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Biomedical subjects

W K Ramp

Publications and source records attributed to W K Ramp.

At least 37 records · Page 2Linked to original sources
PubMed1992

Inhibition of cell metabolism by a smokeless tobacco extract: tissue and species specificity.

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.

Alkaline Phosphatase↗
PubMed1991

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.

Alkaline Phosphatase↗
PubMed1991

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 2,2'dipyridyl 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, 2,2'dipyridyl, 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 2,2'dipyridyl, inhibits the hydroxylation of proline and the synthesis of collagenase-digestible protein.

2,2'-Dipyridyl↗
PubMed1991

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

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 [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/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 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.

Alkaline Phosphatase↗
PubMed1990

Electrical stimulation of bone and its implications for endosseous dental implantation.

Applied electrical potentials can alter cellular movement, stimulate production or destruction of cells, and change the chemical concentration and composition of both soft tissue and bone. These actions vary depending on the microamperage and duration of the applied current and whether it is continuous or pulsed, the type of metallic electrode, and the types of cells or tissues involved. Relative to skeletal effects, researchers have accumulated data from bone cell cultures, embryonic and adult animal bone, and human clinical studies. This article reviews the historical use of electric current for fracture healing, the piezoelectric effect found in tissues, and the possible deleterious effects of electrical stimulation. The types of electric current presently used for treating extremity fractures and reports where electric current has been used on tissues in the oral cavity are discussed. Alternate sources of energy to stimulate bone and possible implications for use of electrical stimulation to augment attachment of endosseous dental implants are also included.

Bone Regeneration↗
PubMed1989

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.

Animals↗
PubMed1988

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.

Adult↗
PubMed1988

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 Ringer's 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 Ringer's 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.

Animals↗
PubMed1987

Hydrogen peroxide inhibits glucose metabolism and collagen synthesis in bone.

Effects of H2O2 on bone were evaluated in an organ culture system. Tibiae from chick embryos were incubated for up to 3 days in culture medium containing 0.07 to 20 mM H2O2. Glucose metabolism was monitored by measuring lactate production and oxygen consumption, and collagen synthesis was determined by hydroxylation of proline. In addition to markedly inhibiting these parameters, H2O2 also decreased bone weight and alkaline phosphatase activity. Multiple exposures to H2O2 were somewhat more effective than a single exposure. Since H2O2 inhibits bone at low concentrations in vitro, the results suggest that the potential for harmful effects of H2O2 in the oral cavity should be investigated.

Animals↗
PubMed1986

Effects of vitamin D deprivation and 1,25-dihydroxyvitamin D3 treatment on ion release from rat tibiae in vitro.

Skeletal effects of dietary vitamin D and of 1,25-dihydroxyvitamin D3[1,25(OH)2D3] in vivo and in vitro were investigated using bones from vitamin D-deficient suckling rats. Tibiae from 19- to 21-day-old pups were incubated for up to 8 h in a defined medium and the net releases of Ca, inorganic phosphate (Pi) and Mg into and from the bones were determined. The pups were delivered and nursed by mothers fed either a diet containing vitamin D3 (+D) or a D-free diet (-D) starting on the sixth day of pregnancy. Effects of metabolic inhibition with iodoacetic acid (IAA, 1 mM); of elevated medium Pi concentration (2 mM); and of 1,25(OH)2D3 in vitro (2 and 10 ng/ml of medium) or in vivo (500 ng/mother ip, 2x) were evaluated. Although no effects of vitamin D on glucose metabolism or net Pi release were seen, tibiae from -D pups showed a marked decrease in net Ca release (up to 70%), while net Mg release was increased by 27%. IAA completely inhibited net Ca release in this system, had no effect on net Pi release, and reduced lactate production and net Mg release by 86 and 33%, respectively. Elevating the medium Pi concentration from 1 to 2 mM did not affect the differences between +D and -D bones. Treatment with 1,25(OH)2D3 in vitro and in vivo returned ion releases by the -D tibiae partly or completely to the +D levels. The results support the following conclusions: (1) vitamin D deficiency during suckling reduces net Ca release and increases net Mg release from bone which may contribute to the changes in circulating concentrations of these ions seen in D deficiency; (2) these effects on bone appear to be specific for Ca and Mg, since the vitamin has little or no effect on net Pi release; (3) net Ca and Mg release from rat bone in vitro presumably are energy-dependent processes, while net Pi release is not; and (4) 1,25(OH)2D3 can affect net Ca and Mg release from -D bones by a direct action.

Animals↗
PubMed1985

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

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.

Alkaline Phosphatase↗
PubMed1984

Inhibition of net calcium efflux from bone by ethanol in vitro.

Ethanol administered to animals is known to cause hypocalcemia. This investigation was undertaken to evaluate direct early effects of ethanol on the net fluxes of Ca2+ and inorganic phosphate (Pi) into or out of bone using tibiae from 13-day-old chick embryos and 8-day-old suckling rats in vitro. When chick bones were incubated with ethanol in the medium (10 microliters/ml), net Ca2+ efflux was decreased 19, 22, 27, and 31% at 2, 4, 6, and 8 h, respectively. Metabolically inhibited bones showed a net influx rather than efflux of Ca2+ and were not further affected by ethanol. The ethanol had no consistent effect on net Pi efflux and slightly reduced lactate production. At three doses of ethanol (3, 10, and 30 microliters/ml of medium) the reduction of Ca2+ efflux was dose related in both chick and rat bones. Ethanol completely inhibited parathyroid hormone-stimulated, net Ca2+ efflux from chick bones, even at ethanol levels that by themselves did not affect Ca2+ efflux. These findings are consistent with the hypothesis that the hypocalcemia in ethanol-treated animals may be due in part to inhibition of net Ca2+ efflux from bone.

Animals↗
PubMed1982

Magnesium promotes both parathyroid hormone secretion and adenosine 3',5'-monophosphate production in rat parathyroid tissues and reverses the inhibitory effects of calcium on adenylate cyclase.

Reduced extracellular Ca2+ is known to promote PTH secretion, while severe Mg2+ depletion has the opposite effect. We have correlated the effects of Mg2+ and Ca2+ on parathyroid hormone (PTH) secretion and cAMP accumulation by rat parathyroid tissues in vitro with the effects of these two metals on adenylate cyclase activity in broken membrane preparations. PTH secretion was maximal at 0.5 mM Ca2+, falling to low levels as the Ca2+ concentration was increased to 2.5 mM. Deletion of Mg2+ from the medium resulted in a marked decrease in PTH secretion at any given Ca2+ concentration. At a constant Ca2+ concentration of 1 mM, both PTH secretion and cAMP production rose to maximal rates as the Mg2+ concentration was increased from 0 to 2 mM. The adenylate cyclase of rat parathyroid membranes was stimulated by both GTP and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p]. EDTA-treated membranes could not be stimulated by Gpp(NH)p. Repletion with Mg2+ was more effective than repletion with Ca2+ in restoring responsiveness to the guanine nucleotide. When membranes were maximally preactivated by Gpp(NH)p and then assayed in the presence of variable concentrations of metal ions, enzyme activity was directly inhibited by Ca2+ and stimulated by Mg2+. Adenylate cyclase sensitivity to Ca2+ inhibition was dependent upon the Mg2+ concentration; in the presence of 0.6 mM Mg2+ a 50% inhibition was produced by 0.05 mM Ca2+, while in the presence of 8 mM Mg2+ a 10-fold higher Ca2+ concentration was required for a similar inhibitory effect. The results suggest that Ca2+ may decrease PTH secretion at least in part by a direct inhibition of adenylate cyclase. Mg2+ may promote PTH secretion either by enhancing the activation of adenylate cyclase by endogenous guanine nucleotides or by competing with Ca2+ for binding to a distinct regulatory site on the enzyme.

Adenylyl Cyclase Inhibitors↗
PubMed1981

Hypocalcemic, hypophosphatemic rickets in rat pups suckling vitamin D-deprived mothers.

The effects of maternal vitamin D deprivation on rat pups were examined in terms of serum levels of Ca, phosphorus (P), and 25-hydroxyvitamin D (250HD), ash content, and histological appearance of long bones. In each of 10 experiments, litters of 8--10 young were examined at 2 or 3 different ages within the range of 5--57 days. Pregnant mothers were fed a diet containing 0.4% Ca, and 0.4% P and either no vitamin D(--D) or 5 IU D3/g diet (+D). Mothers fed the --D diet developed marked hypocalcemia (4--5 mg/dl) during lactation, but no change in milk Ca or P. The earliest changes in pups suckled by mothers on --D diets compared to pups suckled by mothers on +D diets were significant reductions at 8 days in serum P (6--12% in 3 of 5 experiments) and in serum 250HD3. By the 15th day, serum 250HD3 was undetectable, body weight was reduced by 26%, serum Ca was reduced by 9%, and serum P was reduced by 15%. During the following 10 days, when gradual weaning took place, serum Ca decreased progressively, serum P returned to normal, serum Mg became elevated, bone ash (as percent of dry weight of tibia) was decreased, and decalcified sections of the proximal end of the tibia revealed irregularity and widening (1.5- to 3-fold) of the hypertrophic cartilage layer and the metaphyseal trabeculae. All of these changes were also prominent in 8-week-old --D rats. The results indicate that rat pups suckling vitamin D-deprived mothers can develop biochemical evidence (including hypophosphatemia) as well as histological evidence of vitamin D deficiency similar to that of human vitamin D deficiency rickets. This is in contrast to rats deprived of vitamin D after weaning, who do not develop hypophosphatemia or characteristic histological evidence of rickets unless deprived of P as well as vitamin D.

25-Hydroxyvitamin D 2↗