Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP‐dependent protein kinase (PKA)‐regulated Cl^? channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A‐kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co‐localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.     

Immunocytochemical analysis of cyclic AMP receptor proteins in the developing rat parotid gland

Previous studies showed that regulatory subunits of type II cyclic AMP-dependent protein kinase (RII) are present in adult rat parotid acinar cells, and are secreted into saliva. If the synthesis and intracellular distribution of RII exhibit developmental specificity, then RII can be an indicator of secretory and regulatory activity of salivary glands. OBJECTIVE: To determine the expression and distribution of RII in the rat parotid at specific ages representing defined developmental stages. METHODS: Parotid glands of fetal, neonatal and adult rats were prepared for morphologic and immunocytochemical study. The cellular distribution of RII was studied using light microscopic immunogold silver staining with anti-RII, and its intracellular distribution using electron microscopic immunogold labeling. RESULTS: In utero, parotid RII levels were low; 5-18 days after birth, labeling of secretory granules and cytoplasm rose to a peak, followed by a rapid decrease in both compartments at 25 days. At 60 days, granule labeling increased to levels near those at 18 days, whereas cytoplasmic labeling remained low. Nuclear labeling was highest during the first 3 weeks after birth, and then declined. CONCLUSIONS: The higher nuclear and cytoplasmic labeling during the neonatal period may reflect RII involvement in acinar cell differentiation. The accumulation of RII in secretory granules is similar to the pattern of the major salivary proteins, amylase and PSP. The redistribution of RII in these compartments during development may reflect changing gene expression patterns, and may be useful for identification of genetic or metabolic abnormalities.     

The inhibition of DNA synthesis by prostaglandin E2 in human gingival fibroblasts is independent of the cyclic AMP-protein kinase A signal transduction pathway

In this study we attempted to clarify the mechanism of the inhibitory effects of PGE₂ on DNA synthesis in Gin-1 (fibroblasts derived from healthy human gingiva) from the aspect of the cyclic AMP-dependent protein kinase signal transduction pathway. PGE₂ upregulated intracellular cyclic AMP accumulation and inhibited DNA synthesis in Gin-1 in a dose-dependent manner. When the PGE₂-induced intracellular cyclic AMP accumulation was further enhanced by treatment with the cyclic AMP-phosphodiesterase inhibitor, IBMX, the inhibitory effect of PGE₂ on DNA synthesis was also enhanced. Furthermore, when we examined the effects of forskolin, an activator of cyclic AMP production, on intracellular cyclic AMP accumulation and DNA synthesis, similar results were obtained. However, inhibitors of cyclic AMP-dependent protein kinase (protein kinase A) such as HA1004 did not diminish the inhibitory effect of PGE₂ on DNA synthesis in Gin-1. These results suggest that in Gin-I, PGE₂-induced cyclic AMP accumulation may not lead to the activation of protein kinase A or protein kinase A activity may not relate directly to the growth inhibitory effect of PGE₂, and that PGE₂ does not inhibit DNA synthesis through the cyclic AMP-protein kinase A signal transduction pathway in Gin-1.     

Expression of secretory proteins in oral fluid after orthodontic tooth movement.

Orthodontic treatment alters the expression of secretory proteins at the local level in bone and the oral cavity, but its systemic effects are not well understood. Total secretory proteins and a cyclic adenosine monophosphate (AMP)-dependent protein kinase subunit (RII) were measured in saliva and gingival crevicular fluid (GCF) after the placement of orthodontic separators to determine if mechanical force applied to teeth affects protein secretion. Whole saliva and GCF were collected before and 1 day after treatment. Electrophoresis and Western blotting were carried out to establish the banding patterns of total proteins and to measure the isotype and amount of RII that serves as an apparent stress indicator. Digitized image files were used for densitometric analyses of the relative concentrations of RII and total protein. Individual protein values showed no statistically significant changes in saliva or GCF. Western blots, however, showed a dramatic difference in RII after the placement of separators: the 50-to-55 kilodaltons (kd) band virtually disappeared and was replaced by a fragment in the 20-kd range. These results suggest that although the expression of total proteins is not altered by mechanical force applied to teeth, a systemic response via the cyclic AMP signaling pathway might have been activated.     

Differential response of type I and type II cyclic AMP-dependent protein kinases in submandibular gland of isoproterenol-treated rats

The cytosolic protein kinase isozymes were studied in relation to the secretion of saliva and the proliferative process induced by isoproterenol. At the same time as salivary secretion began, cytosolic protein kinase was activated and, in the absence of cyclic AMP, increased to a maximum level at 10 min after administration of the drug. The active state of the enzyme was maintained for 1–2 h. Chromatographic analysis of the cytosol enzyme revealed that in an activated state type II kinase easily dissociates into the catalytic and regulatory subunits while type I kinase hardly dissociates. Thus we conclude that type I kinase, at least in submandibular gland, is less dissociable in the presence of cyclic AMP than type II kinase but highly active in a ternary complex (R₂-4 cAMP-C₂). The presence of cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase II, was demonstrated by use of a heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase. The level of protein kinase I decreased to its lowest value, about half that of the control value, at 10 h after isoproterenol injection and then increased slightly at 15 h, whereas type II kinase remained virtually unchanged, suggesting that the two protein kinase isozymes are independently regulated in the proliferating cells.     

Comparison of cyclic AMP-dependent protein kinases from salivary glands of four species

Cyclic AMP-dependent protein kinase activity, subcellular distribution, and isozyme profile were compared in rabbit, rat, guinea pig, and mouse in both parotid and submandibular glands. Glands were homogenized under hypotonic conditions and the following fractions isolated: 600 g pellet, 27,000 g pellet, and 27,000 g supernatant. The specific activity of the enzyme was similar in the eight glands and was highest in the 27,000 g supernatant. The average activity in the 27,000 g supernatant was approximately 75% of the total gland activity, although there was considerable variability between tissues and species. After being washed with isotonic buffer, this percentage was increased to an average of 84%. When isozyme patterns of the kinase were examined, the rabbit parotid was unique in that it contained a high percentage of isozyme I as isolated on DEAE cellulose columns.     

TGF-β1受体对ClC-3氯通道在正畸骨代谢中表达的影响

目的观察TGF-β1受体（TGF-β1 receptor,TβR）对成骨细胞中ClC-3氯通道表达的影响,初步探索TβR在ClC-3氯通道调控细胞成骨分化中的作用。方法通过CCK-8试剂盒法和Real-Time PCR法检测TβR抑制剂在MC3T3-E1细胞中对TβRI、TβRII表达的最佳抑制方式;然后观察阻断TβR对ClC-3氯通道表达的影响,以及通过siRNA基因转染技术阻断ClC-3氯通道观察对TβRI、TβRII表达的影响;最后观察TβR对成骨相关基因（Alp、Runx2）表达的影响。结果浓度为2μM的TβR抑制剂作用MC3T3-E1细胞24h对TβRI、TβRII表达抑制作用较好,并且对细胞的毒性作用较小。抑制TβR表达可促进ClC-3基因和蛋白的表达(P<0.05),同样下调ClC-3氯通道的表达,TβRI和TβRII基因的表达升高（P<0.05）,TRβII升高程度强于TβRI。阻断TβR可促进成Alp、Runx2的表达（P<0.05）。结论TβR可抑制骨代谢过程中ClC-3氯通道在成骨细胞中的表达,TRβII对ClC-3氯通道的表达更敏感。     

Regulation of osteoprotegerin gene expression in dental follicle cells

Colony-stimulating factor-one (CSF-1) and parathyroid-hormone-related protein (PTHrP) down-regulate osteoprotegerin (OPG) gene expression in the dental follicle of the rat first mandibular molar. To examine this regulation at the signal transduction level, we treated cultured dental follicle cells with either phorbolmyristate acetate (PMA) or dibutyryl cyclic AMP (dbcAMP) to activate either protein kinase C (PKC) or protein kinase A (PKA). Our results demonstrate that PMA up-regulates OPG gene expression and down-regulates the expression of CSF-1 and the PTHrP receptor (PTHrP-R). Conversely, dbcAMP down-regulates OPG expression and up-regulates CSF-1 and PTHrP-R expression. Immunostaining shows that PMA also increases the steady-state levels of protein. Thus, treatment with agents that affect protein kinase activity also enhance the steady-state mRNA and protein levels of OPG, as well as decreasing the mRNA levels of CSF-1 and PTHrP-R. The PKC-alpha isoform may be critical in OPG regulation because PKC-alpha gene expression is enhanced by PMA and reduced by either CSF-1 or PTHrP.     

Cyclic AMP-dependent protein kinase-independent angiotensin II-induced inhibition of calcium current in hamster submandibular ganglion neurons

In a previous study, we demonstrated that angiotensin II (AngII) inhibited voltage-dependent calcium channels (VDCCs) currents (ICa) in hamster submandibular ganglion (SMG) neurons. In sinoatrial node cells, it has been reported that AngII inhibits ICa by suppressing cyclic AMP production. In this study, to investigate the possible involvement of a cyclic AMP-cyclic AMP dependent protein kinase (PKA) pathway in the AngII-induced inhibition of ICa, effects of AngII were examined in SMG neurons after treatment with an activator and inhibitor of PKA. Neither pretreatment of neurons with membrane permeable cyclic AMP nor intracellular dialysis of PKA blocker attenuated the AngII-induced inhibition of ICa. These results indicate that AngII inhibited ICa via a cyclic AMP-PKA-independent mechanism in SMG neurons.     

Kinetic properties of a cyclic AMP-dependent protein kinase from pig parotid gland.

Protein kinase from pig parotid gland exhibited kinetic properties similar to protein kinases from other tissues. The pH optimum for the enzyme was 6.6-6.7 in the presence of cyclic AMP and 7.9 in its absence. With histone as substrate, maximum activity of the enzyme was approximately 21 nmoles/mg protein/minute. The apparent K_m for ATP was 16 μM in the absence of cyclic AMP and 18–20 μM in its presence. Cyclic AMP was associated with nearly a two-fold decrease in the apparent K_m for histone. Half-maximal stimulation of kinase activity occurred at approximately 0.1 μM cyclic AMP as opposed to 0.8 and 5 μM for cyclic IMP and cyclic GMP respectively. Of the protein kinase substrates tested, histone IIA was the superior substrate. The enzyme was essentially inactive in the absence of magnesium and calcium was unable to substitute for magnesium. Maximum enzyme activity occurred at 5 mM magnesium. Half-maximal inhibition of enzyme activity by calcium in the presence of 10 mM magnesium occurred at approximately 2 mM. These results offer a basis for studying a possible role for protein kinase in salivary gland secretory function. Protein kinase activity could be partly regulated in vivo by high calcium levels in the gland.     

Effect of electric currents on gingival cyclic nucleotides in vivo

Electric currents have been shown to affect a variety of tissues. The objective of this investigation was to determine whether the application of minute electric currents, non-invasively, to cat gingival tissues would alter the immunohistochemical staining pattern of gingival cells for cyclic AMP and cyclic GMP, substances associated with cell stimulation. Three groups of 4 cats each were treated with an electric device delivering 15 ± 2 μamperes d. c. current to the gingiva near one maxillary canine for 1, 3 and 7 days, respectively. Horizontal sections of each maxilla were stained immunohistochemically for either cyclic AMP or cyclic GMP and intensely stained cells near the cathode and anode were counted. Near the cathode the number of gingival cells intensely stained for cyclic nucleotides was doubled by days 1 and 7 but not at day 3. Near the anode larger increases for both cyclic nucleotides were observed at all time periods. Changes were observed in the cellular staining patterns for cyclic AMP and cyclic GMP as a result of the electric stimulation suggesting activation of nuclear protein kinases. These results demonstrate that the stimulation of gingival cells by electric currents involves cyclic nucleotide fluctuations similar to those observed following the application of hormones and drugs.     

The effects of calmodulin antagonists on prostaglandin E2-induced responses in rat calvarial bone cells

Osteoclastic (OC) and osteoblastic (OB) cells were isolated by sequential collagenase digestions of new-born rat calvaria. Prostaglandin E2(PGE2) did not alter total calmodulin levels after a 5 or 60 min incubation. The calmodulin antagonists, trifluoperazine (TFP) at 10-50 microM and W-7 (50 microM) inhibited PGE2-induced increases in calcium uptake by OC cells, but had no effect on control OC or OB calcium levels. W-5 (50 microM), a chlorine-deficient analogue of W-7 with weak anti-calmodulin activity, had no effect. Compound 48/80 (100-500 micrograms/ml), a highly effective calmodulin antagonist in other systems, had no effect on PGE2-induced calcium levels or control calcium uptake. There was inhibition of PGE2-induced increases in cyclic AMP by compound 48/80 (100 micrograms/ml) in both OC and OB cells but no effect on control levels. TFP at 50 microM inhibited both control and PGE2-induced increases in cyclic AMP but at 10 microM it lessened only the hormone-induced effect. W-7 (100 microM) inhibited PGE2-induced increases in OC and OB cyclic AMP but had no effect on control levels; W-5 (50 microM) had no effect on either of these. Dibutyryl cyclic AMP had no effect on control calcium uptake, PGE2-induced increases or W-7 inhibition of the PGE-2 effect on calcium uptake. The calmodulin antagonists, at doses which had affected only PGE2-induced increases in calcium uptake and/or cyclic AMP production, had no effect on leucine uptake by OC or OB cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of cyclic-AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

AIMS: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. MATERIALS AND METHODS: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. RESULTS: Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). CONCLUSION: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway.     

Cyclic-AMP-dependent protein phosphorylation in the soluble fraction of parotid glands from rats with drug-induced hypothyroidism

Cyclic-AMP-dependent protein kinase from the soluble fraction of parotid glands of hypothyroid rats was partially purified. Its isozyme distribution and kinetic properties were similar to those of euthyroid rats. Electrophoresis of 100 microliters portions at 20 mA per slab revealed that an endogenous protein (mol. wt 68,000) was specifically phosphorylated in hypothyroidism; this protein was not found in euthyroid rats. In the presence of cyclic AMP, there was stimulated phosphorylation of euthyroid-soluble proteins with molecular weights of 115,000, 98,000, 57,000, 50,000, 44,000, 33,000 and 19,000, and of proteins from hypothyroid rats with weights of 115,000, 98,000, 60,000, 50,000, 33,000 and 19,000. Tolbutamide reduced incorporation of 32Pi into soluble proteins from both groups. However, cyclic AMP still induced phosphorylation in euthyroid preparations in the presence of tolbutamide, but its effect was markedly reduced in the hypothyroid state. These differences in endogenous protein phosphorylation may have different effects on amylase release induced by beta-adrenergic stimulation.     

Nitric oxide production by a human osteoblast cell line stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide

Background/aim:  Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells).
Methods:  Cells were stimulated directly with A. actinomycetemcomitans lipopolysaccharide or pretreated with the following l -NIL (an iNOS inhibitor), anti-CD14, Toll-like receptor 2 (TLR2), or TLR4 antibody before stimulation with A. actinomycetemcomitans lipopolysaccharide. The role of the cyclic nucleotides was assessed by pretreating the cells with the following; ODQ (a guanylyl cyclase inhibitor); SQ22536 (an adenylyl cyclase inhibitor); db-cAMP (a cyclic adenosine monophosphate analog); br-cGMP (a cyclic guanosine monophosphate analog); forskolin (an adenylyl cyclase activator), IBMX [a non-specific phosphodiesterase (PDE) inhibitor], or KT5720 [a protein kinase A (PKA) inhibitor]. The cells were also preincubated with genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], BPB [a phospholipase A2 (PLA2) inhibitor], and NDGA (a lipoxygenase inhibitor). The iNOS activity and nitrite production in the cell cultures were determined spectrophotometrically.
Results:  The results showed that A. actinomycetemcomitans lipopolysaccharide stimulated both iNOS activity and nitrite production by HOS cells; this was reduced by l -NIL, anti-CD14, or anti-TLR4 antibody, SQ22536, KT5720, genistein, bisindolylmaleimde, BPB, and NDGA, but was enhanced by db-cAMP, IBMX, and forskolin.
Conclusion:  These results therefore suggest that A. actinomycetemcomitans lipopolysaccharide may induce the production of NO by HOS cells via a CD14–TLR4 molecule complex, a cAMP–PKA pathway, as well as by a PTK, PKC, PLA2, and lipoxygenase-dependent mechanism.     

Inductive effect of Porphyromonas gingivalis fimbriae on differentiation of human monocytic tumor cell line U937

This study used the human monocytic tumor cell line U937 to examine whether Porphyromonas gingivalis fimbriae induce differentiation of monocyte/macro-phage progenitors. When the progenitor cells were incubated with P. gingivalis fimbriaè, the incubation resulted in increased Fc rosette formation and increased CDllb production by the cells. The presence of a protein kinase C inhibitor, H7 or calphostin C, in the medium eliminated the stimulatory effects of P. gingivalis fimbriae on Fc rosette formation. Furthermore, CD11b expression was inhibited by calphostin C. In contrast, HA1004, an inhibitor of cyclic nucleotide-dependent protein kinase, had no effect on P. gingivalis fimbriae-induced Fc rosette formation or CD11b expression. These results demonstrate that P. gingivalis fimbriae are a potent inducer of the differentiation of the monocyte/macrophage tumor cell line U937, most probably via cyclic nucleotide-independent protein kinase C.     

Cyclic AMP levels in alveolar bone of orthodontically-treated cats.

Specific biochemical changes in mechanically stressed bone were studied in cats, orthodontically treated for periods of time ranging from 1 hr to 28 days. Alveolar bone samples were obtained from compression and tension sites surrounding tipping maxillary and mandibular canines. The samples were extracted with water, boiled, homogenized and the supernatants were assayed for cyclic AMP by a protein-binding method. It was found that cyclic AMP levels in treated sites initially decreased, then increased after 1–2 days and remained elevated to the end of the experimental period. It was suggested that the initial decrease in cyclic AMP at the compression sites was due to periodontal ligament cell-necrosis, while at the tension sites the decrease was due to a rapid increase in the cell population. The elevation in the level of cyclic AMP observed 2 weeks after the initiation of treatment was probably due to increased bone remodelling activity by a maturing cell population with a slow rate of growth. The level of cyclic AMP in bone after application of orthodontic forces may be the result of increased phosphodiesterase activity, and/or the involvement of a relatively small number of cells. It is concluded that mechanical forces can act as a first messenger and facilitate bone formation and resorption by activation of the cyclic nucleotide system.     

The role of cyclic‐AMP on arginase activity by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Aims: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8‐bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS‐stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS‐stimulated cells in the presence of 3‐isobutyl‐1‐methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results: Arginase activity in A. actinomycetemcomitans LPS‐stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS‐stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen‐stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP‐PKA‐dependent pathway.     

Characterization of the binding of delmopinol to salivary precipitates.

The aim of this study was to quantify the amount of delmopinol recovered in different molecular weight ranges of salivary proteins in the supernatant and pellet of delmopinol-saliva mixes. Unstimulated whole saliva was collected from 5 subjects and mixed with radiolabeled delmopinol to obtain a final drug concentration of 9.7 mM. The salivary-delmopinol solutions were incubated and then centrifuged. The resulting pellets and supernatants were studied in an electrophoresis assay. Each individual sample was run in three different electrophoretic gel lanes. The first lane was stained with silver dyes; the second lane was used for electrophoretic blotting and autoradiography; the third lane was cut in standard slices that were dissolved and analyzed with scintillation counting. The scintillation results demonstrated that higher radioactivity levels were detected at high molecular weight ranges (700-600 kDa). Furthermore, pellet samples were found to contain the highest amounts of delmopinol. The autoradiography results confirmed that delmopinol was bound to proteins of high molecular weight (700-600 kDa).     

Cholera toxin and forskolin stimulate formation of osteoclast-like cells in mouse marrow cultures and cultured mouse calvarial bones

Osteoclasts are hematopoietic in origin and formed by proliferation, differentiation and fusion of osteoclast progenitor cells. However, the signal transducing mechanisms involved in generation of osteoclasts are not clear. We have used two well-known adenylate cyclase stimulators to examine the effect of cyclic AMP (cAMP) on the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in cultured mouse calvarial bones and in mouse bone marrow cultures. The effects of forskolin and cholera toxin were compared with those of parathyroid hormone (PTH) and 1,25(OH)2vitaminD3 (1,25(OH)2D3). PTH, as well as forskolin and cholera toxin, increased the number of osteoclast profiles/mm bone in 24-h and 120-h cultures of mouse calvarial bones. In mouse bone marrow cultures, 1,25(OH)2D3 or PTH stimulated formation of TRAP-positive multinucleated cells. Moreover, forskolin or cholera toxin produced dose-dependent stimulation of these cells at a range of concentrations correlating with their effect on cAMP production. The osteoclastic phenotype of the TRAP-positive cells was demonstrated by autoradiography of 125I-labelled calcitonin binding and by the bone-resorbing activity of the cells. The sustained presence (0-9 d) of forskolin or PTH was required to obtain maximal formation of osteoclasts. However, the presence of 1,25(OH)2D3 was required only for the last 3 d of culture for maximal osteoclast formation. We conclude that PTH may stimulate osteoclast generation using the adenylate cyclase cAMP system as a signal transduction mechanism.