Warren K. Ramp

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 μg/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 μg nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 μg/ml. Release of [3H]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 [3H] thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 μg/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.

Smokeless tobacco contains a nonnicotine inhibitor of bone metabolism

The effects of smokeless tobacco on bone were investigated using tibiae from chick embryos. The bones were cultured in nicotine (15-1500 micrograms/ml) or in smokeless tobacco extract (STE, 15 mg tobacco/ml culture medium) and the effects on bone glucose metabolism (oxygen consumption and lactate production) and collagen synthesis ([3H]proline hydroxylation) were tested in vitro. Only the highest concentration of nicotine tested produced substantial effects as demonstrated by decreased oxygen consumption and [3H]hydroxyproline content (82 and 90%, respectively) and by a 15% increase in lactate production. Results with STE, which contained nicotine concentrations ranging from 104 to 125 micrograms/ml, showed similarly reduced [3H]hydroxyproline content (90%), but oxygen consumption was only reduced 36%, while lactate production was elevated 60%. The ability of the bones to recover from treatment with STE was demonstrated by increased oxygen consumption and [3H]hydroxyproline content (18 and 78%, respectively) and decreased lactate production (47%) compared to the STE-treated bones. These findings suggest the following conclusions: (1) both nicotine and STE at concentrations found in the saliva of smokeless tobacco users stimulate glycolysis and markedly inhibit bone collagen synthesis and mitochondrial activity; (2) effects of STE on bone are not due to nicotine; and (3) under the conditions studied, bone partially recovers from the effects of STE.

Inhibition of Cell Metabolism by a Smokeless Tobacco Extract: Tissue and Species Specificity

Abstract Smokeless tobacco contains a nonnicotine inhibitor of posttranslational modification of collagen (hydroxylation of [3H]proline) by cultured chick embryo tibias and osteoblasts. This study was undertaken to determine whether a methanol extract of smokeless tobacco (STE) containing the inhibitor has similar effects on collagen-producing cells and tissues other than bone. Its effects on DNA synthesis and cell proliferation (incorporation of [3H]thymidine) were also determined. Frontal bone, aorta, and cartilage were incubated for 2 days in medium containing STE. Glycolysis (lactate production) was stimulated by 80% in cartilage, but was not affected in the other tissues; medium alkaline phosphatase activity was unaffected. In frontal bone and cartilage, [3H] hydroxyproline content was decreased 88% and 57%, respectively, and [3H]proline content was decreased 68% and 37%, respectively; neither was affected in the aorta. Confluent cultures of collagen-producing mouse fibroblasts or primary osteoblasts obtained from chick embryo calvarias were incubated for 2 days in medium containing increasing concentrations of STE. Glycolysis and DNA synthesis were not affected. Cell proliferation was unaffected in fibroblasts, but was inhibited (34%) at the highest STE concentration in osteoblasts. AIPase activity was not detectable in fibroblast medium, but was decreased up to 72% in osteoblast medium. Inhibition of collagen synthesis by STE was concentration related in both cell types. At the highest concentration, [3H] hydroxyproline and [3H]proline contents in the cell layers were decreased to the following respective values: fibroblasts 56% and 45% and osteoblasts 50% and 29%, respectively. When incubation with STE was discontinued for 1 day, recovery did not occur. These findings suggest that inhibition of collagen synthesis by STE is not specific for bone, that collagen-producing cells are directly affected, and that recovery is not immediate. This inhibitor could contribute to the periodontal disease often seen in users of smokeless tobacco. Its identification and removal would produce a safer product.

Intracellular Processing and Secretion of Parathyroid Gland Proteins

Publisher Summary This chapter focuses on parathyroid hormone (PTH), its synthesis, intracellular processing, and secretion, and provides current information on the chemistry and biology of secretory protein-I (SP-I) and its possible relationship to PTH. The parathyroid gland is important in fields related to calcium metabolism because of the role played by its hormone PTH plays in the regulation of body calcium. The chapter also focuses on the gland because of the realization that it also synthesizes and secretes a major glycoprotein termed SP-I that is similar to chromogranin A (CGA), a protein co-secreted with epinephrine by the adrenal. SP-I/CGA appears to be present in a large number of endocrine, but not exocrine, cells. PTH is unglycosylated and generally does not contain modified amino acids. PTH exhibits a complex conformation in solution that appears to reorder at different pHs. Images of PTH examined by dark-field electron microscopy suggest that the molecule consists of two domains connected by a short stalk. The fundamental mechanisms responsible for secretory control are at least as complex as those regulating intracellular formation and processing of the hormone.

A serum substitute promotes osteoblast-like phenotypic expression in cultured cells from chick calvariae

The effects of medium supplements were tested on embryonic chick calvarial cells in culture. Isolates were divided among four treatment groups: Nu-Serum, chicken serum, fetal bovine serum, or calf serum. Expression of the osteoblastic phenotype was assessed by cell morphology, DNA content, [3H]thymidine incorporation, lactate production, cellular and medium alkaline phosphatase activities, and collagen synthesis. Cells grown in Nu-Serum demonstrated increased alkaline phosphatase activity and a six-fold higher rate of collagen synthesis compared to chicken serum. These cells displayed a polygonal profile, abundant rough endoplasmic reticulum, Golgi apparati, and elaborated an extensive matrix of banded collagen which was well mineralized by day 10 of culture. Although highly mitogenic, chicken serum promoted a more fibroblastoid morphology. Compared to the sera tested, Nu-Serum preferentially promoted the osteoblast-like phenotype in chick calvarial cells in culture.

Short-term storage of freshly harvested bone

An in vitro study was designed to test effects of various graft storage media on glucose metabolism and collagen synthesis of embryonic chick tibiae, using these variables as indices of bone cell viability. Normal saline solution, distilled water, and 5% dextrose in lactated Ringers solution were evaluated after a 5-hour incubation and again after a 3-day recovery period in a complete culture medium. The study suggests that bone grafts may be stored in normal saline solution or 5% dextrose in lactated Ringers solution for up to 5 hours. Normal saline solution is recommended because of fewer deleterious effects. Distilled water should not be used as a storage medium for bone grafts.

Comparison of the effects of smokeless tobacco extract with the effects of prolyl hydroxylase inhibitors on collagenous and noncollagenous protein synthesis by osteoblasts.

The effects of smokeless tobacco extract (STE) and prolyl hydroxylase inhibitors on protein synthesis by isolated osteoblast-like cells were compared. STE and 2,2dipyridyl markedly inhibited alkaline phosphatase (Alpase) and [3H]proline hydroxylation without affecting glycolysis (lactate production). However, pyridine 2,5-dicarboxylate (2,5-PDC) did not inhibit [3H] proline hydroxylation, Alpase activity, or glycolysis at moderate concentrations. The [3H]hydroxyproline to [3H]proline ratio in the cell layers demonstrated a concentration-dependent decrease with increasing STE and inhibitor concentrations. In the cell layers, the collagenous protein (CP) content was decreased after exposure to STE, 2,2dipyridyl, and 2,5-PDC and the noncollagenous protein (NCP) content was decreased after exposure to STE and 2,5-PDC. However, the effects on CP were at least twofold greater than on NCP. Similar results were observed regarding protein released to the culture medium. These data demonstrate that STE, like 2,2dipyridyl, inhibits the hydroxylation of proline and the synthesis of collagenase-digestible protein.

1,25-Dihydroxyvitamin D3 decreases alkaline phosphatase activity in cultures of embryonic chick tibiae.

Abstract The influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the alkaline phosphatase (AlPase) activity in cultures of chick embryo tibiae was determined. A dose-related, decreased release (30-47%) of AlPase from the bones was seen with the metabolite at 0.05-0.5 ng/ml of medium with a similar effect on the bone content of enzyme. The highest dose (1 ng/ml) decreased the bone content by 38% without further effect on AlPase release. Combining a low level of 1,25(OH)2D3 (0.05 ng/ml) with parathyroid hormone (PTH, 1 U/ml) reduced release of enzyme additively, but caused no greater decrease in bone content of activity than PTH alone. No effects of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3, 0.5 ng/ml] on release or bone content of AlPase were found when this metabolite was added alone or in combination with PTH; however, 24,25(OH)2D3 did prevent the inhibition of release of AlPase when added with 1,25(OH)2D3, After a 1-day exposure to 1,25(OH)2D3, continued incubation in metabolite-free medium resulted in an 89% increase in bone content of AlPase. The results suggest that 1,25(OH)2D3, as well as PTH, may have regulatory roles in bone growth through their effects On AlPase.

Smokeless tobacco contains an inhibitor of prolyl hydroxylase activity

The effects of smokeless tobacco extract (STE) and various constituents of STE on prolyl hydroxylase activity were determined using enzyme extracted from chick embryos. STE inhibited prolyl hydroxylase activity in a concentration-dependent manner, but nicotine and anabasine had essentially no effect. Enzymatic activity was inhibited by zinc, but not by the other inorganic elements in STE; however, the zinc concentration in STE was not high enough to produce the observed inhibition. The inhibition by STE was diminished by increasing concentrations of 2-oxoglutarate, but not by increasing concentrations of other cofactors. Thus, STE contains an inhibitor of prolyl hydroxylase which may be competitive with 2-oxoglutarate.

Interrelationships of vitamin D, bone metabolism and blood calcium concentration in the chick.

It is not known if the effects of vitamin D deficiency on chick bone are due to direct actions of the vitamin or if they are secondary to other changes, such as hypocalcemia. Day-old cockerels were fed either a rachitogenic diet containing no Ca (-D-Ca), 1.4% Ca (-D), or 3% Ca (-DHiCa) and given corn oil (-D groups) or vitamin D3 in corn oil (+D and +D-Ca) p.o. for up to 21 days. Radii were harvested and incubated for 6-8 h in a defined medium. Medium samples were taken every 2 h and analysed for Ca, Pi and lactate. Some bones were incubated in a respirometer to measure O2 consumption. Compared to +D, -D birds showed evidence of D deficiency by decreased plasma Ca concentration (35%), bone and body weight (43%) and Ca release from bone (70%) and by histological changes in bone characteristic of rickets. Increases were seen in total and bone alkaline phosphatase activity in plasma (270 and 706%, respectively), Pi release (23%), O2 consumption (23%) and lactate production (52%) by the -D radii. The marked hypocalcemia seen in the -D chicks did not occur in -DHiCa birds. Nevertheless, bone and body weights were decreased in this group and bone lactate production, O2 consumption and total and bone alkaline phosphatase in plasma were increased. Rachitic bone lesions were only partially corrected by the high-Ca diet. Release of Ca and Pi from the -DHiCa bone was not different than from +D radii. Comparing +D-Ca and -D-Ca groups with +D chicks, both were hypocalcemic with decreased bone weight, body weight and bone Ca release, while showing elevated lactate production and Pi release. The only difference between the +D-Ca and -D-Ca groups was a 50% decrease in Ca release by -D-Ca bone. The results suggest that in chicks: (1) some, but not all, of the effects of vitamin D deficiency on bone can be corrected by normalizing plasma Ca and (2) evaluation of the effects of vitamin D deficiency on bone may require hypocalcemia, since some responses are masked by normocalcemia.