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We investigated the role of histamine H1 receptors in mediating the anorectic effect of intraperitoneally injected amylin (5 and 20 microg/kg), the amylin agonist salmon calcitonin (sCT; 10 microg/kg), leptin (1.3 mg/kg), and cholecystokinin (CCK; 20 microg/kg). The experiments were performed with mice lacking functional H1 receptors (H1Rko) and wild-type (WT) controls. The mice were also injected with the H3 antagonist thioperamide (20 mg/kg), which reduces feeding by enhancing the release of endogenous histamine through presynaptic H3 receptors. The feeding-suppressive effect of thioperamide was abolished in H1Rko mice. The anorectic effects of amylin and sCT were significantly reduced in 12-h food-deprived H1Rko mice compared with WT mice [1-h food intake: WT-NaCl 0.51 +/- 0.05 g vs. WT-amylin (5 microg/kg) 0.30 +/- 0.06 g (P < 0.01); H1Rko-NaCl 0.45 +/- 0.05 g vs. H1Rko-amylin 0.40 +/- 0.04 g; WT-NaCl 0.40 +/- 0.09 g vs. WT-sCT (10 microg/kg) 0.14 +/- 0.10 g (P < 0.05); H1Rko-NaCl 0.44 +/- 0.08 g vs. H1Rko-sCT 0.50 +/- 0.06 g]. The anorectic effect of leptin was absent in ad libitum-fed H1Rko mice, whereas CCK equally reduced feeding in WT and H1Rko animals. This suggests that the histaminergic system is involved in mediating the anorectic effects of peripheral amylin and sCT via histamine H1 receptors. The same applies to leptin but not to CCK. H1Rko mice showed significantly increased body weight gain compared with WT mice, supporting the role of endogenous histamine in the regulation of feeding and body weight.

The absorption of water soluble compounds with different molecular weights, such as phenol red (MW 354), trypan blue (MW 960), fluorescein isothiocyanate dextrans, (MW 4400 and 9100) was studied in the lung, nasal cavity, buccal cavity, small and large intestine of rats. For all the compounds, maximal absorption was observed when administered to the lung. The rank order of absorption of each compound from various administration sites was lung>small intestine> or =nasal cavity> or =large intestine> or =buccal cavity. In addition, the relationship between logarithm absorption % of the compounds from various administration sites and logarithm molecular weights of these compounds was examined. The absorption of compounds gradually decreased with increasing molecular weight for each site of administration. Moreover, the absorption of [Asu1.7]-eel calcitonin (ECT) from these sites and the effect of 10 mM sodium glycocholate, an absorption enhancer, on its absorption were also investigated in rats. When ECT alone was administered into these sites, the lung had the best absorption site of ECT, followed by the nasal cavity, the large intestine, the small intestine and the buccal cavity. Therefore, the absorption of ECT was also dependent on the administration site, although the rank order of absorption % of ECT was different from the other compounds. Sodium glycocholate (NaGC) remarkably increased ECT absorption from the small intestine, while we found marginal increase in its absorption from the lung even in the presence of NaGC. These findings provided useful fundamental information that might aid in the selection of administration routes for drugs of differing molecular weights including peptide drugs as far as the degree of drug absorption is concerned.

The present study was planned to assess the safety, tolerability and efficacy on bone mineral density (BMD), pain, quality of life and fracture risk of alendronate, calcitonin and calcium treatments. A total of 151 postmenopausal women with lumbar spine BMD 2 SD or more below the young adult mean were randomly assigned to one of three groups: 51 patients received oral alendronate 10 mg and calcium 1000 mg (alendronate group), 50 patients intranasal salmon calcitonin 100 IU and oral calcium 1000 mg (calcitonin group), and 50 patients oral calcium 1000 mg (calcium group) daily for one year. BMD was assessed by dual energy X-ray absorbtiometry, pain by a visual analogue scale, and quality of life by the Nottingham health profile. Significant increases in BMD at all sites were obtained in the calcitonin and alendronate groups, but not in the calcium group. Pain and quality of life improved significantly in both the calcitonin and alendronate groups, but not in the calcium group. New vertebral fractures were seen in 31.58% of the alendronate, 37.5% of the calcitonin, and 40% of the calcium groups, representing no statistical difference. No serious side-effects were seen in any of the patients during follow-up.

Two isoforms of the calcitonin receptor are expressed in rabbit: the common C1a isoform and the calcitonin receptor Delta e13 isoform, which has a deletion in the seventh transmembrane domain. Using microphysiometry, we investigated the effects of calcitonin on proton efflux from HEK293 cells stably transfected with C1a, calcitonin receptor Delta e13, or empty vector. In C1a-expressing cells only, calcitonin rapidly induced a biphasic elevation in proton efflux consisting of an initial transient and a sustained plateau, accompanied by an increase in lactate efflux. Inhibitors of Na(+)/H(+) exchange abolished only the initial transient, whereas removal of extracellular glucose abolished only the sustained plateau. These data suggest that activation of Na(+)/H(+) exchange mediates the initial transient, whereas increased glucose metabolism underlies the sustained plateau. Because both receptor isoforms activate adenylyl cyclase, the lack of effect of calcitonin on proton efflux from calcitonin receptor Delta e13-expressing cells argued against involvement of cAMP in activating proton efflux. Similarly, studies involving elevation or buffering of cytosolic free Ca(2+) concentration argued against involvement of Ca(2+). Activation of PKC mimicked the plateau phase of calcitonin-induced proton efflux from C1a cells, whereas inhibition or depletion of PKC suppressed it. Activation of proton transport and production are novel cellular responses to calcitonin, mediated selectively by the C1a receptor isoform via a mechanism involving PKC.

We synthesized seven O-glycosylated calcitonin derivatives, each with a single GalNAc residue attached to either Ser or Thr, and studied their three-dimensional structure and biological activity to examine site-dependent effects of O-glycosylation. The CD spectra in an aqueous trifluoroethanol solution showed that the GalNAc attachment at Thr6 or Thr21 reduced the helical content of calcitonin, indicating that the O-glycosylated residue functions as a stronger helix breaker than the original amino acid residue. Only the GalNAc attachment at Ser2 or Thr21 retained the hypocalcemic activity of calcitonin. This result corresponded well to that of the calcitonin-receptor binding assay. The GalNAc attachment other than Ser2 or Thr21 perturbed the interaction with the receptor, resulting in the loss of the hypocalcemic activity. The biodistribution did not change much among the seven derivatives, but some site dependency could also be observed. Thus, we can conclude that the O-glycosylation affects both the conformation and biological activity in a site-dependent manner.

The effects of calcitonin, alendronate and fluorophosphate preventive treatment on ovariectomized rat femur were studied by comparing densitometric, mechanical, mineralogical and histomorphometric data. Sixty retired breeder female Sprague-Dawley rats, aged 10 months, were randomly divided into six groups. A group (baseline) was euthanized at the beginning of the study as a baseline group; four groups were ovariectomized and one was sham-operated (sham) and considered as a sham-aged group. A group of ovariectomized rats was used as a sham-therapy control (OVX) and received only deionized drinking water, while the other three received: a) salmon calcitonin (SCN) at a dose of 2 IU/kg/d s.c. (OVX + SCN); b) alendronate sodium salt (ALN) at a dose of 6 microg/kg/d administered by gavage (OVX + ALN); and c) L-glutamine monofluorophosphate (G-MFP) and calcium at a rate of 1:30 F/Ca at a dose of 0.21 mg F/6.30 mg Ca per kg/d by gavage (OVX + MFP). Significant increases (P < 0.05) of about 15 and 27% in femoral proximal epiphysis bone mineral density (BMD) of the OVX + ALN group were observed versus healthy groups and the OVX group, respectively. The OVX + ALN group also showed significant increases in femoral mid-diaphysis BMD when compared to OVX (18%, P < 0.001), OVX + SCN (14%, P < 0.05) and OVX + MFP (18%, P < 0.001) groups. In the OVX + MFP group, the three-point bending test demonstrated significant increases (P < 0.05) in maximal load of 21 and 22% when compared to the OVX and OVX + SCN groups, respectively. Also, stiffness data showed significant increases of the OVX + MFP (17%) and sham (14%) groups in comparison with the OVX group. A decrease in Mg (42%, P < 0.05), and increases in Ca (15%, P < 0.0001) and PO4 (8%, P < 0.005) content were found by comparing OVX + MFP and OVX groups. Trabecular bone volume results showed significant increases by comparing OVX + ALN and OVX groups (12.20%, P < 0.0005), as well as control groups. Tested agents were able to reduce the bone loss due to estrogen deficiency, but this did not always produce an increase in strength of the treated bone. Alendronate treatment prevented a decrease in bone mineral density and maintained bone mechanical properties after ovariectomy without impairment of bone mineralization in aged rats.

One of the causes of insensitivity to androgen ablation therapy in prostate cancer is thought to be attributable to elevated neuropeptides secreted by neuroendocrine cells in the tumor mass. Calcitonin (CT), one of these neuropeptides, is reported to be associated with the growth of prostate cancer. There is an increase in mitogen-activated protein (MAP) kinase activation as prostate cancer progresses to a more advanced and androgen-independent disease. We examined the effect of CT on signal transduction and the relation between CT and early-response genes in the human androgen-insensitive prostate cancer cell line, DU145. The basal phosphorylation level of extracellular signal-regulated kinase 1/2, which is a key kinase in the mediation of growth factor-induced mitogenesis in prostate cancer cells, was constitutively up-regulated. N-[2-(4-bromocinnamyl) aminoethyl]-5-isoquinoline-sulfonamide (H89), a specific inhibitor of protein kinase A, potentiated the effects of more increased phosphorylation of extracellular signal-regulated kinase 1/2. CT induced the inhibition of this MAP kinase phosphorylation, and this effect was completely abolished by pretreatment with H89. Our findings demonstrate that CT caused the inhibition of constitutive MAP kinase phosphorylation in a protein kinase A-dependent manner in DU145. The transient increase of c-fos expression was detected after CT treatment, whereas expression of c-jun RNA was down-regulated after CT treatment. These results suggest that CT may regulate early-response genes, c-fos and c-jun, via a MAP kinase cascade. In conclusion, these findings suggest that DU145 might be a useful model as a therapeutic approach of neuropeptides in androgen-independent prostatic carcinoma.

Amylin and calcitonin gene-related peptide (CGRP) are homologous 37 amino acid peptides that are found in the circulation. Both peptides belong to the calcitonin family. Similar to calcitonin, amylin and CGRP inhibit osteoclast activity, although they are much less potent than calcitonin. Calcitonin is known to act on the latter stages of osteoclast development, inhibiting the fusion of committed preosteoclasts to form mature multinucleated cells; however, whether or not calcitonin acts earlier in the formation of the precursor osteoclasts is controversial. The question of osteoclast development has never been examined with respect to amylin and CGRP. These issues are addressed in the present study. We studied the effects of calcitonin (salmon and rat), amylin (human and rat), and CGRP (human and rat) in mouse bone marrow cultures stimulated to generate osteoclasts using 1alpha,25-dihydroxyvitamin D3. Calcitonin dose-dependently decreased the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as well as TRAP-positive mono-/binucleated cells at concentrations >10(-13) mol/L. Amylin and CGRP showed similar effects at concentrations >10(-9) mol/L. In addition, calcitonin substantially reduced the ratio of TRAP-positive multinucleated to mono-binucleated cells, indicating an effect on fusion of osteoclast precursors. The present data establish that this family of peptides not only acts on mature osteoclasts but also inhibits their development in bone marrow cultures. This activity is shared by amylin and CGRP. The much greater potency of calcitonin than amylin and CGRP is consistent with the action of these peptides being mediated by calcitonin receptors.

It has been demonstrated that calcitonin-binding sites are present in a variety of tissue types, including in the pituitary gland. Interleukin-6 (IL-6) is also produced in the pituitary and it regulates the secretion of various hormones. In this study, we examined the expression of the calcitonin receptor and the mechanism of IL-6 production induced by calcitonin in the pituitary folliculo-stellate cell line (TtT/GF). The mRNA of calcitonin receptor subtype C1a, but not that of C1b, was detected by RT-PCR in TtT/GF cells and in the normal mouse pituitary. Calcitonin increased cAMP accumulation and IL-6 production in a concentration-dependent manner in TtT/GF cells. As calcitonin activates the PKA and PKC pathways, we investigated the contributions of PKA and PKC to IL-6 production. IL-6 production was only slightly increased by either 8-bromo-cAMP (1 mM) or phorbol 12-myristate 13-acetate (100 nM) alone. However, IL-6 was synergistically induced in the presence of both 8-bromo-cAMP (1 mM) and phorbol 12myristate 13-acetate (100 nM). Furthermore, calcitonin-induced IL-6 production was completely suppressed by H-89 (PKA inhibitor) or GF109203X (PKC inhibitor), indicating that the activation of both PKA and PKC is necessary for calcitonin-induced IL-6 production. On the other hand, pertussis toxin (G(i)/G(o) signaling inhibitor) treatment achieved an approximately 9-fold increase in calcitonin-induced IL-6 production. These results show that calcitonin-stimulated IL-6 production is mediated via both PKA- and PKC-signaling pathways, whereas calcitonin also suppresses IL-6 production by activating G(i)/G(o) proteins in folliculo-stellate cells.

Objective: To investigate the effect of sequential application of calcitonin and parathyroid hormone (PTH) on bone remodeling process., Methods: Twelve female rats were divided equally into 2 groups: experimental group and control group. 1 U/100 g of calcitonin was injected intramuscularly to 6 rats for two days, and rrPTH in dosage of 30 micrograms was injected intramuscularly in the fourth and fifth days. At the sixth day their lumbar vertebral bodies were resected. Undecalcified and decalcified bone sections were made to be observed by optical microscopy and electronic microscopy. Bone histomorphological parameters were measured. Normal saline and solvent of the same volume were injected intramuscularly to the controls., Results: The OS/BS (%), O. Th (micron), N.Ob.S (cell/mm), and Vos/TV (%) were 19.9% +/- 6.2%, 3.4 microns +/- 0.4 micron, 37.6 cells/mm +/- 4.6 cells/mm, and 1.8% +/- 0.6% respectively in the experimental group, significantly higher than those in the control group (P < 0.05). After the PTH administration, more and plumper osteoblasts were observed. They were rich in rough endoplasmic reticulum, mitochondria, and Golgi apparatus., Conclusion: Sequential application of calcitonin and parathyroid hormone obviously promotes the bone formation process. Both sites of bone remodeling formation and sites of bone modeling formation, especially the latter, increase.

Although 3',5'-cyclic AMP (cAMP) is known to regulate oligodendrocyte development in vitro, little is known about the identity of agonists that induce cAMP synthesis in oligodendroglia. To identify such agonists, we used a novel immunohistochemical method of visualizing cAMP within single cells to screen compounds that are known to activate cAMP synthesis in other cellular systems. Calcitonin, corticotropin-releasing hormone, and vasoactive intestinal peptide elevated cAMP in oligodendroglial cells but not in other cell types present in the cultures (i.e., astrocytes and microglia). In contrast, prostaglandins and the beta-adrenergic agonist isoproterenol, which have previously been reported to induce modest increases in oligodendroglial cell cAMP from biochemical assay of cell homogenates, did not induce a detectable cAMP response in individually identified oligodendroglial cells but instead induced a robust cAMP response in nonoligodendroglial cells.

Locally secreted growth factors and neuropeptides may play an important role in sustaining the growth of hormone-independent prostate cancer. Our previous studies have shown that calcitonin-like immunoreactive peptide (CTI) is secreted by primary prostate cells in culture, and its secretion from malignant prostate cells is significantly higher than benign cells. Exogenously added calcitonin (CT) induces DNA synthesis in serum-starved prostate cancer LNCaP and PC-3M cells. Present studies extended these findings by cloning cDNAs for CT and CT receptor (CT-R) from prostate cancer cells and studying the expression of CT and CT-R mRNA in prostate cancer cell lines and primary prostate tumor specimens. The results have shown that PC-3 cells expressed CT, and not CT-R, mRNA, whereas CT-R, but not CT, mRNA was expressed by LNCaP cells. Conditioned media from PC-3 cells induced DNA synthesis of LNCaP cells, and this mitogenic response was abolished by anti-CT serum. Highly aggressive PC-3M cells co-expressed CT and CT-R mRNAs. CT also induced a twofold increase in DNA synthesis of primary prostate cells and anti-CT serum caused a 56% decline. In-situ hybridization histochemistry of archival prostate specimens has selectively localized CT and CT-R mRNA in basal epithelium of benign and low grade PC specimens, and these mRNAs were not detected in either luminal epithelium or stroma. In contrast, CT and CT-R mRNA were detected throughout the luminal epithelium of moderate and high-grade PC specimens. Most epithelial cells of low and moderately differentiated tumors expressed either CT or CT-R mRNA, suggesting that CT may serve as a paracrine factor. In contrast, CT and CT-R mRNAs were co-expressed by most tumor cells in advanced PC specimens. The cells expressing CT-R mRNA in primary tumors also co-expressed PCNA. These results, when combined with mitogenic actions of CT on primary prostate cells as well as PC cell lines, strongly support the role for CT in sustaining the growth of cancer cells.

Aim: To study the effect of repeated systemic injections of elcatonin (a synthetic analog of eel calcitonin) on the responses of rat femoral artery preparation to vasoactive drugs and to determine subtypes of muscarinic cholinoceptors involved in acetylcholine (ACh)-induced vasorelaxation in elcatonin-treated rats., Methods: Spontaneously hypertensive rats (SHR) were treated sc with elcatonin, 0.5 and 5 U/kg, 3 times a week for 2 weeks. Responses to vasoactive drugs were determined in helically cut strips of femoral arteries of these rats. Schild plot data for muscarinic cholinoceptor antagonists were obtained on these vascular strips, using ACh as an agonist., Results: Elcatonin did not alter systemic blood pressure and contractile responses of the femoral artery to KCl, norepinephrine, 5-hydroxytryptamine, and prostaglandin F(2alpha). Elcatonin attenuated isoproterenol-induced relaxation, increased ACh- and ATP-induced relaxations, and did not change relaxant responses to sodium nitroprusside and cromakalim in the femoral artery. Nitro L-arginine in the combination with tetraethylammonium (or charybdotoxin) completely abolished the relaxant response to ACh in the control but not in the elcatonin-treated arteries. The muscarinic cholinoceptor subtype involved in the ACh-induced relaxation was M3 in the elcatonin-treated as well as control SHR., Conclusion: Elcatonin decreases beta-adrenoceptor-mediated relaxation and increases M(3) cholinoceptor-mediated relaxation in the SHR femoral artery. Although the ACh-induced relaxation is explained by stimulated releases of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in the SHR artery, a NO-and EDHF-independent mechanism in addition to NO and EDHF is responsible for the response to ACh in the femoral artery from the elcatonin-treated SHR.

The aim of the present study was to investigate and to compare the effect of calciotropic hormones, human calcitonin gene related peptide (CGRP) I and II, salmon calcitonin (CT) and human amylin on the adenylate cyclase activity in abalone gill membranes. In addition to human CGRPI, human CGRPII and salmon CT stimulated the adenylate cyclase activity. No effect was observed with amylin. The higher effect was observed with human CGRPI and II that induced a 160-170% increase of the enzyme activity. Fifty percent of the maximal activity was observed with 3 and 8 nM of CGRP I and II, respectively. Salmon CT induced a lower effect: the maximal activity was obtained with a hormone concentration of 266 nM and represented a 130% stimulation of the basal activity. In the presence of CGRP 8-37, a competitive antagonist of CGRP action, the stimulation observed with CGRPI was abolished and returned to the basal level. This study points out that, in invertebrates, CGRP receptors present in gill membranes are linked to an adenylate cyclase system similar to that described in vertebrates. In addition, these data are in favour of a role for CGRP in branchial function both in non-mammalian vertebrates and invertebrates

We recently reported the direct inhibitory effect of adrenomedullin on caecal circular smooth muscle cells via cAMP system. This study was designed to determine whether the structurally related peptides to adrenomedullin (i.e.; calcitonin gene-related peptide (CGRP), calcitonin, and amylin) can inhibit the cholecystokinin octapeptide (CCK-8)-induced contractile response by exerting a direct action on guinea-pig caecal circular smooth muscle cells, and to compare the inhibitory potency of these peptides. In addition, to elucidate each intracellular mechanisms, the effects of an inhibitor of cAMP-dependent protein kinase, inhibitors of particulate or soluble guanylate cyclase on the each peptide-induced relaxation were investigated. Adrenomedullin, CGRP, calcitonin, and amylin inhibited the contractile response produced by CCK-8 in a dose-dependent manner, with IC50 values of 0.14 nM, 0.37 nM, 5.4 nM, and 160 nM, respectively. An inhibitor of cAMP-dependent protein kinase significantly inhibited the relaxation produced by all of these peptides. On the contrary, inhibitors of particulate or soluble guanylate cyclase did not have any significant effect on the relaxation produced by these peptides. In this study, we demonstrated the direct inhibitory effects of the structurally related peptides to adrenomedullin (i.e.; CGRP, calcitonin, and amylin) on the isolated caecal circular smooth muscle cells via cAMP system. The order of potency was as follows; adrenomedullin falling dots CGRP > calcitonin > amylin.

Using the pulse depolarization-iontophoresis (PDP-IP) system, salmon calcitonin (sCT), a drug for the treatment of osteoporosis, was transdermaly administered in rats. While absorption of sCT was not observed after passive transdermal administration, the serum sCT concentration was confirmed at a dose of 0.2-4 microg when the PDP-IP system was employed. The results indicated that PDP-IP could enhance transdermal absorption of peptide drugs. Also noted was the increased amount of absorption of sCT along with an increase in the dose. We investigated the influence of electrical parameters (current, frequency) in PDP-IP on the transdermal absorption of sCT. An optimal current for drug absorption was found within the range of transported current (0.1-1.0 mA) employed for PDP-IP. In comparison with the results obtained at 0.1 mA, the drug absorption increased, along with an increase in transported current, when the current was set at 0.5 mA, while the drug absorption decreased at 1.0 mA in comparison. The decrease in drug absorption was assumed to be attributable to the structural destruction of skin by application of excessive current. There was no change in skin resistance attributable to the frequency; nor was there any influence of the frequency on the amount of drug absorption.

Calcitonin (CT), a 32-amino acid peptide hormone secreted mainly from the thyroid gland, plays an important role in maintaining bone homeostasis. To discover non-peptide small molecules with biological actions similar to those of CT, a cell-based screening of an in-house chemical library was performed and a pyridone derivative (SUN B8155) was identified. Like CT, it elevated cyclic AMP (cAMP) levels in T47D and UMR106-06 cells which endogenously express human and rat CT receptor, respectively. SUN B8155 also stimulated cAMP formation in cells expressing recombinant human CT receptor, but not in those expressing human parathyroid hormone/parathyroid hormone-related peptide receptor. Accumulation of cAMP in T47D cells was blocked by a selective antagonist of CT receptor, salmon CT(8-32), whereas SUN B8155 did not displace the specific binding of [(125)I]CT to the receptor. Our results suggested that the compound selectively interacts with the CT receptor by a mechanism similar to but probably different from that of CT itself. In rats, intraperitoneal administration of SUN B8155 significantly lowered serum calcium levels, like CT. Our results demonstrate, for the first time, that the biological activities of the newly identified small molecule can mimic that of CT, acting via the CT receptor.

Fertilization-promoting peptide (FPP) regulates the adenylyl cyclase (AC)/cAMP pathway to elicit capacitation-dependent responses, stimulating capacitation in uncapacitated spermatozoa and then arresting it in capacitated cells, thereby inhibiting spontaneous acrosome reactions. Like FPP, calcitonin and angiotensin II are found in seminal plasma and so might affect sperm function; this study investigated responses in uncapacitated and capacitated mouse spermatozoa to these three peptides. Both calcitonin (5 ng/ml) and angiotensin II (1 and 10nmol/l), like FPP (100nmol/l), significantly stimulated capacitation, assessed using chlortetracycline (CTC) fluorescence and fertilization in vitro analyses. Combinations of two or three peptides, at high and low, non-stimulatory concentrations, were more stimulatory than the individual peptides, suggesting that they may act on the same signalling pathway, plausibly AC/cAMP; preliminary data indicate that calcitonin does stimulate cAMP production. In capacitated cells, FPP and calcitonin elicited pertussis toxin-sensitive inhibition of spontaneous acrosome loss, suggesting involvement of inhibitory G proteins; angiotensin II had no detectable effect. When all three peptides were used, angiotensin II did not interfere with inhibitory responses to FPP/calcitonin. These results suggest that angiotensin II, calcitonin and FPP may somehow modulate the AC/cAMP signal transduction pathway, but the precise mechanisms involved have yet to be elucidated.

Currently, no effective therapy exists for patients suffering from progressive medullary thyroid carcinoma (MTC), a calcitonin (CT)-secreting C cell tumor. As CT, which arises from the precursor protein preprocalcitonin (PPCT), is expressed by almost all MTC cases, these molecules may represent target antigens for immunotherapy against MTC. In our study we investigated whether DNA immunization is able to induce cellular and humoral immune responses against human PPCT (hPPCT) in mice. Antigen-encoding expression plasmids were delivered intradermally by gene gun. One group of mice received DNA encoding hPPCT only. Two groups were coinjected with mouse cytokine genes. We observed in lymphocyte proliferative assays substantial proliferation against hPPCT in mice coinjected with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, in contrast to mice vaccinated with hPPCT expression plasmid only. In addition, codelivery of the GM-CSF gene augmented the frequency of anti-hPPCT antibody seroconversions in sera of immunized animals, as shown by enzyme-linked immunosorbent assay. These results illustrate that cellular and humoral immune responses against hPPCT can be generated by DNA immunization and increased by coinjection of the GM-CSF gene. Our findings may have implications for the use of DNA immunization as a potential novel immunotherapeutic treatment for patients suffering from progressive MTC.

The plasma levels of procalcitonin (PCT) are increased in patients with severe bacterial infections. Its cellular origin and potential pathophysiological function in sepsis is, however, unclear. White blood cells have recently been described to express both PCT mRNA and protein. The aim of this study was to determine whether PCT has any influence on the surface expression of receptors, relevant in inflammation, on human whole blood leukocytes under normal and septic conditions. Venous blood from healthy donors was incubated with PCT (40 ng/ml or 1200 ng/ml) alone or in combination with lipopolysaccharide (LPS, 10 ng/ml) or peptidoglycan (PepG, 10 micrograms/ml) for 6 h. The surface expression of CD14, CD54, CD64, CD80, CD86 and HLA-DR was determined by flow cytometry. We could not detect any influence of PCT on the expression of these receptors. Further studies on potential effects on other cell types during infection seem warranted.

Cathepsin K is a lysosomal cysteine proteinase (LCP) predominantly expressed in osteoclasts. This study was conducted to evaluate the importance of human cathepsin K for osteoclastic bone resorption relative to that of other LCPs. To accomplish this, we quantitatively determined the expression levels of major LCPs (cathepsins B, K, L, and S) in human osteoclastic cells by using competitive RT-PCR. Giant cell tumor of bone (GCT) was used as a source of human osteoclastic cells, since the tissue was shown to contain a large number of cells satisfying the criteria for typical osteoclasts. The involvement of LCPs in the bone-resorption process by the GCT cells was confirmed by showing that trans-epoxysucciny-L-leucylamido-(4-guanidino) butane (E-64), a nonselective cysteine proteinase inhibitor, exerted an inhibitory effect on the pit formation. We isolated osteoclast-like cells (OLCs) positive for tartrate-resistant acid phosphatase (TRAP) and cathepsin K from the GCT tissue to a degree of almost 95% purity. In these cells, the expression of cathepsin K was shown to be approximately 20-, 130-, and 410-fold stronger than that of cathepsins B, L, and S, respectively. A similar result was obtained when human bone marrow cells in culture were used as another source of OLCs. Further, we found that cathepsin K was expressed in OLCs far more strongly than in several human nonosteoclastic cells including osteoblastic cell lines. The abundant and selective expression of cathepsin K in OLCs relative to that of other LCPs suggests that cathepsin K is mainly responsible for osteoclastic degradation of human bone matrix.

Bone healing around titanium implants has already been evaluated; however, the effect of drugs such as calcitonin during the period of bone maturation around titanium implants has not yet been investigated. The purpose of this study was to evaluate the effects of calcitonin administration on the late period of bone healing following titanium implant insertion. Twenty-seven adult New Zealand rabbits received one implant in each femur. Thirteen animals were randomly selected as the test group (2 IU/kg--calcitonin) and fourteen animals served as control (saline). The animals were sacrificed 6, 8, 12 and 18 weeks after surgery. Endosteal/periosteal bone length (EB/PB), endosteal/periosteal bone area (EBA/PBA) and total cortical length (TCL) around the implants were analyzed. After 6, 8, 12 and 18 weeks, a positive time effect was strongly observed (P < 0.05). Considering the treatment factor, there was a positive effect of calcitonin on EBA and EB variables at 12 weeks and TCL at 18 weeks. In conclusion, the administration of salmon calcitonin to healthy animals may improve bone mass at the later stages of bone healing following titanium implant insertion.

Background/aims: Both calcium and calcitonin have been involved in mediating gastrointestinal motility. Using a thyroidectomized rat model the present study tried to determine which one of them was essential for normal small intestinal transit., Methodology: Adult Sprague-Dawley male rats simultaneously received thyroidectomy or a sham operation plus a laparotomy for duodenal tubing. After two-week housing, a small intestinal transit study was conducted via geometric center computation of radioactivities of the intestinal segments counted from intraduodenal feeding of radiochromium. Before feeding, these rats were i.p. injected with saline or human calcitonin in the doses of 0.1, 1 and 10 microM/kg, respectively. Another group of Tx rats received i.v. infusion of saline or CaCl2 for 30 min before motility study., Results: Thyroidectomization effectively delayed transit compared to the sham-operated controls (3.59 +/- 0.15 vs. 2.98 +/- 0.32, P < 0.05). High doses of calcitonin treatment in thyroidectomized rats further inhibited transit (P < 0.01), whereas calcitonin did not restore the suppressed calcium level. Delayed transit in thyroidectomized rats was restored after calcium infusion (2.95 +/- 0.22 vs. 3.78 +/- 0.42, P < 0.05) while their hypocalcemia was already corrected., Conclusions: Thyroidectomization disturbs small intestinal transit, while peripheral calcitonin replacement further inhibits transit. Since calcium replacement enables to restore small intestinal transit, it is apparent that calcium rather than calcitonin is essential for normal small intestinal transit.

Objective: Serum procalcitonin (PCT) concentration was recently introduced as valuable diagnostic marker for systemic bacterial infection and sepsis. At present, the cellular sources and biological properties of PCT are unclear. During sepsis and septic shock, inducible nitric oxide synthase (iNOS) gene expression is stimulated followed by the release of large amounts of nitric oxide (NO). We investigated the possible association between PCT and iNOS gene expression in an in vitro cell culture model., Design: Prospective, controlled in vitro cell culture study., Setting: University research laboratories., Interventions: Confluent rat vascular smooth muscle cells (VSMC) were incubated for 24 hrs and 48 hrs with PCT (1 ng/mL, 10 ng/mL, 100 ng/mL, 1,000 ng/mL, 5,000 ng/mL) alone or with the combination of tumor necrosis factor-alpha (TNF-alpha, 500 U/mL) plus interferon-gamma (IFN-gamma, 100 U/mL). iNOS gene expression was measured by qualitative as well as quantitative polymerase chain reaction analysis, NO release was estimated by the modified Griess method., Measurements and Main Results: PCT in increasing concentrations had no effect on iNOS gene expression and nitrite/nitrate release for 24 hrs and 48 hrs, respectively. However, PCT ameliorated TNF-alpha/IFN-gamma-induced iNOS gene expression in a dose-dependent manner (maximal inhibition at PCT 100 ng/mL by -66% for 24 hrs and -80% for 48 hrs). This was accompanied by a significantly reduced release of nitrite/nitrate into the cell culture supernatant (maximal reduction at PCT 100 ng/mL by -56% and -45% for 24 hrs and 48 hrs, respectively)., Conclusions: We conclude that recombinant PCT inhibits the iNOS-inducing effects of the proinflammatory cytokines TNF-alpha/ IFN-gamma in a dose-dependent manner. This might be a counter-regulatory mechanism directed against the large production of NO and the concomitant systemic hypotension in severe sepsis and septic shock.

Calcitonin is a hormone that decreases plasma calcium through inhibition of osteolysis. It is used in the treatment of osteoporosis and other bone disorders. Salmon calcitonin is typically utilized in individuals for whom use of estrogen is contraindicated, for example, women at high risk for breast cancer. In addition to actions on bone, calcitonin may have effects on the central nervous system. Receptors for calcitonin are present on central neurons, and salmon calcitonin has been shown to alter neuronal activity. Since salmon calcitonin is used clinically, and it can have actions on neurons, the present studies were designed to determine whether salmon calcitonin could alter death of cortical neurons. The effects of salmon calcitonin on neuronal death induced by exposure of murine cortical cultures to serum deprivation, staurosporine, oxygen-glucose deprivation, kainate, and NMDA were tested. Salmon calcitonin had no effect on apoptotic cell death in cortical cultures. However, acute treatment with salmon calcitonin (1-1000 nM) caused significant potentiation of neuronal death induced by oxygen-glucose deprivation. Similarly, salmon calcitonin potentiated cell death induced by exposure to kainate. In contrast, it did not potentiate cell death induced by exposure to NMDA. In fact, addition of a high concentration (1000 nM) of salmon calcitonin attenuated NMDA toxicity. These results indicate that calcitonin is not a survival factor for cortical neurons; however, it can alter excitotoxic cell death. The most interesting, and disturbing, effect is the ability of low concentrations of salmon calcitonin to potentiate oxygen-glucose deprivation-induced cell death.

Antidepressants are used in the treatment of a variety of pain syndromes. Most of them act by blocking noradrenaline (NA) and serotonin (5-HT) reuptake. It is also well known that the serotonergic system is also involved in calcitonin (CT) analgesia. Taking these two evidences into account, the modification of the analgesic effect of nortriptyline, amitriptyline, and paroxetine in the presence of salmon CT (s-CT) was examined in mice. The forced-swimming test was carried out in order to choose doses of each drug that did not induce an antidepressant effect under our experimental conditions (nortriptyline: 0.2-5 mg/kg ip, amitriptyline: 2.5-20 mg/kg ip, and paroxetine: 5-30 mg/kg ip). The analgesic effect of each antidepressant was then evaluated using the acetic acid test. At the doses tested, the antidepressants induced a dose-dependent analgesic effect. When mice were pre-treated with a subanalgesic dose of s-CT (2.5 IU/kg), the analgesic effect of amitriptyline and paroxetine was significantly increased though no modification was found for nortriptyline. In summary, s-CT was able to increase the analgesic effect of the antidepressant drugs that reduce the uptake of 5-HT, suggesting that the joint administration of antidepressants and CT may be an interesting alternative in pain management.

In the present study the relationship between calcitonin and kappa-opioid system was analysed. The possibility that salmon calcitonin (S-CT) exerts analgesic activity in mice as well as the influence of salmon calcitonin on the analgesic effect of kappa-opioid agonist, U-50, 488H were examined. The writhing test in mice was used as analgesic test. S-CT given alone intraperitoneally at a high dose produced antinociceptive effect. S-CT given at a subanalgesic dose increased the antinociceptive effect of kappa-opioid agonist, U-50, 488H. The obtained results suggest that the increase in the effectiveness of kappa-opioid receptor agonist may be one of the mechanisms involved in the analgesia induced by salmon calcitonin.

The discovery of osteoprotegerin (OPG), osteoprotegerin ligand (OPGL), and RANK has elucidated the mechanism by which osteoblasts and stromal cells regulate osteoclastic differentiation and function and mediate the effects exerted by other hormones and cytokines. We have investigated the effects of these novel cytokines on the preosteoclastic cell line FLG 29.1. We show that OPGL alone and in combination with macrophage colony-stimulating factor (CSF-1) dramatically reduced replication and increased tartrate-resistant acid phosphatase activity. However, although FLG29.1 cells appear to adhere to the bone surface, they are not able to form resorption lacunae. OPG and calcitonin completely abolished the differentiation induced by OPGL. RANK was detectable in FLG 29.1 and the number of positive cells was increased by OPGL/CSF-1 treatment while reduced by calcitonin. We propose that calcitonin could interact with the OPG/OPGL, and its effects on RANK may explain in part the action of this hormone in suppressing bone resorption., (Copyright 2000 Academic Press.)

We examined the effects of PTH, calcitonin (CT) and parotin subunit on the intracellular Ca2+ of odontoblasts using a chelate reagent, FURA2-AM. Rat CT (rCT), at a final concentration of 0.01 microM, induced a gradual lowering of Ca2+, and addition of ATP, in the presence of CT, resulted in a partial and short-lasting recovery of Ca2+. At higher concentrations, it caused a rapid decrease of Ca2+. CTs of other animal species showed similar effects. Human PTH (hPTH) added at concentrations of 0.01, 0.1 and 1 microM, caused no significant changes in intracellular Ca2+. Parotin subunit caused a rapid lowering of Ca2+ which was seen already at 0.01 microM. rCT added after treatment with hPTH caused an immediate decrease of Ca2+ to zero level, showing that CT action was enhanced by pretreatment with hPTH. This enhancement was also confirmed by addition of hPTH after rCT, where at 1 microM, it caused further acute decrease in Ca2+. After intracellular Ca2+ was lowered by CT pretreatment, parotin, at 0.1 microM, induced a further but gradual decrease of Ca2+. The present results, together with our previous study indicating that hPTH increased cAMP production and that CT inhibited the PTH action, made it clear that all the hormones affect odontoblasts, and that CT and parotin act via Ca-related signal transduction system, while PTH acts via cAMP-PKA-related cascade. Possible crosstalk of both systems was also suggested.

Clinical and experimental observations, together with immunohistochemical findings, suggest that neuro-osteogenic interactions may occur in the skeleton. In this study, we have examined the effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in bone, on the activity of the bone-resorbing osteoclast. Effects on bone resorption were assessed by counting the number of pits formed by rat osteoclasts incubated on devitalized slices of bovine cortical bone. Under conditions with an initially sparse density of stromal cells/osteoblasts, VIP caused a rapid cytoplasmic contraction and decreased motility of osteoclasts. This was coupled with a decrease in the number of resorption lacunae and a decrease in the total area resorbed by the osteoclasts in 48-h cultures. Time-course experiments revealed that the inhibitory effects on contraction and motility were transient and that the cells gradually regained their activity, such that, when culture time was prolonged to 120 h, a stimulatory effect by VIP on bone resorption was observed. When osteoclasts were incubated on bone slices, in the presence of an initially large number of stromal cells/osteoblasts, VIP treatment increased the number of resorption pits and total bone area resorbed in 48-h cultures. Using atomic force microscopy, we provide direct evidence that both osteoclasts and stromal cells/osteoblasts bind VIP. Also, VIP was shown to cause a rapid rise of intracellular calcium in osteoclasts and in a proportion (20%) of stromal cells/osteoblasts. Taken together, these data suggest that differentiated osteoclasts are equipped with receptors for VIP that are linked to a transient inhibition of osteoclast activity and, in addition, that stromal cells/osteoblasts have VIP receptors coupled to a delayed stimulation of osteoclastic resorption.

The quantitative comparison of the relative potency of agonists is a standard method of receptor and agonist classification. If agonist potency ratios do not correspond in two given tissues, this is used as presumptive data to conclude that the receptors in those two tissues are different. This article presents data to show that a single receptor can demonstrate varying agonist potency ratios in different host cells. These data are described in terms of the production of more than one agonist-selective receptor active state and the interaction of these different active states with multiple G proteins in the membrane to produce cellular response. Stable host human embryonic kidney 293 cells with enhanced quantities of the respective Galpha-protein were created. Wild-type and Galpha-subunit enriched cells were then transiently transfected with human calcitonin receptor type 2 (hCTR2). Binding did not detect differences in the G protein-enriched cells versus wild-type cells. In contrast, functional studies did show differences between the host cell lines and Galpha-subunit enriched cell lines. The relative potency of eight calcitonin agonists was measured in studies of calcium fluorescence in transfected cells containing human calcitonin receptor type 2 by comparing pEC(50) (-log molar concentration producing half-maximal response) values. In Galphas-enriched cells, the relative order of potency of the agonists changed. The host-cell dependent differences in potency ratios ranged from 2-fold to more than 46-fold. This finding is not consistent with the idea that all of the agonists produce response in the same manner (i.e., through a common active state of the receptor). These data are consistent with the idea that these different agonists produce arrays of active states that differentially use G proteins. This idea is discussed in terms of the design of stimulus-bias assay systems to detect agonist-selective receptor active states with resulting potential for increased selectivity of agonists.

We have previously shown that in a HEK-293 cell line that overexpresses the C1a isoform of the calcitonin receptor (C1a-HEK), calcitonin induces the tyrosine phosphorylation of the focal adhesion-associated proteins HEF1 (a p130(Cas)-like docking protein), paxillin, and focal adhesion kinase and that it also stimulates the phosphorylation and activation of Erk1 and Erk2. We report here that cell attachment to the extracellular matrix, an intact actin cytoskeleton, and c-Src are absolutely required for the calcitonin-induced phosphorylation of focal adhesion-associated proteins. In contrast to the phosphorylation of paxillin and HEF1 in cells attached to fibronectin-coated dishes, calcitonin failed to stimulate the phosphorylation of paxillin and HEF1 in suspended cells, in cells attached to poly-d-lysine-coated dishes, and in attached cells pretreated with the RGD-containing peptide GRGDS. Overexpression of wild-type c-Src increased calcitonin-induced paxillin and HEF1 phosphorylation, whereas overexpression of kinase-dead Src or Src lacking a functional SH2 domain inhibited the calcitonin-stimulated tyrosine phosphorylation of these proteins. Overexpression of Src lacking the SH3 domain did not affect the calcitonin-induced phosphorylation of paxillin and HEF1. In contrast to the regulation of paxillin and HEF1 phosphorylation, the calcitonin-induced phosphorylation of Erk1 and Erk2 did not appear to involve c-Src and was only partially dependent on cell adhesion to the extracellular matrix and an intact actin cytoskeleton. Furthermore, inhibition of Erk1 and Erk2 phosphorylation had no effect on the calcitonin-induced phosphorylation of paxillin and HEF1. Thus, in C1a-HEK cells, the calcitonin receptor is coupled to the tyrosine phosphorylation of focal adhesion-associated proteins and to Erk1/2 phosphorylation by mechanisms that are in large part independent.

PTH regulates calcium homeostasis through direct actions on its cognate type I receptor in the kidney and bone. PTH inhibits phosphate transport in renal proximal (PCT) tubules and stimulates calcium absorption by distal convoluted tubules (DCT). We examined PTH activation of the mitogen-activated protein kinase (MAPK) cascade raf-MEK-ERK in PCT and DCT cells and its effects on calcium transport and signaling. In DCT cells, PTH stimulates phosphorylation of ERK2 and activation of ERK2 kinase and is blocked by the MEK inhibitor PD98059. In DCT cells, stimulation of calcium entry with ionomycin did not activate ERK2 or augment PTH-stimulated ERK2 activity, indicating that MAPK activation lies upstream of calcium entry. ERK2 activation by PTH was blocked by the protein kinase C inhibitor calphostin-C but was unaffected by the protein kinase A inhibitor Rp-cAMPs. PD98059 abolished the increase of intracellular calcium induced by PTH demonstrating that ERK2 activation is directly involved in the increase of intracellular calcium activated by PTH in the DCT. Thus, PTH- stimulated ERK2 activation is PKC dependent and calcium independent. PTH also induced ERK2 phosphorylation in PCT cells. However, this effect is not involved in the transient rise of intracellular calcium because PD98059 did not inhibit the PTH-stimulated rise of intracellular calcium but abolished ERK2 activation. In conclusion, PTH activates MAPK in both distal and proximal renal tubule cells. However, the rise of [Ca2+]i depends upon MAPK activation only in distal cells. Thus, a common PTH1R exhibits differential signaling along the nephron that contributes to the ability to regulate distinct physiological actions of PTH.

Neuroendocrine cells are present in normal and tumoral prostate tissue, the neuropeptides secreted by this cells have a biological functions that have not been fully elucidated. The presence of neuroendocrine cells in prostatic carcinoma have been shown to increase tumor progression. We characterized the in vitro proliferative influence of bombesin and calcitonin in androgen-insensitive, PC-3 and DU-145, and androgen-sensitive, LNCaP, cell lines of human prostate cancers. The influence of these neuropeptides on proliferation were assessed using the colorimetric XTT assay and by cells counts with a hemocytometer. The growth of PC-3 and DU-145 cell lines is stimulated by bombesin and calcitonin but exerted any stimulatory effect on the proliferation of the LNCaP cell line. This indicate that bombesin and calcitonin can modulate proliferation of androgen-insensitive human prostate cell lines "in vitro" and may be potential paracrine growth promoters in stablished androgen irresponsive human prostatic carcinoma cells.

Amylin is a hormone belonging to the calcitonin protein family of peptides. To facilitate receptor screening studies, alternatively radiolabeled and biologically active amylin and salmon calcitonin analogues were synthesized by reductive methylation. Free amino groups of amylin and salmon calcitonin were methylated by reaction of peptides with formaldehyde and sodium [(3)H]borohydride. Radioactively labeled peptides were purified by size exclusion chromatography followed by HPLC. Analysis by MALDI-TOF mass spectrometry of purified amylin and salmon calcitonin peptides revealed incorporation of both two and four tritiated methyl groups per peptide molecule. Specific activities of 22.6 and 23.2 GBq/mmol were measured for amylin and salmon calcitonin, respectively. Methylation of rat amylin and salmon calcitonin did not affect their biological activities as both retained their potency to inhibit insulin-stimulated glycogen synthesis in isolated rat soleus muscle. The synthesis of these tritiated analogues provides an alternative chemically stable radiolabeled ligand which may be useful in exploring receptor interactions within the calcitonin peptide family., (Copyright 2000 Academic Press.)

The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is induced in many cell types in response to a variety of extracellular signals and causes cell cycle arrest in both the G1 and G2/M phases of the cell cycle. We reported previously that calcitonin (CT) receptor (CTR)-mediated growth inhibition of HEK-293 cells stably transfected with the human CTR is accompanied by a rapid and sustained induction of p21 and cell cycle arrest in G2. In the present study we have shown that CT stimulates transcription from a p21 promoter-luciferase construct. Using deletion and mutation analysis of the p21 promoter we have demonstrated that transcriptional activation of p21 by CT is p53-independent and is mediated through specific activation of Sp1-binding sites in a region of the promoter between -82 and -69, relative to the transcription start site. CTR-mediated transcriptional activation of p21 was specific for the insert-negative isoform of the human CTR. Butyrate, which was shown previously to activate the same Sp1 site, synergised with CT to increase further p21 promoter activity. These results provide the first demonstration that CTR can induce gene transcription through the constitutively expressed transcription factor Sp1, and define a mechanism of cell growth suppression that may have implications for other members of the seven-transmembrane domain G protein-coupled receptor superfamily.

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

Using mouse osteoclast-like cells (OCs), we have shown that short exposure to calcitonin (CT) resulted in prolonged reduction of CT binding by inhibiting de novo CT receptor (CTR) synthesis. Additionally, CT-treated OCs demonstrated resistance to CT rechallenge on the inhibitory effect of CT in osteoclastic bone resorption. There is, however, scant information on CT effects on human osteoclasts. In this study, we examined the features of CTR down-regulation and its recovery after short exposure to CT of human OCs. OCs were prepared by treatment of peripheral blood mononuclear cells in vitro with osteoclast differentiation factor and macrophage colony-stimulating factor. Treatment of OCs with salmon CT (sCT) and human CT (hCT) resulted in a dose-dependent reduction in [125I]sCT binding capacity. Continued receptor occupancy by ligand was excluded by using a glycine-acid washing procedure. Treatment with sCT reduced CTR messenger RNA expression, suggesting that CTR down-regulation is, at least partly, attributable to an inhibition of de novo CTR synthesis. To investigate the intracellular signal transduction pathways that mediate these effects, we examined the effects of activation of the protein kinase (PK)A, PKC, and Ca2+-calmodulin-dependent kinases. Treatment with PKC activators mimicked CT, whereas neither activation of PKA nor elevation of intracellular Ca2+ did so. We further investigated the intracellular signaling pathways responsible for the inhibitory effects of CT on bone resorption, which showed that treatment with PKC activators reproduced the effects of CT. These data suggest that the PKC pathway plays an important role in homologous CTR down-regulation, as well as inhibition of bone-resorbing activity by CT, in human OCs. Short exposure of OCs to CT (10(-9) M, 1 h) reduced [125I]sCT-specific binding for a prolonged period, as we have shown previously in mouse OCs. The reduced specific binding, CTR messenger RNA levels, and CT-sensitive adenylate cyclase responsiveness returned to the control levels by 96 h after removal of CT. These results strongly support the notion that escape from CT inhibition of osteoclastic bone resorption in humans is attributable to the development of resistance by OCs to CT. This study also showed that even short exposure to CT induced prolonged desensitization to CT rechallenge, although the OCs eventually regained responsiveness to sCT rechallenge.

Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.

Recently we reported that calcitonin (CT) induces growth arrest at the G2 stage of the cell cycle in HEK-293 cell lines expressing the most abundant, insert-negative, isoform of the human CT receptor (insert -ve hCTR). The present study investigates the involvement of the MAPK signalling pathway in the anti-proliferative actions of CT and compares the activity of an isoform of the hCTR that contains a 16 amino acid insert in the first putative intracellular loop (insert +ve hCTR). Comparison of HEK-293 cells stably transfected with the insert -ve or the insert +ve hCTR, showed that accumulation of cAMP and intracellular free calcium in response to CT were specific for the insert -ve receptor isoform. However, a novel acidification of the extracellular medium was mediated by both isoforms. Treatment with CT of cells expressing the insert -ve hCTR, caused a decrease in cell growth associated with an induction of p21(WAF1/CIP1). Analysis by fluorescence-activated cell scanning showed that growth inhibition was associated with an accumulation of cells in G2. CT treatment of cells expressing the insert -ve, but not insert +ve hCTR, induced the phosphorylation of Erk1/2 MAPK, which persisted for at least 72 h. Treatment of cells expressing the insert -ve hCTR with the MAPK kinase (MEK) inhibitor, PD-98059, inhibited the phosphorylation of Erk1/2 and abrogated the growth inhibitory effects of salmon CT, the accumulation of cells in G2, and the associated induction of p21(WAF1/CIP1). These data suggest that activation of Erk1/2 are downstream effectors of the insert -ve hCTR in modulating cell cycle progression.

The current studies were intended to determine whether the anabolic effects of calcitonin (CT) on human osteoblast-line cells were (1) unique to osteosarcoma cells or also evident in osteoblast-line cells derived from normal human bone; and/or (2) associated with effects on several insulin-like growth factor (IGF) system components. Preliminary studies identified several osteoblastic cell lines, derived from normal human bone, which showed calcitonindependent increases in cell proliferation, alkaline phosphatase activity, and/or (45)Ca uptake (P < 0.05-P < 0.001). Two of these cell lines-(human vertebrae) HBV-155 and HBV-163-were included with the human osteosarcoma cell line, SaOS-2, in most of our subsequent studies of calcitonin effects on selected IGF system components: IGF-II, IGF-I, and IGF binding proteins -3, -4, and -5. The results of those studies revealed that a 48 hour exposure to salmon CT caused a dose-dependent (0.03-3 mU/ml) increase in the net extracellular level of IGF-II (r = 0.96, P < 0.01) in serum-free cultures of SaOS-2 cells, with a maximal 60% increase at the highest tested dose (P < 0.02). Similar effects were seen with HBV-163 cells (r = 0.90, P < 0.01) and HBV-155 cells (r = 0.55, P < 0.02). The effect of calcitonin on the extracellular level of IGF-II was biphasic with respect to time: it decreased at 6 hours (P < 0.005 and P < 0.001, for SaOS-2 cells and HBV-163 cells, respectively) and increased at 24 hours (P < 0.02 and P < 0.05). These calcitonin-dependent increases in the extracellular level of IGF-II were associated with parallel increases in IGF-I (P < 0.005 for SaOS-2 cells and P < 0.03 for HBV-163 cells), but calcitonin did not affect the extracellular level of transforming growth factor (TGF)-beta. The calcitonin-dependent changes in IGF-II were not associated with changes in the extracellular levels of IGF binding proteins -3, -4, or -5. Finally, our studies showed that two other members of the CT superfamily-CT gene-related peptide and amylin-did not mimic the effect of CT to increase the extracellular level of IGF-II. Together, these data demonstrate that human osteoblast-line cells derived from normal human bone can respond to CT, and that those responses can include CT dose- and time-dependent increases in the extracellular levels of IGF-I and IGF-II.

As the population ages, osteoporosis becomes a growing public health concern. Current treatments provide patients with limited clinical improvement, numerous side effects, and no cure. The naturally-occurring peptides calcitonin and parathyroid hormone, which regulate bone metabolism, offer alternative treatment options. Clinical studies indicate the usefulness of calcitonin and parathyroid hormone in osteoporosis and Paget's disease of bone. For the peptides to become viable therapies, formulations must be developed that bypass the need for injection. Pulmonary delivery of calcitonin and parathyroid hormone appears likely in the near future.

Chronic treatment with calcitonin in osteoporotic patients alleviates the pain associated with this condition by an unknown mechanism. In ovariectomized rats that develop osteoporosis and hyperalgesia, we examined whether a functional change in serotonergic systems in the spinal dorsal horn was involved, using whole-cell recordings from substantia gelatinosa neurons in spinal cord slices and [(3)H]8-hydroxy-2(di-n-propylamino)tetralin ([(3)H]8-OH-DPAT) binding. Hyperalgesia could be attributed to the elimination of presynaptic inhibition by 5-HT of glutamatergic primary C-afferent terminals and an associated decrease in the density of [(3)H]8-OH-DPAT binding sites whose receptors are neither 5-HT(1A)- nor 5-HT(7)-subtype. These changes in serotonergic systems were restored after chronic treatment with calcitonin. Reversal of 5-HT receptor changes by calcitonin treatment may provide an explanation for its analgesic actions in patients.

Treatment with NO-releaser NOC18 significantly promoted apoptosis in murine osteoclast-like cells, with a transient increase in caspase-3-like protease activity. In contrast, the apoptosis was protected against by caspase inhibitors, most efficiently with the broadly acting caspase specific inhibitor z-Asp-CH2-DCB, indicating involvement of multiple caspases in progression of the apoptosis. Among osteoclast survival factors examined, calcitonin completely protected against morphologically defined-apoptosis and the increase of caspase-3-like protease activity. The effect of calcitonin was mimicked by treatment of cells with (Bu)2cAMP and forskolin, and abolished by protein kinase-A inhibitor H-89. Independently from the PKA activation, colony stimulating factor-1, interleukin-1beta and the receptor activator of NF-kappaB ligand also protected against the apoptosis but were less effective than calcitonin. All survival factors investigated inhibited conversion of procaspases-3 and -9 to their mature forms in the cells. Thus, downstream antiapoptotic signaling activity from each factor overlapped in inhibition of caspases. However, how this was attained seemed to be different from each other. Typically, only colony stimulating factor-1 up-regulated expression of endogenous caspase inhibitor protein, X-linked inhibitor of apoptosis (XIAP), in the osteoclast-like cells.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter candidate colocalized with acetylcholine in efferent fibers innervating hair cell organs. We have used the Xenopus laevis lateral line organ to investigate the responses of a hair cell organ to the CGRP family of peptides. Two isoforms of CGRP, r alpha-CGRP and r beta-CGRP, and a human analog of alpha-CGRP, h(Tyro) alpha-CGRP, produced dose-dependent increases in afferent nerve fiber discharge rate with EC50 values of approximately 1 microM. Rate increases were 31.2, 18.9, and 10.3%, respectively. The peptide fragment rCGRP8-37 a selective CGRP1 receptor antagonist, competitively inhibited the response to r alpha-CGRP. Diacetoamidomethyl cysteine CGRP (r[Cys(ACM)2,7]alpha-CGRP), a CGRP2 agonist, did not change discharge rate. Rat amylin did not increase rate until very high concentrations, and then the change was less than 7%. Rat adrenomedullin produced no increase in rate. Responses to r alpha-CGRP developed after metamorphosis. No change in spontaneous discharge rate was observed until postmetamorphic day 6, and then it was only a fraction of the maximal response. This response progressively increased until postmetamorphic day 28, when it reached its maximal value. The most straightforward interpretation of our results is that the effect of CGRP is mediated by the CGRP1 receptor and that CGRP, of the peptides presently known to exist in the CGRP family, is the most likely endogenous peptide mediating these effects.