RESUMO
Hypocalcemia and resistance to exogenous parathyroid hormone have been reported in several clinical states associated with magnesium deficiency. On the basis of such observations, it has been suggested that magnesium depletion per se may result in impaired responsiveness of the adenyl cyclase-adenosine 3',5'-monophosphate (3',5'-AMP) system. To test this hypothesis, 4 wk old male parathyroidectomized rats were maintained on normal or magnesium-deficient diets for 4 wk and their responses to parathyroid hormone compared. Serum magnesium and calcium fell progressively in the magnesium-deficient group. Despite clinical and biochemical evidence of severe magnesium deficiency in these animals, renal production and excretion of 3',5'-AMP in response to parathyroid hormone was normal both in vitro and in vivo. Additionally, administration of either dibutyryl 3',5'-AMP or parathyroid extract to fasting magnesium-depleted rats produced a normal increase in serum calcium. Parathyroid hormone infusion studies demonstrated normal renal and skeletal responsiveness as measured by urinary excretion of calcium, magnesium, phosphate, and hydroxyproline. These data show that the effect of parathyroid hormone on 3',5'-AMP formation and excretion, the responsiveness of skeletal tissue to 3',5'-AMP, and the renal and skeletal system responses to parathyroid hormone are not altered by pure magnesium deficiency in the parathyroidectomized rat.
Assuntos
Deficiência de Magnésio/fisiopatologia , Hormônio Paratireóideo/farmacologia , Monofosfato de Adenosina/urina , Animais , Osso e Ossos/efeitos dos fármacos , Cálcio/sangue , Cálcio/urina , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Fêmur , Nucleotídeos de Guanina/urina , Hidroxiprolina/urina , Rim/metabolismo , Magnésio/sangue , Magnésio/urina , Masculino , Glândulas Paratireoides/cirurgia , Fosfatos/sangue , Fosfatos/urina , RatosRESUMO
Parameters of mineral and bone metabolism were studied in 17 patients treated chronically with supraphysiologic doses of glucocorticoids. When compared to 15 matched normal subjects, the patient group exhibited similar serum 25-hydroxyvitamin D (25-OHD) levels, decreased intestinal 47Ca absorption, increased serum immunoreactive parathyroid hormone, and decreased forearm bone mass. Iliac crest bone biopsies revealed a decreased bone formation rate and increased osteoclast number. Treatment with 25-OHD (mean dose 4.03 micrograms/d) and calcium (500 mg/d) in nine patients produced a 46% increase in 47Ca absorption (P less than 0.001) and a 54% decrease in serum immunoreactive parathyroid hormone (P less than 0.001) by 3 mo. In addition, by 12 mo the treatment group exhibited (a) a 13.2 +/- 5.1% increase in metaphyseal (P less than 0.001) and a 2.1 +/- 0.4% increase in diaphyseal (P less than 0.05) forearm bone mass, and (b) significant decreases in cortical and endosteal osteoclast number. Biochemical and bone mass changes persisted through 18 mo. No significant changes in any parameter occurred in eight control patients administered calcium 100 mg/d. It is concluded that treatment with 25-OHD and calcium can significantly improve parameters of mineral and bone metabolism in patients with glucocorticoid-induced osteopenia.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/uso terapêutico , Reabsorção Óssea/metabolismo , Cálcio/metabolismo , Glucocorticoides/efeitos adversos , Esteroide Hidroxilases/uso terapêutico , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/sangue , Reabsorção Óssea/sangue , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cálcio/sangue , Cálcio/uso terapêutico , Feminino , Humanos , Absorção Intestinal , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/imunologiaRESUMO
The metabolic fate of intravenously injected vitamin D(3)-1,2-(3)H (D(3)-(3)H) was studied in two normal individuals on chronic phenobarbital therapy. Silicic acid column chromatography of lipid-soluble plasma extracts obtained serially for 96 hr after D(3)-(3)H injection demonstrated a decreased plasma D(3)-(3)H half-life and increased conversion to more polar metabolites. The polar metabolites formed included several with chromatographic mobility similar to known biologically inactive vitamin D metabolites and one with chromatographic mobility identical to 25-hydroxycholecalciferol. Disappearance of this latter material was also accelerated. A child with rickets and a normal volunteer studied before and after a 2 wk course of phenobarbital therapy demonstrated similar alterations in D(3)-(3)H metabolism. When liver microsomes from 3-wk-old Sprague-Dawley rats treated with phenobarbital were incubated with D(3)-(3)H, polar metabolites were produced with chromatographic mobility similar to the plasma D(3)-(3)H metabolites from phenobarbital-treated humans. Similar incubations employing 25-hydroxy-cholecalciferol-26-27-(3)H as the substrate also demonstrated an increased conversion to polar metabolites. The data suggest that the reported increased incidence of osteomalacia observed in patients on chronic anticonvulsant therapy may be the result of an accelerated conversion of vitamin D and its active metabolite, 25-hydroxycholecalciferol, to polar metabolites by druginduced liver microsomal enzymes.
Assuntos
Colecalciferol/metabolismo , Fenobarbital/farmacologia , Adulto , Animais , Anticonvulsivantes/efeitos adversos , Colecalciferol/sangue , Cromatografia em Camada Fina , Indução Enzimática , Feminino , Meia-Vida , Humanos , Hidroxicolecalciferóis/metabolismo , Taxa de Depuração Metabólica , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , Oxigenases de Função Mista/metabolismo , Osteomalacia/induzido quimicamente , Fenobarbital/uso terapêutico , Fenitoína/farmacologia , Ratos , TrítioRESUMO
Chronic administration of high doses of anticonvulsant drugs frequently produces classic osteomalacia with bone histologic changes characteristic of increased parathyroid hormone (PTH) effect in man. However, several reports have documented defects in calcified tissue metabolism suggestive of an end-organ resistance to PTH after chronic anticonvulsant drug therapy. To examine the direct action of anticonvulsant drugs on bone resorption, we investigated the effects of diphenylhydantoin (phenytoin) (DPH) (100-200 mug/ml) and phenobarbital (10-400 mug/ml) on basal and hormonally mediated resorption 5-day cultures of fetal rat forelimb rudiments. In this system both drugs significantly inhibited basal and PTH-stimulated (45)Ca and [(3)H]hydroxyproline release, as well as 1,25-dihydroxyvitamin D(3)-stimulated (45)Ca release. The effects of DPH and phenobarbital were additive, with DPH exhibiting a several-fold more potent inhibitory effect than phenobarbital. Whereas DPH exhibited a striking synergism with the inhibitory effects of human calcitonin (HCT) on PTH-induced resorption, the effect of phenobarbital was merely additive to that of HCT. PTH and PTH plus HCT-induced increases in bone cyclic AMP (cAMP) content were significantly inhibited by DPH but not by phenobarbital. However, in contrast to effects on (45)Ca release, DPH inhibition of cAMP generation was not accentuated in the presence of HCT. It is concluded that: (a) both DPH and phenobarbital can directly inhibit basal and hormonally stimulated bone resorption, with DPH being much more potent in this regard; (b) DPH appears to inhibit bone resorption via a cAMP-independent mechanism and has an additional suppressive effect on PTH-induced cAMP generation; and (c) the synergistic interaction of DPH and HCT in inhibiting (45)Ca release occurs at a site independent of cAMP generation.
Assuntos
Reabsorção Óssea/efeitos dos fármacos , Hormônios/farmacologia , Fenobarbital/farmacologia , Fenitoína/farmacologia , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Calcitonina/farmacologia , Cálcio/metabolismo , AMP Cíclico/biossíntese , Di-Hidroxicolecalciferóis/antagonistas & inibidores , Sinergismo Farmacológico , Técnicas In Vitro , Hormônio Paratireóideo/antagonistas & inibidores , RatosRESUMO
The effect of dietary calcium intake on calcium metabolism was studied in eight normal volunteers by multicompartmental analysis of radiocalcium and balance data. In paired studies of six normal subjects on normal and high or low calcium intakes, necessary and sufficient criteria were used to determine changes in calcium metabolic parameters produced by alterations in dietary calcium. These changes involved gastrointestinal calcium absorption rate, renal and endogenous fecal rate constants, and bone resorption rate. Bone accretion rate and compartment sizes need not change between the paired studies. The changes of parameters involving kidney, gut, and bone were in a direction to support calcium homeostasis and were compatible with the pattern of changes produced by parathyroid hormone. However, the source of the stimulus for hormone secretion was not apparent since plasma calcium concentrations showed no significant difference between paired studies. The implications of these findings relative to control of hormone secretion, calcium regulatory mechanisms, and metabolic bone disease are discussed.
Assuntos
Cálcio da Dieta , Cálcio/metabolismo , Reabsorção Óssea , Cálcio/urina , Isótopos de Cálcio , Fezes/análise , Homeostase , Humanos , Absorção Intestinal , FósforoRESUMO
Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD3) and 1, 25-dihydroxycholecalciferol (1,25-(OH)2D3). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD3-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport alpha-globulin. A cathodal, 1, 25-(OH)2D3-binding protein, which sedimented at 3-4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.
Assuntos
Proteínas de Transporte/metabolismo , Di-Hidroxicolecalciferóis/metabolismo , Hidroxicolecalciferóis/metabolismo , Adenoma/metabolismo , Proteínas de Transporte/isolamento & purificação , Eritrócitos/metabolismo , Humanos , Linfócitos/metabolismo , Músculos/metabolismo , Especificidade de Órgãos , Neoplasias das Paratireoides/metabolismoRESUMO
High levels of interleukin-6 (IL-6) have been detected in synovial fluid from patients with inflammatory arthropathies associated with local bone resorption, suggesting a role for IL-6 as a local regulator of bone resorption and remodeling. In the present study we examined the effects of IL-6 on [3H]thymidine ([3H]TdR) incorporation, collagen synthesis, and alkaline phosphatase activity in UMR-106-01 rat osteoblastic osteosarcoma cells. IL-6 stimulated a dose-dependent increase in [3H]TdR incorporation that was maximal at 1000 U/ml (-147% of basal, p less than 0.005) in osteoblastlike cells that were in a logarithmic phase of growth. The increase in [3H]TdR incorporation was maximal between 12 and 24 h and was neutralized by pretreatment with the polyclonal rabbit antibody to IL-6. IL-6 also increased cell number and the secretion of prostaglandin E2 in UMR-106-01 cells in logarithmic growth phase. The stimulation of [3H]TdR incorporation and release of PGE2 into the culture medium by IL-6 was inhibited by indomethacin. A 24 h exposure of the osteoblastlike cells to 1000 U/ml of IL-6 reduced [3H]proline incorporation into collagenase-digestible (CDP) protein to 73% of control values (p less than 0.01). Noncollagen protein (NCP) synthesis was inhibited to 80% of control values (p less than 0.01) by 1000 U/ml of IL-6. The inhibitory effect was relatively greater on CDP than on NCP and consequently resulted in a decrease in the percentage of collagen synthesis. Alkaline phosphatase activity was not altered in these cells after a 24 h exposure to 1-1000 U/ml of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Interleucina-6/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Colágeno/biossíntese , Dinoprostona/metabolismo , Indometacina/farmacologia , Osteoblastos/metabolismo , Fenótipo , Proteínas/metabolismo , Ratos , Timidina/metabolismo , Trítio , Células Tumorais CultivadasRESUMO
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.
Assuntos
Insulina/farmacologia , Osteoblastos/enzimologia , Hormônio Paratireóideo/farmacologia , Proteína Quinase C/metabolismo , Animais , Membrana Celular/enzimologia , Citosol/enzimologia , Osteoblastos/efeitos dos fármacos , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
UMR-106-01 osteoblast-like cells respond to high concentrations of parathyroid hormone (PTH) in vitro by decreasing thymidine incorporation, a marker of DNA synthesis and cell proliferation. This response is different from in vivo conditions, such as primary and secondary hyperparathyroidism, in which high PTH levels are associated with an increased number of osteoblasts. When the response of UMR-106-01 cells to PTH is evaluated in vitro, however, these cells are exposed to only a single hormone. The present study was designed to evaluate the combined effects of two hormones, PTH and insulin, on the DNA synthesis of UMR-106-01 cells. PTH is known to decrease and insulin to increase thymidine incorporation by UMR-106-01 cells. To examine the interaction of these hormones, acute studies, defined as a 24 h exposure to hormone, and chronic studies, defined as a 7 day exposure to hormone, were performed. Both acute and chronic exposure to 10(-9) M PTH decreased thymidine incorporation by UMR-106-01 cells, with suppression ranging from 27 to 81% (P < 0.05). Both acute and chronic exposure to 10(-8) M insulin (INS) increased thymidine incorporation by UMR-106-01 cells; this ranged from 26 to 58% (P < 0.05). However, chronic exposure to 10(-9) M PTH followed by an acute exposure to 10(-8) M INS resulted in a 710% increase in thymidine incorporation (P < 0.01). Reversing the sequence by chronically exposing UMR-106-01 cells to 10(-8) M INS followed by acute exposure to 10(-9) M PTH resulted in a 53% decrease in thymidine incorporation (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
DNA/biossíntese , Insulina/farmacologia , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Análise de Variância , Animais , Divisão Celular/efeitos dos fármacos , Osteoblastos/metabolismo , Ratos , Timidina/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Iodeto Peroxidase/metabolismo , Osteoblastos/fisiologia , Osteossarcoma/patologia , Tri-Iodotironina/farmacologia , Fosfatase Alcalina/metabolismo , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Osteoblastos/enzimologia , Osteossarcoma/enzimologia , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/metabolismo , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/patologiaRESUMO
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)
Assuntos
Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Receptor de Insulina/metabolismo , Animais , Glucose/análogos & derivados , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Ratos , Temperatura , Células Tumorais Cultivadas/metabolismoRESUMO
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)
Assuntos
Osso e Ossos/metabolismo , Desoxiaçúcares/metabolismo , Desoxiglucose/metabolismo , Insulina/farmacologia , Osteoblastos/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Animais Recém-Nascidos , Transporte Biológico Ativo/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Calcitonina/farmacologia , AMP Cíclico/metabolismo , Cinética , Monossacarídeos/farmacologia , Técnicas de Cultura de Órgãos , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Florizina/farmacologia , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
Osteoblasts arise from partially differentiated osteogenic progenitor cells (OPCs) which in turn arise from undifferentiated marrow stromal mesenchymal stem cells (MSCs). It has been postulated that age-related defects in osteoblast number and function may be due to quantitative and qualitative stem cell defects. To examine this possibility, we compared osteogenic stem cell number and in vitro function in marrow cells from 4-month-old and 24-month-old male BALB/c mice. Histologic studies demonstrated that these mice undergo age-related bone loss resembling that seen in humans. In primary MSC cultures grown in media supplemented with 10 nM dexamethasone, cultures from older animals yielded an average of 41% fewer OPC colonies per given number of marrow cells plated (p < 0.001). This implies that for a given number of marrow cells there are fewer stem cells with osteogenic potential in older animals than there are in younger animals. The basal proliferative rate in cultures from older animals, as measured by 3H-thymidine uptake, was more than three times that observed in cultures from young animals (p < 0.005). However, the increase in proliferative response to serum stimulation was 10-fold in the younger cultures (p <0.001) and insignificant (p <0.4) in the older cultures. Colonies in both age groups became alkaline phosphatase positive at the same rate, and virtually all colonies were positive after 12 days of culture. Cultures from both age groups produced abundant type I collagen. These studies suggest that defects in the number and proliferative potential of MSCs may underlie age-related defects in osteoblast number and function.
Assuntos
Envelhecimento/fisiologia , Osteoblastos/fisiologia , Osteoporose , Células-Tronco/fisiologia , Animais , Contagem de Células , Divisão Celular , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco/patologiaRESUMO
Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with > 80% type I collagen and < 5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%-0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.
Assuntos
Cartilagem/metabolismo , Regulação da Expressão Gênica/fisiologia , Células-Tronco/metabolismo , Animais , Cartilagem/citologia , Cartilagem/ultraestrutura , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Transformada , Colágeno/biossíntese , Pró-Colágeno/genética , RNA Mensageiro/biossíntese , Ratos , Receptores de Hormônios Paratireóideos/análise , Células-Tronco/citologiaRESUMO
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.
Assuntos
Adenilil Ciclases/metabolismo , Insulina/farmacologia , Osteoblastos/enzimologia , Hormônio Paratireóideo/farmacologia , Sistemas do Segundo Mensageiro , Fosfolipases Tipo C/metabolismo , Animais , Cálcio/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Fosfatos de Inositol/biossíntese , Nifedipino/farmacologia , Osteossarcoma , Ratos , Células Tumorais CultivadasRESUMO
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.
Assuntos
Hidrocortisona/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Citratos/metabolismo , Ácido Cítrico , Colágeno/biossíntese , DNA/metabolismo , Descarboxilação , Tamanho do Órgão/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.
Assuntos
Dinoprostona/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Genes fos/genética , Genes jun/genética , Proteínas Imediatamente Precoces , Mitose/efeitos dos fármacos , Osteoblastos/patologia , Hormônio Paratireóideo/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias Ósseas , Cálcio/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Fosfatos de Inositol/metabolismo , Mitose/fisiologia , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteossarcoma , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radioimunoensaio , Ratos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Timidina/metabolismo , Fatores de Transcrição/genética , Trítio , Células Tumorais Cultivadas , Dedos de ZincoRESUMO
The UMR 106-06 rat osteosarcoma osteoblast-like cell line possesses calcitonin (CT) receptors in addition to expressing PTH receptors and a highly osteoblast-like phenotype, and may represent an intermediate developmental stage between early osteoblast precursors and mature osteoblasts. Therefore, we examined the effects of CT and PTH on second messenger generation and osteoblastic function in these cells. In UMR-106-06 cells, 10-1000 nM CT produced a dose-dependent stimulation of intracellular free calcium concentration ([Ca2+]i), which reached a plateau between 2-3 min. This stimulatory effect was abolished in the absence of extracellular Ca2+ ([Ca2+]o) and was mimicked by forskolin and (Bu)2cAMP. One hundred nanomolar CT also produced a slight but significant increase in inositol triphosphate production (13%, P less than 0.05) but did not produce a rapid, transient increase in [Ca2+]i. In contrast, PTH produced a rapid, transient increase in [Ca2+]i, which reached a maximum within 30 sec. This stimulatory effect of PTH on [Ca2+]i signal was dose-dependent and accompanied by a parallel stimulation of inositol triphosphate production. PTH, forskolin, and (Bu)2cAMP all produced a marked dose-related suppression of both DNA and collagen synthesis, which paralleled their stimulatory effects on intracellular cAMP levels. In marked contrast, CT only minimally reduced DNA and collagen synthesis despite producing comparable increases in intracellular cAMP. One hundred nanomolar CT also stimulated alkaline phosphatase specific activity by 33% (P less than 0.05). Thus, CT stimulates cAMP, [Ca2+]i, and inositol phosphate second messengers in UMR 106-06 cells. However, in contrast to other agents which elevate intracellular cAMP levels, CT does not suppress DNA synthesis. These results suggest that the linkage of CT receptor second messengers to effects on cell function differ from those of PTH and/or that CT may produce additional second messenger(s) which antagonize the antiproliferative effect of increased cAMP levels in UMR-106-06 cells.
Assuntos
Calcitonina/farmacologia , Cálcio/metabolismo , AMP Cíclico/biossíntese , Osteoblastos/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Animais , Colforsina/farmacologia , Colágeno/biossíntese , DNA/biossíntese , Relação Dose-Resposta a Droga , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Hormônio Paratireóideo/farmacologia , Fosfatidilinositóis/metabolismo , Ratos , Receptores da Calcitonina , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/fisiologia , Células Tumorais CultivadasRESUMO
PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.
Assuntos
Cartilagem/metabolismo , DNA/biossíntese , Proteínas da Matriz Extracelular , Prostaglandinas/farmacologia , Proteoglicanas/biossíntese , Sistemas do Segundo Mensageiro , Agrecanas , Alprostadil/farmacologia , Animais , Bucladesina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Dinoprosta/farmacologia , Dinoprostona/farmacologia , Lectinas Tipo C , Proteína Quinase C/metabolismo , Ratos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The effect or oral glucose ingestion on renal phosphate reabsorption was studied in 13 patients with the inherited form of vitamin D-resistant rickets (VDRR) and 5 normal subjects. In contrast to the normal subjects, glycosuria developed in six VDRR patients after glucose ingestion and resulted in a further 43% decrease in renal phosphate reabsorption. This was accompanied by a 33% increase in phosphate clearance. This was not attended by differences in fasting glucose or phosphorus levels between groups, or in their respective values 1 h after glucose ingestion. Baseline renal phosphate reabsorption was less and baseline phosphate clearance was greater in those VDRR subjects who developed glycosuria. The accumulated data suggest that excessive glucose ingestion by some patients with VDRR may add an additional insult to the phosphaturia characteristic of this disorder. This, in turn, would further compromise the response of circulating phosphate to therapeutic attempts at oral phosphate supplementation, thereby reducing the efficacy of oral phosphate therapy on skeletal growth and development in this disorder.