Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Giant cell tumors of bone obtained from 7 patients were dispersed with clostridial collagenase and trypsin and adherent cells were maintained in culture. Early cultures contained both mononucleated and multinucleated cells presumably derived from the stromal and giant cells of the original tumor. The original multinucleated cells did not survive for greater than 7-10 days whereas the mononucleated cells persisted and could be passaged by trypsinization. In 5 of 7 early cultures exposed to parathyroid hormone (PTH) there was a rise in cAMP within 5-10 min in both cells and medium which averaged approximately 12-fold. None of the cells responded to calcitonin and a variable rise in cAMP was seen after incubation with prostaglandin E2. In cells cultured from 3 tumors the PTH response disappeared with passage of the cells, but in the remaining 2, PTH response persisted through multiple passages. The presence as well as the magnitude of the PTH-induced cAMP response in these cells is consistent with a skeletal origin.
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PMID:Response to hormones of cells cultured from human giant cell tumors of bone. 8

Six populations of bone cells (populations 1-6) were obtained by sequential digestion of mouse calvaria with collagenase and trypsin. After release from the tissue, each cell population was cultured for seven days. Parathormone, but not calcitonin, elicited an increase in intracellular cyclic AMP in the cells of populations 4, 5, and 6. In contrast, both hormones elicited increases in cyclic AMP in populations 2 and 3 but had no effect on population 1. When the cells of population 2 were exposed to a Falcontissue culture polystyrene surface for periods of time up to 5 min, many cells adhered. The nonadhering cell population contained a lesser proportion of cells responsive to calcitonin, whereas the adhering population contained a greater proportion responsive to this hormone. Conversely, when the cells of population 2 were exposed to an acid-treated nylon surface, the nonadhering cells contained a larger proportion of those responsive to calcitonin and a smaller proportion responsive to parathormone. When those cells that were enriched for calcitonin responsiveness were examined, we found an increased proportion that exhibited an asymmetric bipolar morphology. These differed from large amorphous, often binucleate, cells which predominated in those populations that responded exclusively to parathormone. These results establish that bone contains at least two types of target cells--one that responds to parathormone but not calcitonin, the other that responds predominantly to calcitonin.
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PMID:Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. 17 56

Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately two-fold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activites were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activites was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in te PT cells. Parathormone stimulated acid phosphatase and hyaluronate synthesis by 100-200% only in the CT cells; in inhibited alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase by 75-90% only in the PT cells. Calcitonin alone had no effect on any of these activities other than cAMP production, but in inhibited the action of parathormone in the CT cells. The sensitivities, time courses of development,and magnitudes of these hormonal effects were similar to those observed previously in intact calvaria, indicating that the isolated cell system is a reliable model for the study of bone metabolism. Based on the metabolic responses of the cells, we postulate that the CT type of populations is enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is enriched in osteoblasts.
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PMID:Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts. 18 58

Five different cell populations, designated I to V, were isolated from minced newborn rat calveria by 5-sequential 20 min incubations with an enzyme mixture containing collagenase, elastase and DNAse. In primary culture, all five populations responded to parathyroid hormone (PTH) to a different degree, population IV giving the highest increase in cyclic-3'5'-adenosine monophosphate (cAMP) level. None of the five populations gave any response to calcitonin. Upon subsequent subcultures, all populations, except population IV, either lost or considerably decreased their response to PTH. Population IV gave a two to three-fold increase in cAMP concentration in response to PTH up to the third subculture. No morphological differences could be observed among the five populations. The third subculture of population IV cells that had been stored in 10% glycerol at -80C for four months was subsequently thawed and subcultured to the sixth subculture. These cells still responded to PTH with an increase in cAMP level. In a second experiment, 5 different cell populations designated I to V were isolated in a similar way by incubation with collagenase and DNAse. The maximum response to PTH was found in population 3. The preservation of the PTH-responsiveness of this population, after subculturing, freezing, storing in 10% glycerol at -80 C and subsequent subculturing, was likewise demonstrated. The hormone-responsiveness of cells from the sixth subculture of previously frozen and thawed population IV cells was further analyzed. These cells responded to PTH at a concentration of 0.1 U/ml to 5U/ml and to prostaglandin E1 (PGE1) at a concentration of 0.1 microng/ml to 10 microng/ml. The time course of action on population IV of PTH was found to be different from that of PGE1, suggesting a possible difference in the regulation of intracellular cAMP levels by these hormones.
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PMID:Parathyroid hormone- and prostaglandin E1-response in a selected population of bone cells after repeated subculture and storage at -80C. 19 Dec 37

In order to explore the distribution of hormone-responsive cells in skeletal tissues, we have examined the effects of synthetic bovine parathyroid hormone N-terminal peptide (bPTH 1-34) and salmon calcitonin (sCT) on cyclic AMP levels in periosteum-free rat calvaria, segments of periosteum, and in isolated cells dispersed from each tissue by collagenase digestion. Synthetic bovine PTH increased cyclic AMP levels to a greater degree in calvaria and in isolated bone cells than in the periosteal segments and cells, whereas sCT was more effective in the periosteal than in the bone systems. Primary cultures prepared from bone and periosteal cell populations exhibited progressive increases in their responsiveness to bPTH (1-34) and progressive decreases in responsiveness to sCT. After six days in the culture, bone cells failed to respond to sCT, and sCT did not modify their response simultaneously added bPTH (1-34). Six-day periosteal cell cultures exhibited residual sCT responsivity and an additive response upon simultaneous exposure to high concentrations of bPTH (1-34) and sCT suggesting separate sites of hormone action. Adenosine, a known stimulator of bone cell adenylyl cyclase, caused a greater increase in periosteal cell than in bone cell cyclic AMP. bPTH (1-34)-responsive cells which enrich periosteum-free bone may be osteoblasts, in view of their histological prominence in this tissue and in the bone cell isolates. Periosteal cells which responded to sCT and to adenosine preferentially are unidentified. Although periosteal segments contained numerous fibroblast-like cells, skin fibroblasts cultured from the same fetuses were sCT-insensitive. Growth in primary culture appears to alter the number of hormone-responsive cells or responsiveness of existing cells to each hormone, or both.
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PMID:Evidence for preferential effects of parathyroid hormone, calcitonin and adenosine on bone and periosteum. 19 Dec 42

We have previously shown that bone cells possess glucocorticoid receptors and that, in addition to being inhibitory to cell growth, glucocorticoid treatment potentiates the ability of parathyroid hormone (PTH) to stimulate cyclic AMP (cAMP) formation. This study extends those observations to specific subpopulations of bone cells and explores the mechanism of the cAMP augmentation. Subpopulations of cultured bone cells derived from 20-d-old fetal rat calvaria were enriched for "osteoblast-like" (OB) and "osteoclast-like" (OC) cells by sequential collagenase digestion. OC cells released during the first 30 min of collagenase digestion were characterized by low alkaline phosphatase activity, a cAMP response to salmon calcitonin (CT), but only a small cAMP response to bovine PTH. In contrast, OB cells released between 30 and 120 min of collagenase digestion, possessed high alkaline phosphatase activity, responded with a large cAMP rise to PTH, but exhibited no response to CT. Glucocorticoid receptors, with similar properties, were demonstrated in both populations (K(d) congruent with 5 nM, N(maximum) congruent with 400 fmol/mg cytosol protein). Dexamethasone equivalently inhibited cell growth and alkaline phosphatase activity in both populations. Dexamethasone potentiation of cAMP generation occurred after PTH but not CT stimulation. A greater enhancement of cAMP generation observed in OB cells appears to result from two glucocorticoid actions: (a) stimulation of adenylate cyclase and (b) inhibition of phosphodiesterase. Only the latter mechanism was found in OC cells. Dexamethasone-treated cells showed an increase in both sensitivity and maximal response of cAMP to PTH. The possible relationship of these actions to the mechanism of glucocorticoid-induced osteopenia is discussed.
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PMID:Glucocorticoid receptors and actions in subpopulations of cultured rat bone cells. Mechanism of dexamethasone potentiation of parathyroid hormone-stimulated cyclic AMP production. 22 Feb 82

1. The addition of heparin to the culture fluid of mouse tibiae or calvaria did not cause any significant resorption of bone collagen or mineral. However, heparin (or analogue sulfated polyanions), enhanced greatly the amount of latent, trypsin-activatable collagenase (i.e. procollagenase) released by the bones in the medium without influencing that of directly active collagenase which was always very low. Heparin appeared to act by increasing the production of the enzyme which is immediately excreted. Procollagenase and collagenase are not stored in bone tissue, even under conditions where it is in active resorption. 2. Parathyroid hormone induced in the explants a resorption of both mineral and collagen that was inhibited by calcitonin. These hormones, however, had no influence on the release of procollagenase or collagenase either in the presence or in the absence of heparin. 3. Once activated, bone collagenase digested the collagen of the bone explants, and more extensively after their demineralization. Thus the latent collagenase that accumulates around non-resorbing bones has to be considered as a precursor, (and not as a residue), of active enzyme. 4. Active collagenase added to incipient cultures of bones disappeared with a half-life of 24 h. The lost enzyme could, however, not be reactivated by trypsin and thus was not transformed into latent procollagenase.
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PMID:Collagenase, procollagenase and bone resorption. Effects of heparin, parathyroid hormone and calcitonin. 22 39

The effects of parathyroid hormone (PTH) on bone collagen synthesis were assessed in organ cultures of fetal rat calvaria by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and non-collagen protein (NCP) using purified bacterial collagenase. 1) PTH decreased the incorporation of labeled proline into CDP at concentrations similar to those which stimulate bone resorption in vitro. 2) This effect was observed in bones treated for 6 h, but not for 3 h; it was maximal at 24 h and was maintained for 96 h. Bones treated with PTH for 48 h and transferred to control media for 48 h showed recovery of CDP labeling to control values. 3) the effect was specific for bone collagen. There was little alteration in the incorporation of proline into NCP, and incorporation into collagen was not inhibited. 4) The effect could be ascribed to decreased collagen synthesis and not to changes in amino acid uptake, precursor pool size, or degradation of newly synthesized CDP. In 3 hour experiments, PTH did increase the labeling of CDP and NCP, but only at tracer concentration of proline in the medium, compatible with an early stimulation of amino acid uptake. 5) Similar inhibition was observed with purified bovine (1-84) PTH and synthetic bovine PTH (1-34) as well as with crude homologous PTH obtained from rat parathyroid gland culture fluid. Human (hCT) and salmon (sCT) calcitonin did not inhibit the effect of PTH on the labeling of CDP nor did they stimulate CDP labeling directly at concentrations which inhibited bone resorption. Dibutyryl cyclic-3',5'-adenosine monophosphate (D3cAMP) inhibited labeling of CDP at concentrations of .03 to .3 mM, thus mimicking the action of PTH. However, in this system DBcAMP inhibited 45Ca release, thus mimicking CT. We conclude that the direct effect of PTH on bone collagen synthesis is a slow reversible inhibition, not opposed by CT. This effect may be mediated by cAMP formation in bone cells.
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PMID:Hormonal control of bone collagen synthesis in vitro: effects of parathyroid hormone and calcitonin. 94 52

The adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] sensitivity to salmon calcitonin in 11 different segments of the rabbit nephron was investigated using a micromethod for enzyme activity measurements in samples, each containing a single piece of tubule. The required segments were isolated by microdissection from collagenase-treated rabbit kidneys. The results were expressed as femtomoles of adenosine 3':5'-cyclic monophosphate formed per mm of tubular length per 30 min of incubation time. In the presence of 0.1 mug/ml of synthetic salmon calcitonin, it was found that eight segments exhibited no hormonal sensitivity whereas maximal responses were induced in three segments, the "bright" portion of the distal convoluted tubule, the cortical and the medullary portions of the thick ascending limb of the loop of Henle (stimulated over control activity ratios were 32, 11, and27). The very high sensitivity to calcitonin of the adenylate cyclase contained in these three segments (0.01 ng/ml of salmon calcitonin inducing a 2-fold stimulation; half-miximal stimulation corresponding to about 0.3 ng/ml of salmon calcitonin) suggests that the distal convoluted tubule, as well as th cortical and medullary portions of the thick ascending limb of the loop of Henle represent physiological target structures of calcitonin action within the kidney.
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PMID:Distribution of calcitonin-sensitive adenylate cyclase activity along the rabbit kidney tubule. 106 74

We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.
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PMID:Preparation and characterization of a mouse osteoclast-like multinucleated cell population. 128 5


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