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Title: Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

Abstract

Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of ({sup 3}H)proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of ({sup 3}H)hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of ({sup 3}H)thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred inmore » nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.« less

Authors:
; ;  [1]
  1. (Univ. of Louisville, KY (USA))
Publication Date:
OSTI Identifier:
5642964
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the Society for Experimental Biology and Medicine; (USA); Journal Volume: 197:1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ALKALINE PHOSPHATASE; ENZYME ACTIVITY; BONE CELLS; DNA REPLICATION; COLLAGEN; BIOSYNTHESIS; NICOTINE; BIOLOGICAL EFFECTS; CHICKENS; DOSE-RESPONSE RELATIONSHIPS; EMBRYOS; INHIBITION; PROLINE; THYMIDINE; TOBACCO PRODUCTS; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ALKALOIDS; AMINES; AMINO ACIDS; ANIMAL CELLS; ANIMALS; AUTONOMIC NERVOUS SYSTEM AGENTS; AZINES; AZOLES; BIRDS; CARBOXYLIC ACIDS; CONNECTIVE TISSUE CELLS; DRUGS; ENZYMES; ESTERASES; FOWL; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROGEN COMPOUNDS; HYDROLASES; ISOTOPE APPLICATIONS; NUCLEIC ACID REPLICATION; NUCLEOSIDES; NUCLEOTIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PARASYMPATHOLYTICS; PARASYMPATHOMIMETICS; PHOSPHATASES; PROTEINS; PYRIDINES; PYRIMIDINES; PYRROLES; PYRROLIDINES; RIBOSIDES; SCLEROPROTEINS; SOMATIC CELLS; SYNTHESIS; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Ramp, W.K., Lenz, L.G., and Galvin, R.J. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells. United States: N. p., 1991. Web. doi:10.3181/00379727-197-43221.
Ramp, W.K., Lenz, L.G., & Galvin, R.J. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells. United States. doi:10.3181/00379727-197-43221.
Ramp, W.K., Lenz, L.G., and Galvin, R.J. 1991. "Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells". United States. doi:10.3181/00379727-197-43221.
@article{osti_5642964,
title = {Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells},
author = {Ramp, W.K. and Lenz, L.G. and Galvin, R.J.},
abstractNote = {Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of ({sup 3}H)proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of ({sup 3}H)hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of ({sup 3}H)thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.},
doi = {10.3181/00379727-197-43221},
journal = {Proceedings of the Society for Experimental Biology and Medicine; (USA)},
number = ,
volume = 197:1,
place = {United States},
year = 1991,
month = 5
}
  • Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP hasmore » no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.« less
  • We have investigated the actions of human PTH (hPTH-(1-34)) on the association of 45Ca2+ with two human (SaOS-2 and MG-63) and two rat (ROS 17/2.8 and UMR-106) osteoblast-like cell types. In SaOS-2 cells, hPTH-(1-34) binds to specific membrane receptors to activate adenylate cyclase. Treatment of SaOS-2 cells with hPTH-(1-34) resulted in an increase in 45Ca2+ uptake, in a dose-dependent fashion, up to 2- to 4-fold above control values. The increase was first evident at 10 min and persisted for at least 30 min. Treatment with nimodipine, a calcium channel antagonist, was without effect on the stimulatory action of PTH. Amore » similar enhancement of cell-associated 45Ca2+ was observed when the cells were incubated with vasoactive intestinal peptide, which acts via different receptors to activate adenylate cyclase in SaOS-2 cells. Treatment with (Bu)2cAMP also induced an increase in cell-associated 45Ca2+. Pretreatment of SaOS-2 cells with hPTH-(1-34) for 4 h, which induced homologous desensitization to a second challenge with the same peptide for stimulation of cAMP production, did not attenuate the further enhancement of cell-associated 45Ca2+ by a second treatment with hPTH-(1-34). We then examined a possible relationship between alkaline phosphatase (ALPase) and 45Ca2+ uptake. SaOS-2 cells contained high levels of alkaline phosphatase activity and continuously released the enzyme into the medium. Release was enhanced by treatment with hPTH-(1-34) for 10 min. Incubation of cells with levamisole (an inhibitor of the liver/bone/kidney type of ALPase) resulted in a rapid decrease in basal and PTH-stimulated 45Ca2+ uptake, while treatment with L-Phe-Gly-Gly was without effect. Treatment of the cells with ALPase (bovine kidney) enhanced 45Ca2+ uptake. In MG-63 cells, a stimulatory effect of hPTH-(1-34) on cell-associated 45Ca2+ was also observed; however, hPTH-(1-34) did not stimulate cAMP production in MG-63 cells.« less
  • Alkaline phosphatase (orthophosphoricmonoester phosphohydrolase (alkaline pH optimum), EC 3.1.3.1) purified from a Burkitt lymphoma cell line (Daudi) and Moloney-virus-induced murine leukemia (YAC) showed unique catalytic properties in substrate specificity and inhibition by cysteamine-S-phosphate. It migrated on polyacrylamide gel electrophoresis in a single activity band. Alkaline phosphatase with similar properties was found in several human lymphoblastoid cell lines, in chronic lymphatic leukemic cells, in organs of leukemic mice, and in sera of patients with certain lymphoproliferative disorders. The unique kinetic properties of this enzyme were established using two kinds of substrate, namely, the monoesters of orthophosphoric acid (Type I) and themore » S-substituted monoesters of thiophosphoric acid (Type II). The enzyme catalyzed the hydrolysis of Type I, but failed to hydrolyze Type II. The enzyme probably interacts directly either in catalysis or in binding with the --P--O-- or --P--S-- bonds. Thus, in these respects it differs from the established enzymatic mechanism of other known alkaline phosphatases, and therefore can be considered a new enzyme. This enzyme was designated N-alkaline phosphatase. N-alkaline phosphatase was absent in untreated and mitogen-stimulated human blood lymphocytes, in murine lymph nodes, and pig thymus (even after mitogen stimulation), and in blast cells of acute myeloid leukemic patients. The appearance of N-alkaline phosphatase only in certain types of proliferating lymphocytes and in the sera of patients with lymphoproliferative disorders may be exploited as a cell marker and as a diagnostic tool.« less
  • It has recently become apparent that a number of hormones and growth factors modulate cytosolic pH (pH{sub i}) and there is some evidence that this in turn may influence cell growth. The authors have examined the effects of parathyroid hormone (PTH) on both these parameters in an osteoblast-like cell line, UMR 106. Preliminary studies, using the pH-sensitive fluorescent probe 2{prime},7{prime}-bis(2-carboxyethyl)-5,(6)-carboxyfluorescein indicated that these cells regulate pH{sub i} by means of an amiloride-inhibitable Na{sup +}-H{sup +} exchanger. Rat PTH-(1-34) (rPTH) caused a progressive dose-related decrease in pH{sub i} with a half-maximal effect at 10{sup {minus}11} M. The diacylglycerol analogue, phorbol 12-myristatemore » 13-acetate, increased both pH{sub i} and ({sup 3}H)thymidine incorporation, and amiloride reduced both indexes. However, rPTH remained a potent inhibitor of ({sup 3}H)thymidine incorporation in the presence of amiloride, even though it did not affect pH{sub i} in these circumstances. It is concluded that PTH decreases pH{sub i} and growth in UMR 106 cells but that these changes can be dissociated. Depression of pH{sub i} may have other important effects on bone metabolism, such as reducing cell-cell communication, and may be associated with alkalinization of the bone fluid compartment.« less