Search PubMed⌕ Search

Biomedical subjects

T J Hahn

Publications and source records attributed to T J Hahn.

At least 37 records · Page 2Linked to original sources
PubMed1991

Insulin acutely suppresses parathyroid hormone second messenger generation in UMR-106-01 osteoblast-like cells: differential effects on phospholipase C and adenylate cyclase activation.

Insulin modifies the effects of PTH on osteoblast-like cells. However, the basis for this effect is unknown. In bone and kidney cells, the effects of PTH on cellular function are mediated by second messengers generated through both the phospholipase C and adenylate cyclase systems. Therefore, we examined the effects of insulin on PTH second messenger generation in UMR-106-01 rat osteoblastic osteosarcoma cells. PTH produced a rapid, transient increase in intracellular free calcium concentration ([Ca2+]i) which was maximal at 30 sec and was only minimally reduced in the absence of extracellular calcium. Inositol-triphosphate (IP3) production was increased in parallel. PTH stimulation of [Ca2+]i was concentration-dependent from 0.5-1,000 nM, with half-maximal stimulation at approximately 50 nM PTH. A 30-sec exposure to 50 nM PTH produced 32% and 23% increases in IP1 and IP3 production, respectively (both P less than 0.05). Although insulin alone did not significantly alter basal [Ca2+]i, a 1-min exposure to 1-100 nM insulin produced a concentration-dependent suppression of the PTH-stimulated transient increase in [Ca2+]i and IP3 generation. 100 nM insulin decreased 50 nM PTH stimulation of [Ca2+]i and IP3 levels by 84% (P less than 0.02) and 80% (P less than 0.001), respectively. Preexposure to insulin also decreased PTH stimulation of intracellular cAMP levels, but to a lesser degree. A 1-min exposure to 100 nM insulin produced a 32% (P less than 0.01) decrease in PTH-stimulated cAMP generation, but lower insulin concentrations were without significant effects. These results demonstrate that in UMR-106-01 cells, insulin suppresses PTH stimulation of second messengers generated through both the phospholipase C and adenylate cyclase systems, but has a more marked effect on the former.

Adenylyl Cyclases↗
PubMed1989

Characterization of insulin binding in the UMR-106 rat osteoblastic osteosarcoma cell.

The correlation of insulin receptor occupancy with classic insulin effects, such as stimulation of glucose uptake, have not been examined in osteoblastlike cells. Accordingly, we characterized insulin binding and examined its relationship to stimulation of glucose analog transport in the UMR-106 rat osteoblastic osteosarcoma cell line. Insulin binding in UMR-106 cells was found to be pH sensitive, temperature dependent, saturable, and specific. Proinsulin was 100-fold less effective than insulin in displacing specific [125I]insulin binding in these cells, whereas IGF-I at concentrations between 0.1 and 10 nM produced no displacement of [125I]insulin but did produce significant displacement of insulin binding at 100 and 1000 nM. Insulin receptor downregulation was observed after exposure to 100 nM insulin for 6 h at 37 degrees C and was temperature dependent. Insulin binding was reversible after 24 h at 4 degrees C. Insulin binding correlated directly with stimulation of 2-deoxyglucose uptake at insulin concentrations between 0.1 and 100 nM, with a half-maximal concentration (ED50) of 0.9 nM for both [125I]insulin binding displacement and stimulation of 2-deoxyglucose uptake. Hence, there was no evidence for spare insulin receptors with regard to stimulation of glucose analog transport. Scatchard analysis of insulin binding kinetics yielded a curvilinear plot, suggesting negative cooperativity. Analysis of insulin binding kinetics using a two-site model yielded a KD of 0.9 nM for the apparent high-affinity binding site and an estimated 80,000 high-affinity binding sites per cell. These findings demonstrate that osteoblastlike cells exhibit a relationship between insulin binding and glucose transport stimulation that is similar to that in liver cells and other insulin-sensitive tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Thyroid hormone 5'-deiodinase activity, nuclear binding, and effects on mitogenesis in UMR-106 osteoblastic osteosarcoma cells.

The hyperthyroid state in vivo is associated with an increase in osteoblast number and activity, suggesting that thyroid hormone may stimulate osteoblast replication and function. We therefore examined the effects of T3 (16-1170 pM) on replication rate as assessed by cell counts in UMR-106 osteoblastic osteosarcoma cells cultured for 5-10 days in medium supplemented with 10% hormone-stripped fetal calf serum (FCS). Despite the virtual absence of thyroid hormone in the control medium (total T3 concentration, 0.02 ng/ml), the addition of T3 in concentrations to 1000 pM did not increase the cell replication rate. At higher T3 concentrations, a slight decrease in growth rate was observed. No significant 5'-monodeiodinase activity was detected in UMR-106 cell homogenates. However, nuclear binding of T3 was demonstrated in intact cells. A high-affinity nuclear binding component was identified with a Ka of 2.6 x 10(10) M-1 and a maximum binding capacity of 7.7 pg T3 per mg DNA, equivalent to 51 binding sites per cell nucleus. A lower affinity nuclear T3 binding component with a Ka of 1.8 x 10(9) M-1 was also identified. Thus, despite the presence of nuclear T3 receptors, UMR-106 cells do not exhibit a mitogenic response to T3.

Alkaline Phosphatase↗
PubMed1989

Protein kinase C activity in UMR-106-01 cells: effects of parathyroid hormone and insulin.

The calcium and phospholipid-dependent protein kinase C (PKC) system appears to play an important role in mediating hormonal effects in various tissues including bone. Accordingly, we characterized PKC activity in the UMR-106-01 rat osteosarcoma osteoblastlike cell line and examined its hormonal regulation. UMR-106-01 cells were found to possess a classic, phorbol ester-activated PKC system, which was highly calcium and phospholipid dependent. A 30 s exposure to 10 nM bovine parathyroid hormone (PTH) (1-34) increased cytosolic and membrane-bound PKC activity by 12 and 157%, respectively, resulting in a 2.2-fold increase in the membrane-bound to cytosolic (MB/C) activity ratio (all p less than 0.01). The MB/C activity ratio was highest at 20 min, exhibiting a 2.8-fold increase over the control values (p less than 0.01). In contrast, 10 nM insulin increased cytosolic PKC activity but decreased membrane-bound activity, resulting in a 61% decrease in the MB/C activity ratio at 20 min (p less than 0.02). Moreover, insulin reduced PTH stimulation of the PKC activity ratio by 42 and 62% at 30 s and 20 min, respectively (p less than 0.02). Thus, PTH and insulin have opposing effects on the PKC activity ratio in UMR-106-01 cells.

Animals↗
PubMed1989

Glucose transport system in UMR-106-01 osteoblastic osteosarcoma cells: regulation by insulin.

Insulin is a potent stimulator of collagen synthesis and other osteoblastic cell functions. In various insulin-sensitive tissues, stimulation of glucose transport and glycolytic metabolism are hallmarks of insulin action and may play a role in insulin regulation of cellular function. However, the effects of insulin on glucose metabolism in osteoblast-like cells have not been defined. We therefore characterized 2-deoxy-D-glucose (2-DG) transport in UMR-106-01 rat osteoblastic osteosarcoma cells and examined its regulation by insulin. 2-DG (0.1 mM) uptake was shown to be linear with time over 45 minutes, temperature-sensitive, and inhibited by phloridzin. Competitive inhibition studies against other hexoses demonstrated a transport system stereospecificity for 2-DG similar to that previously demonstrated in fat and muscle cells. Kinetic analysis of 15 minute 2-DG uptake at 25 degrees C demonstrated a saturable transport mechanism with a Km (1.9 mM) similar to that observed for 2-DG transport in other tissues. Insulin stimulated 2-DG transport in a dose-related manner, with significant stimulation observed at 0.5 nM and maximal effect observed at 50 nM insulin. The stimulatory effect of insulin was reversibly inhibited by cytochalasin B (50 microM). Insulin stimulation of 2-DG transport was associated with a 1.7-fold increase in Vmax, while Km remained constant. When insulin effects on glucose transport were inhibited by the addition of 5 mM phloridzin, stimulatory effects on DNA and collagen synthesis were diminished, suggesting that stimulation of glucose transport may play a role in insulin effects on replication and function in osteoblast-like cells.

Animals↗
PubMed1988

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells.

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Glucose transport in osteoblast-enriched bone explants: characterization and insulin regulation.

Insulin has potent effects on osteoblast function both in vivo and in vitro. In various insulin-sensitive tissues, stimulation of glucose transport and metabolism are hallmarks of insulin action, and have been postulated to play a role in insulin regulation of cellular function. However, insulin effects on glucose metabolism in osteoblast-like cells have not been demonstrated. Therefore we examined the in vitro effects of insulin on hexose uptake in an osteoblast-enriched rat bone explant preparation. Uniform 5-mm-diameter punch sections were obtained from the cartilage-free frontal portions of the calvaria of 3-day-old rats, and the periosteum was removed. The resulting sections contained a highly enriched population of osteoblast-like cells as determined by histologic criteria, elimination of calcitonin-stimulatable cAMP generation, and enhancement of PTH-stimulatable cAMP generation per microgram of DNA. Sections were incubated for 24 hr at 37 degrees C in BGJb medium and then transferred to modified glucose-free Krebs-Ringer bicarbonate buffer for 2-deoxy-D-glucose (2-DG) uptake studies. 3H-2-DG uptake was linear with time over 60 min, temperature sensitive, and inhibited by 5 mM phloridzin. Kinetic analysis of 2-DG uptake at 25 degrees C demonstrated a saturable transport mechanism with a Km of 2.2 mM, similar to that observed for 2-DG transport in other tissues. Studies of competitive inhibition by other sugars demonstrated a transport specificity for 2-DG that was comparable to that previously observed in fat and muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1988

Insulin and glucose regulation of glycogen synthase in rat calvarial osteoblastlike cells.

Insulin has been shown to stimulate collagen, noncollagen protein and nucleic acid synthesis in bone cells in vitro. However, the effects of insulin on intermediary carbohydrate metabolism in osteoblasts, and in particular on the key regulatory enzyme glycogen synthase, have not been directly examined. Accordingly, we developed a microassay for glycogen synthase (GS) to examine insulin and glucose regulation of this enzyme in cultured osteoblastlike cells. In osteoblast-enriched rat calvarial bone explants incubated for 24 hours in 5 mM glucose, insulin (0.1-100 nM) produced a dose-related stimulation of GSa (glucose-6-phosphate independent GS activity). The insulin-stimulated increase in GSa ranged from a 30% increase in the presence of 0.1 nM insulin to a 163% increase produced by 100 nM insulin, both significant at P less than 0.01. In contrast, GSb (glucose-6-phosphate dependent GS activity) was significantly increased only at a supraphysiologic insulin concentration (100 nM). The GS activity ratio (GSa/GSb) increased with insulin concentration from 0.1-100 nM. Basal values for GSa and GSb activity did not differ between explants incubated in 5mM or 15 mM glucose for 24 hours. However, incubation in the presence of 15 mM glucose blunted the insulin-stimulated increase in GSa activity, with 100 nM insulin producing only a 75% increase in GSa activity. In contrast, maximal insulin-stimulated GSb levels were not affected by high glucose medium. In explants incubated in glucose-free medium, basal GSa activity was significantly greater than in the presence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels.

Changes in free cytosolic calcium were measured in UMR-106 cells in response to parathyroid hormone (PTH) stimulation. Bovine PTH-(1-34) induced an increase in [Ca2+]i with the contour of the rise in [Ca2+]i occurring in three successive phases: a rapid increase in [Ca2+]i occurring within seconds, rapid decrement in [Ca2+]i to near-resting levels within 1 min, and slow increment in [Ca2+]i. Phase one and phase three increases in [Ca2+]i were dependent on medium calcium. The phase one rise in [Ca2+]i was inhibitable by the calcium channel blockers lanthanum and verapamil. Only the phase one rise in [Ca2+]i was blocked by preincubation of the cells with the phorbol ester, phorbol 12-myristate 13-acetate. This channel was also blocked when cellular cAMP levels were increased prior to PTH stimulation. The phase two decrement of [Ca2+]i was due to the rapid inactivation of the phase one calcium channel. The phase three rise in [Ca2+]i was mediated by cellular cAMP levels. This cAMP-dependent Ca2+ channel was insensitive to pretreatment of the cells with phorbol diesters and showed low sensitivity to Ca2+ channel blockers. It is concluded that UMR-106 cells respond to PTH stimulation by the activation of a cAMP-independent Ca2+ channel. This channel rapidly inactivates. The subsequent PTH-dependent increase in cellular cAMP is followed by activation of a cAMP-dependent Ca2+ channel resulting in a slow rise in [Ca2+]i.

Animals↗
PubMed1985

Evaluation of factors associated with glucocorticoid-induced osteopenia in patients with rheumatic diseases.

In 161 ambulatory rheumatic disease patients receiving long-term prednisone therapy, diaphyseal mass (DM) and metaphyseal mass (MM) of the forearm were measured by single photon absorptiometry, and bone radiographs were reviewed when available. Multivariate analysis of treatment and patient characteristics demonstrated that glucocorticoid-induced osteopenia (defined as an elevated DM:MM ratio) and bone fractures occurred with similar frequency in patients of each sex, in whites and blacks, in patients with various rheumatic diseases, and in patients receiving different regimens of prednisone therapy. However, large cumulative doses of prednisone were associated with elevated DM:MM ratios as well as with bone fractures, and menopause or age greater than or equal to 50 years (males or females) was associated with bone fractures. We conclude that long-term therapy with various prednisone regimens results in glucocorticoid-induced osteopenia and fractures. This affect is cumulative, occurs in all patient groups, and results in more bone fractures in certain groups.

Bone and Bones↗
PubMed1984

Effect of oral 1,25-dihydroxyvitamin D and calcium on glucocorticoid-induced osteopenia in patients with rheumatic diseases.

Twenty-three rheumatic disease patients with glucocorticoid-induced osteopenia (defined by measurement of forearm bone mass) completed an 18-month double-blind, randomized study to assess the effect of oral calcium and 1,25-dihydroxyvitamin D (1,25-OH2D) or calcium and placebo on bone and mineral metabolism. Intestinal 47Ca absorption was increased (P less than 0.05) and serum parathyroid hormone levels were suppressed (P less than 0.01) by 1,25-OH2D (mean dose 0.4 micrograms/day); however, no significant gain in forearm bone mass occurred, and bone fractures were frequent in both groups. In the 1,25-OH2D group, histomorphometric analysis of iliac crest biopsy specimens demonstrated a decrease in osteoclasts/mm2 of trabecular bone (P less than 0.05) and parameters of osteoblastic activity (P less than 0.05), indicating that 1,25-OH2D reduced both bone resorption and formation. We conclude that 1,25-OH2D should not be used for treatment of glucocorticoid-induced osteopenia. Since patients receiving calcium and placebo did not exhibit a loss of forearm bone mass, elemental calcium supplementation of 500 mg daily might be useful to maintain skeletal mass in patients receiving long-term glucocorticord therapy.

Administration, Oral↗
PubMed1984

Cortisol modulation of osteoblast metabolic activity in cultured neonatal rat bone.

The effects of cortisol on basal levels of indices of osteoblast metabolic activity and on PTH regulation of osteoblast activity in vitro were examined in intact bone preparations from neonatal rat calvaria. Uniform punch sections from the frontal portion of calvaria of 3-day-old rats were cultured for 24 h at 37 C in modified BGJb medium. When bone sections were incubated in medium supplemented with cortisol (100 nM) for 24 h, indices of osteoblast metabolic activity, expressed both per total bone section and per micrograms bone DNA, were significantly increased relative to control values. Expressed per micrograms DNA, the following percentage increases were observed in cortisol-treated cultures: alkaline phosphatase activity, +22% (P less than 0.02); [3H] collagen synthesis, +41% (P less than 0.001); and [14C]citrate decarboxylation, + 108% (P less than 0.001). Total DNA per bone section after 24 h was increased by 18% (P less than 0.01), and [3H]thymidine incorporation at 24 h was increased by 26% (P less than 0.01) relative to control values. Stimulation by cortisol occurred in a dose-related manner over concentrations from 1 nM to 1 microM. The stimulatory effects of cortisol were first seen after 6 h of exposure and increased steadily through 24 h of exposure. Incubation in the presence of PTH-(1-34) (100 ng/ml) resulted in significant decrease in alkaline phosphatase activity, collagen synthesis, and citrate decarboxylation after 24 h of exposure (P less than 0.001). The relative order of sensitivity to PTH suppression was identical to the relative sensitivity to cortisol stimulation. In the presence of cortisol (100 nM), the suppressive effect of PTH on all three indices was increased significantly by a factor of 2- to 4-fold. It is concluded that in intact cultured bone, physiological concentrations of cortisol produce both an initial enhancement of indices of osteoblast metabolism and increased osteoblast sensitivity to regulation by PTH.

Alkaline Phosphatase↗
PubMed1982

Comparison of parathyroid hormone and calcium ionophore A23187 effects on bone resorption and nucleic acid synthesis in cultured fetal rat bone.

It has recently been demonstrated that calcium ionophore A23187 mimics certain of the effects of parathyroid hormone (PTH) on bone in vitro, including stimulation of 45Ca release and cAMP formation. To further examine the relative effects of these two agents on bone cell metabolism, we compared the effects of synthetic PTH 1-34 (50 ng/ml) and calcium ionophore A23187 (0.5 micrograms/ml) on 45Ca release, DNA concentration, and nucleic acid synthesis in fetal rat forelimb rudiments cultured for periods up to 120 h. Both agents stimulated 45Ca release; however, the effects of PTH were apparent after a shorter period of exposure. Bone DNA concentration (expressed as microgram DNA/mg bone) was not affected by PTH but was significantly increased relative to control values by exposure to A23187 for 8-120 h of incubation. PTH increased the incorporation of 3H-thymidine into DNA at 30 and 48 h, and increased the incorporation of 14C-uridine into RNA at 48 h, time points which corresponded to a period of accelerated PTH stimulation of 45Ca release. In contrast, 3H-thymidine and 14C-uridine incorporation were both uniformly suppressed by A23187 at all time points examined. Thus the increased DNA concentration observed in A23187-treated rudiments appeared to be the result of a decreased rate of bone maturation and mineralization. The markedly different patterns of nucleic acid synthesis in response to PTH and A23187 suggest that these agents differ significantly in their mechanisms of action on bone cell metabolism.

Animals↗
PubMed1981

Bone loss in adults receiving alternate day glucocorticoid therapy. A comparison with daily therapy.

Bone mineral content was measured by photon absorption densitometry in 25 patients with rheumatic diseases receiving glucocorticoids on an alternate day treatment schedule, and in 25 age-, sex-, and race-matched patients receiving daily steroid therapy. Mean values for cortical (diaphyseal) mass, trabecular (metaphyseal) mass, and the cortical/trabecular mass ratios were not different in the two groups. Steroid-induced osteopenia, defined as an elevated ratio of cortical to trabecular mass, occurred in both therapeutic regimens (8 of 25 on alternate day; 11 of 25 on daily). Therefore, bone loss was demonstrable in individuals on alternate day regimens. Serum levels of calcium, ionized calcium, alkaline phosphatase, and parathyroid hormone were similar in the two groups.

Black or African American↗
PubMed1981

Mixed-sclerosing-bone-dystrophy: report of a case and review of the literature.

We present clinical, laboratory, radiologic, genetic, and pathologic findings in a 49-year-old man with mixed-sclerosing-bone-dystrophy (MSBD), review the six cases previously reported as "MSBD", and examine the nosology of this rare bone dysplasia. Our asymptomatic patient showed radiographic changes consistent with osteopoikilosis, osteopathia striata, and melorheostosis and had widespread osteosclerosis of the axial skeleton. Several previous reports of combined osteosclerotic disorders suggest the latter finding represents osteopetrosis, however, histologic examination of our patient's iliac crest excluded that diagnosis. Limited radiographic surveys of his eight children were unremarkable except for isolated bone islands in two sons. Literature review revealed that "MSBD" has actually been used generically to describe the association of a variety of osteosclerotic bone dysplasias when they occur together in a single patient.

Bone Diseases↗