4.16 to -0.89], P = .0008 and -2.09 [-3.73 to -0.45], Padjust = .0064 compared with vehicle-treated control, respectively). Procalcitonin beginning at concentrations of 0.02 ng/mL reduced endothelial cell migration (0.26 [0.06 to 0.47], Padjust = .0069) and new capillary formation in vitro (0.47 [0.28 to 0.66], Padjust < .0001) contrasting the proangiogenic action of vascular endothelial growth factor. Left ventricular injection of procalcitonin in mice on postoperative day 1, 3, and 5 after induction of ischemia impaired new capillary formation and recovery of hindlimb perfusion in vivo (number of capillaries/mm in the ischemic leg of vehicle-treated versus procalcitonin-treated mice, 852.6 [383.4-1322], Padjust = .0002). Twenty-four-hour incubation with procalcitonin reduced the expression of vascular endothelial-cadherin at 100 ng/mL (0.39 [0.06-0.71], Padjust = .0167) and induced endothelial cell death (apoptosis, -5.4 [-10.67 to -0.13], Padjust = .0431). No alteration in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or extracellular signal-regulated kinase 1/2, and AKT signaling pathways was observed. Genomic analysis revealed regulation of a variety of genes involved in inflammation, angiogenesis, and cell growth. CONCLUSIONS: This study found that procalcitonin itself impaired several aspects of endothelial cell function. Procalcitonin-induced loss of endothelial barrier function may contribute to capillary leakage and therapy-refractory hypotension during sepsis. Anti-angiogenic properties of procalcitonin at low concentrations could also identify procalcitonin as a mediator of vascular disease associated with the metabolic syndrome. Future studies are needed to further test procalcitonin as a potential therapeutic target for preserving vascular dysfunction during acute and chronic inflammatory disorders.

Strategic approaches for enhancement of in vivo transbuccal peptide drug delivery in rabbits using iontophoresis and chemical enhancers.

PURPOSE: To evaluate the feasibility of iontophoresis and the combination effects with chemical enhancers on in vivo hypocalcemic effect of transbuccally delivered salmon calcitonin (sCT). METHODS: N-acetyl-L-cysteine (NAC), sodium deoxyglycocholate (SDGC), and ethanol were used as chemical enhancers; and 0.5 mA/cm(2) fixed electric current was employed as a physical enhancer. sCT hydrogel was applied to rabbit buccal mucosa, and blood samples were obtained via the central auricular artery. Blood calcium level was measured by calcium kit and the conformational changes of buccal mucosa were investigated with FT-IR spectroscopy. Hematoxylin/eosin staining was used for the histological evaluation of buccal mucosa. RESULTS: Iontophoresis groups except iontophoresis-NAC group showed significant hypocalcemic effect compared to negative control, in particular iontophoresis-SDGC combination group showed fast onset of action as well as sustained hypocalcemic effect (p < 0.05). FT-IR result demonstrated the reduction of buccal barrier function, and the histological study showed a decrease in buccal thickness as well as minor damage to the dermal-epidermal junctions in the enhancing method groups; however, the damaged tissues virtually recovered within 24 h after the removal of electrodes. CONCLUSIONS: Iontophoresis and combination with SDGC were found to be safe and potential strategies for transbuccal peptide delivery in vivo.

Low cytotoxic elastic niosomes loaded with salmon calcitonin on human skin fibroblasts.

A low cytotoxic elastic niosomal formulation loaded with salmon calcitonin was developed. The elastic niosomes were prepared from Tween 61 mixed with cholesterol at various concentrations of the edge activators (sodium cholate (NaC) and sodium deoxycholate (NaDC); 0.25, 0.5, 2.5, 5 and 10% mole) or ethanol (10-30% v/v). The effects of the niosomal concentrations (5, 10 and 20 mM) and phosphate buffer at pH 7.0 (5, 10, 20 and 30 mM) on the physical characteristics of niosomes were investigated. The 5 mM elastic niosomes in 5 mM phosphate buffer containing calcitonin 0.22 mg/mL gave the highest elasticity (deformability index (DI)) at 6.79 ± 2.03 determined by the extrusion method. The blank elastic niosomes comprised 2.5% mole NaDC, 5% mole NaC or 20% v/v ethanol showed the highest elasticity. The 5% mole NaC elastic niosomes loaded with calcitonin gave the highest DI (21.59 ± 0.91) and percentages of calcitonin entrapment efficiency (60.11 ± 4.98). This study has demonstrated that this NaC elastic niosome did not only reduce the cytotoxicity of the loaded calcitonin but also gave superior cell viability to the ethanolic elastic niosome as well.

Pulmonary delivery of elcatonin using surface-modified liposomes to improve systemic absorption: polyvinyl alcohol with a hydrophobic anchor and chitosan oligosaccharide as effective surface modifiers.

The aim of this study was to investigate the feasibility of surface-modified liposomes for pulmonary delivery of a peptide. Chitosan oligosaccharide (oligoCS) and polyvinyl alcohol with a hydrophobic anchor (PVA-R) were used as surface modifiers. The effect of liposomal surface modification on the behavior of the liposomes on pulmonary administration and potential toxicity were evaluated in vitro and in vivo. In an association study with A549 cells, PVA-R modification reduced interaction with A549 cells, whereas oligoCS modification electrostatically enhanced cellular interaction. The therapeutic efficacy of elcatonin (eCT) after pulmonary administration to rats was significantly enhanced and prolonged for 48 h after separate administration with oligoCS- or PVA-R-modified liposomes. oligoCS-modified liposomes adhered to lung tissues and caused opening of tight junctions, which enhanced eCT absorption. On the other hand, PVA-R-modified liposomes induced long-term retention of eCT in the lung fluid, leading to sustained absorption. Consequently, surface modification of liposomes with oligoCS or PVA-R has potential for effective peptide drug delivery through pulmonary administration.

Nanoparticles of glycol chitosan and its thiolated derivative significantly improved the pulmonary delivery of calcitonin.

A novel thiomer derivative of glycol chitosan (GCS) was synthesized by coupling with thioglycolic acid (TGA) and evaluated for the pulmonary delivery of peptides. Nanoparticles (NPs) based on GCS and GCS-TGA were obtained by the ionic gelation method and demonstrated a particle size in the range of 0.23-0.33 microm with positive surface charge and high calcitonin entrapment. Fluorescent GCS-TGA NPs resulted in a 2-fold increase in mucoadhesion to lung tissue after intra-tracheal administration to rats as compared to non-thiolated NPs. Evaluation of pulmonary toxicity revealed the biocompatibility of the two nanoparticulate formulations with lung tissue. The efficacy of the prepared NPs to enhance the pulmonary absorption of peptides was evaluated after pulmonary administration to rats using a liquid micro-sprayer technique. Calcitonin-loaded GCS and GCS-TGA NPs resulted in a pronounced hypocalcemic effect for at least 12 and 24 h, and a corresponding pharmacological availability of 27 and 40%, respectively. These findings suggest that both GCS and its thiomer derivative are promising and safe carriers for pulmonary peptide delivery.

Toxicidad ocular, bisfosfonatos y enfermedad de Paget / Ocular toxicity, bisphosphonate and Paget disease

No disponible

No disponible

Quantum dot-carrier peptide conjugates suitable for imaging and delivery applications.

We developed multifunctional fluorescent nanoparticles suitable for the nonviral delivery of negatively charged molecules like RNA. Therefore, we incorporated the recently developed branched hCT-derived carrier peptide hCT(18-32)-k7 on the surface of luminescent quantum dots (QDs). Besides detailed characterization of our QD-peptide conjugates concerning stability, toxicity, and uptake mechanism. we used them for efficient RNA delivery into different cell lines. The results of our studies indicate the involvement of more than one endocytotic uptake pathway in the internalization process. Furthermore, we could show that the QD-peptide bioconjugates exhibit no effect on cell viability and possess high stability inside living cells. The efficacy of our newly designed constructs for oligonucleotide drug delivery is highlighted by the successful intracellular transport of Cy-3 labeled RNA. Moreover, by using the chemotherapeutic chloroquine the efficient release of the assemblies out of endosomes was demonstrated. These results prove that our multifunctional platforms are versatile tools for diagnostic and therapeutic imaging purposes applicable for biologically active siRNA or aptamer sequences.

The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects.

The peptide hormone, calcitonin, which is associated with medullary carcinoma of the thyroid, has a marked tendency to form amyloid fibrils and may be a useful model in probing the role of peptide-membrane interactions in beta-sheet and amyloid formation and amyloid neurotoxicity. Using bovine calcitonin, we found that, like other amyloids, the peptide was toxic only when in a beta-sheet-rich, amyloid form, but was non-toxic, when it lacked an amyloid structure. We found that the peptide bound with significant affinity to membranes that contained either cholesterol and gangliosides. In addition, incubation of calcitonin with cholesterol-rich and ganglioside-containing membranes resulted in significant changes in peptide structure yielding a peptide enriched in beta-sheet and amyloid content. Because the cholesterol- and ganglioside-rich phospholipid systems enhanced the calcitonin beta-sheet and amyloid contents, and peptide amyloid content was associated with neurotoxicity, we then investigated whether depleting cellular cholesterol and gangliosides affected calcitonin neurotoxicity. We found that cholesterol and ganglioside removal significantly reduced the calcitonin-induced PC12 cell neurotoxicity. Similar results have been observed with other amyloid-forming peptides such as beta-amyloid (A beta) of Alzheimer's disease and suggest that modulation of membrane composition and peptide-membrane interactions may prove useful in the control of amyloid formation and amyloid neurotoxicity.

Cellular internalization of human calcitonin derived peptides in MDCK monolayers: a comparative study with Tat(47-57) and penetratin(43-58).

PURPOSE: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model. METHODS: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58). RESULTS: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed. CONCLUSIONS: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.

Immunoneutralization of procalcitonin as therapy of sepsis.

Prior studies have demonstrated that the prohormone, procalcitonin (ProCT), and its component calcitonin precursors (CTpr) are increased in the serum of septic patients, correlate with the severity of the illness, and persist for relatively long periods of time. Animal studies in septic hamsters have revealed that the administration of ProCT is toxic and that immunoneutralization with IgG that is reactive to this molecule significantly improves survival. A large animal model of a very rapidly lethal polymicrobial sepsis has been developed in the pig in order to measure continuous physiological and metabolic parameters and also to compare the effects in this animal of an immunoneutralization, which is performed late in the course of the disease, to an identical, but early, therapy. Based upon the physiological and metabolic parameters, the late therapy, which was initiated during the fourth hour at a time when pigs were nearly moribund, was found to be as beneficial as early therapy. In both late and early therapy, the only animals to survive at the predetermined time of euthanasia were those which had received immunoneutralization therapy.

The role of G protein activation in the toxicity of amyloidogenic Abeta-(1-40), Abeta-(25-35), and bovine calcitonin.

More than 16 different proteins have been identified as amyloid in clinical diseases; among these, beta-amyloid (Abeta) of Alzheimer's disease is the best characterized. In the present study, we performed experiments with Abeta and calcitonin, another amyloid-forming peptide, to examine the role of G protein activation in amyloid toxicity. We demonstrated that the peptides, when prepared under conditions that promoted beta-sheet and amyloid fibril (or protofibril) formation, increased high affinity GTPase activity, but the nonamyloidogenic peptides had no discernible effects on GTP hydrolysis. These increases in GTPase activity were correlated to toxicity. In addition, G protein inhibitors significantly reduced the toxic effects of the amyloidogenic Abeta and calcitonin peptides. Our results further indicated that the amyloidogenic peptides significantly increased GTPase activity of purified Galpha(o) and Galpha(i) subunits and that the effect was not receptor-mediated. Collectively, these results imply that the amyloidogenic structure, regardless of the actual peptide or protein sequence, may be sufficient to cause toxicity and that toxicity is mediated, at least partially, through G protein activation. Our abilities to manipulate G protein activity may lead to novel treatments for Alzheimer's disease and the other amyloidoses.

Calcitonin-induced impairment in conditioning is antagonized by chronic antidepressant drug treatment.

The purpose of this study was to test the effect of calcitonin, when injected into the lateral ventricle, on conditioning behaviour and to see whether antidepressant drug treatment can antagonize calcitonin-induced impairment of this behaviour. Conditioned response by conditional stimulus (CS) was compared in control rat (CO) and in rats that received intraventricular perfusion of calcitonin (CA), acute antidepressant drug treatment (ADa), acute antidepressant drug treatment + calcitonin (ADa + CA), chronic antidepressant drug treatment (21 days) + calcitonin the day after (ADc + CA). Control rats acquired easily the conditioned response, the CA group and ADa + CA had problems in making the correlation between CS and unconditional stimulus (US), and consequently did not acquire a conditioned response, but in the ADc + CA group, rats exhibited more conditioned responses. The results indicate that calcitonin disrupts conditioning processes and chronic but not acute antidepressant drug treatment can reverse the effects of calcitonin.

Mortality is increased by procalcitonin and decreased by an antiserum reactive to procalcitonin in experimental sepsis.

OBJECTIVES: Procalcitonin (ProCT), the precursor to the calcitonin hormone, is abnormally increased in experimental and clinical systemic inflammation, including sepsis. Initially, we investigated the effects of supraphysiologic amounts of ProCT administered to animals with septic peritonitis. Subsequently, we evaluated the efficacy of prophylactic and therapeutic immune blockade of ProCT in this lethal model of sepsis. DESIGN: Prospective, experimental, controlled study. SETTING: Animal research laboratory approved by the American Association for the Accreditation of Laboratory Animal Care at a Veterans Affairs Medical Center. SUBJECTS: Young male Golden Syrian hamsters, weighing 90 to 120 g. INTERVENTIONS: In the first study, serum ProCT concentrations were measured in animals at 0, 3, 6, 12, and 24 hrs after induction of sepsis by intraperitoneal implantation of pellets containing Escherichia coli (5 x 10(8) colony-forming units/pellet). In the second study, with mortality as the end point, 30 microg/kg of isolated, purified human ProCT in 10% hamster serum (experimental) or an equal volume of 10% hamster serum (control) were administered intravenously at the time of the E. coli peritoneal implantation. In the third study, experimental animals received intraperitoneal injections of a multiregion-specific goat antiserum reactive to hamster ProCT 1 hr before and 24 hrs after E. coli implantation, while control animals received nonimmune goat serum at the same time points. In the final study, the same antiserum was administered in five divided doses during the 24 hrs after the insertion of E. coli. MEASUREMENTS AND MAIN RESULTS: In the initial study, ProCT concentrations were increased shortly after induction of sepsis and peaked at 12 hrs. Administration of exogenous ProCT to septic animals significantly increased mortality compared with control animals (93% vs. 43%, p=.02). Prophylactic blockade of ProCT almost completely protected the animals from the lethal effects of sepsis: the 102-hr mortality rate in the experimental group was 6% compared with 62% in the control group (p < .003). In the therapeutic trial, the 102-hr mortality rate was 54% in experimental animals compared with 82% in control animals (p < .045). CONCLUSIONS: These results demonstrate that increased ProCT exacerbates mortality in experimental sepsis, whereas neutralization of ProCT increases survival. Thus, ProCT, in addition to being an important marker of severity of systemic inflammation and mortality, is an integral part of the inflammatory process and directly affects the outcome.

Salmon calcitonin induced hypocalcemia and hyperphosphatemia in an elasmobranch, Dasyatis akajei.

Salmon calcitonin (5 micrograms/kg body wt) was administered in an elasmobranch, Dasyatis akajei, to investigate the effects upon plasma calcium and inorganic phosphate. The hormone produced hypocalcemia and hyperphosphatemia in the stingray. It is concluded that calcitonin may have a role in calcium homeostasis by a mechanism different from that on bones.

Improvement of the pulmonary absorption of (Asu1,7)-eel calcitonin by various absorption enhancers and their pulmonary toxicity in rats.

The effects of absorption enhancers on the pulmonary absorption of (Asu1,7)-eel calcitonin (ECT) and their pulmonary toxicity were examined by means of in situ pulmonary experiments. The absorption of ECT from the lungs was estimated by its hypocalcemic effect. The pulmonary membrane toxicity of absorption enhancers was evaluated by the leakage of Evans Blue from the plasma into the lungs. In the absence of absorption enhancers, a slight hypocalcemic effect was obtained following intrapulmonary administration of ECT. However, we found significant hypocalcemic effects after the ECT administration with 10 mM n-lauryl beta-D-maltopyranoside (LM), 10 mM sodium glycocholate (NaGC), and 10 mM linoleic acid-HCO60 (hydrogenated caster oil) mixed micelle (MM). The plasma calcium levels decreased as the amount of LM coadministered with ECT increased. In contrast, 10 mM EDTA did not improve the pulmonary absorption of ECT. Overall, a correlation between the pulmonary absorption of ECT and local toxicity was observed in the presence of these additives. However, 1 mM LM, 10 mM NaGC, and 10 mM MM improve the pulmonary absorption of ECT with low pulmonary toxicity. These findings suggest that the use of these adjuvants would be a useful approach for improving the pulmonary absorption of ECT.

Complex shape changes in isolated rat osteoclasts: involvement of protein kinase C in the response to calcitonin.

The cytoplasmic spreading of osteoclasts has been used to assess responsiveness to agents such as calcitonin and associated signal transduction mechanisms. Although cyclic AMP and intracellular calcium are known mediators of calcitonin effects in osteoclasts, the role of protein kinase C (PKC) is less clear. We have used time-lapse videomicroscopy of isolated rat osteoclasts to characterize shape changes induced by calcitonin, forskolin, and phorbol 12-myristate-13-acetate (PMA) in the absence and presence of PKC blockers. Treatment with calcitonin reduced cytoplasmic plan area but increased perimeter length, resulting in a characteristic "stellate" appearance, whereas forskolin produced "nonstellate" contraction. The response of osteoclasts to PMA was dose dependent. High concentrations (10(-7)-10(-6) M) produced biphasic responses with transitory, calcitonin-like "stellate" contraction followed by sustained expansion, whereas low concentrations (10(-11)-10(-9) M) produced expansion only. The effects of low-concentration PMA could be prevented by pretreatment with a PKC blocker, whereas the effects of high concentrations were only partially inhibited. The effects of forskolin were unchanged by pretreatment with the PKC blocker. Treatment with calcitonin in the presence of various PKC blockers resulted in paradoxical transient expansion followed by contraction. These results indicate that calcitonin-induced shape change in osteoclasts is a complex process involving protein kinase C in addition to cyclic AMP-dependent mechanisms and possibly other factors.

Calcitonin prevents bone loss but decreases osteoblastic activity in ovariohysterectomized beagle dogs.

The antiresorptive effects of calcitonin are well documented. Recent in vitro and in vivo evidence points to an anabolic effect of calcitonin on osteoblasts. To assess the value of calcitonin in preventing the rapid and early bone loss after cessation of ovarian function and to investigate its effects on osteoblasts in vivo, 32 dogs were ovariohysterectomized (OHX) and 32 dogs were sham-operated (Sham). After the surgeries, half of the OHX and Sham dogs received every-other-day subcutaneous injections of human calcitonin (0.25 mg/dog/d), and the remaining dogs were given vehicle. Half of the animals had a bone biopsy at week 2 and were euthanized thereafter; the other half of the animals underwent a bone biopsy at month 1 and were euthanized at month 4. Blood drawings were done at baseline and at the time of each bone biopsy. Calcitonin prevented the increase in erosion depth seen in OHX animals and prevented the cancellous bone loss observed at 2 weeks and at 1 and 4 months. Calcitonin did not affect bone volume in Sham dogs. However, treatment with calcitonin induced a decrease in mineralizing surfaces and bone formation rates at the bone surface and cell level and an increase in mineralization lag time in both Sham and OHX animals without significantly affecting osteoblast number. This finding indicates that the negative effect of calcitonin on bone mineralization is not solely the result of a decrease in bone turnover. The data show that calcitonin, because of its antiresorptive effects, can prevent bone loss after cessation of ovarian function. However, short-term treatment with calcitonin does not stimulate osteoblast activity; on the contrary, it exerts a negative effect on osteoblastic bone formation and mineralization. Long-term studies are needed to investigate whether this unwanted effect of calcitonin on osteoblasts in vivo represents a transitory or persistent phenomenon.