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

The direct effects of porcine insulin and glucagon on bone collagen and non-collagen protein synthesis have been examined in cultures of calvaria obtained from 21-day fetal rats. Bones were incubated for 24 to 96 h and [3H]proline was added for the last 2 h of culture. Incorporation of the label into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Insulin increased the labeling of CDP by 60 to 115% at concentrations of 10(-9) to 10(-6) M. A smaller stimulatory effect was observed on NCP. The effect on CDP appeared after 12 to 24 h of culture, was maintained for 96 h in the continuous presence of the hormone, but was lost within 3 h of removal of insulin from the culture medium. Insulin appeared to have a direct effect on collagen synthesis and not on collagen breakdown. Insulin did not affect the incorporation of [3H]uridine or [3H]thymidine into the RNA and DNA fractions of bone at 24 h. Insulin opposed the inhibitory effects of parathyroid hormone and dibutyryl cyclic-3',5'-adenosine monophosphate and to a lesser extent, the inhibitory effect of isobutylmethylxanthine on the labeling of CDP. Glucagon did not affect the response to insulin and by itself had small and variable inhibitory effects on proline incorporation.
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PMID:Hormonal control of bone collagen synthesis in vitro. Effects of insulin and glucagon. 40 59

Fetal rat bones were cultured in either growth-inducing or resorption-inducing media to study mineral losses during bone growth and atrophy in vitro. Whole radii and ulnae from 19-day-old fetal rats, prelabeled with 45Ca and/or 3H-tetracycline, were cultured intact or cut, and then digested by collagenase to obtain the calcified portion of the bones. Three- to five-fold more 3H-tetracycline than 45Ca was lost from the calcified portion when the bones were cultured for 4 days in growth-inducing media. Similar small amounts of 45Ca were lost from live and killed bones, but more 3H-tetracycline was lost from live bones than from killed bones. More 3H-tetracycline was released into the growth medium with a low concentration of calcium (0.5 mM) than when the calcium concentration was high (1.0 mM); no significant difference was seen in the release of 45Ca into the medium at different calcium concentrations. Larger amounts of both isotopes were lost when the prelabeled bones were cultured in resorption-inducing media than in growth-inducing media. When parathyroid hormone stimulated bone resorption in a resorption-inducing medium, equal proportions of both isotopes and bone collagen were lost. Greater losses of 3H-tetracycline than of 45Ca suggest that 45Ca was conserved locally during the resorption that accompanies bone growth, but not during resorption that accompanies bone atrophy.
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PMID:Fetal rat bone in organ culture: effect of bone growth and bone atrophy on the comparative losses of 45Ca and 3H-tetracycline. 70 8

The mechanism of congenital osteopetrosis in microphthalmic (mi) mice has been examined in bone organ cultures. Resorption was measured by the release of previously incorporated 45Ca in fetal long bones and newborn calvaria from mi mice and heterozygous or homozygous normal litter mates. Bones from mi mice showed a generalized resorption defect with decreased spontaneous or control resorption and failure to respond to parathyroid hormone (PTH), prostaglandin E2, 1,25 dihydroxy vitamin D3, vitamin A, or osteoclast activating factor (OAF) from human peripheral leukocytes or mouse spleen cells. Bones from heterozygotes showed a smaller response to PTH than bones from homozygous normals. Mutant bones failed to show an increase in lysosomal enzyme release in response to PTH or vitamin A, agents which increased release from bones of homozygous normals. Proline incorporation into collagenase-digestible protein was similar in cultures of normal and mutant bone and was inhibited by PTH and OAF. These results indicate that congenital osteopetrosis in mi mice is due to a generalized defect in the function and hormonal response of osteoclasts and suggests that this cell line is separate from the osteoblast cell line which shows no impairment of hormonal response.
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PMID:Studies on congenital osteopetrosis in microphthalmic mice using organ cultures: impairment of bone resorption in response to physiologic stimulators. 87 Jun 7

Rabbit distal convoluted tubules (DCT) microdissected from collagenase-treated kidneys were observed to contain up to four portions of a different appearance under stereomicroscopic examination: (1) a DCTa portion (generally very short), located right after the macula densa (MD) and resembling the portion of the limb (CAL) located before the MD; (2) a constant, "bright" portion, DCTb; (3) a constant, "granular" DCTg portion which, in most DCT, is connected to a portion of the collecting tubule of a similar "granular" appearance (CCTg); (4) many DCT having contacts with the kidney capsule in the superficial cortex were observed to contain an additional portion of a "light" appearance, DCTl, resembling the portion of the collecting tubule (CCTl) to which these superficial DCT are always branched. The hormone-dependent adenylate cyclase (AC) contained in these different portions was investigated by sectioning microdissected distal structures into successive samples according to the above-mentioned criteria, and by measuring with the help of a previously described micromethod, the enzyme activity contained in each single sample under one of the following conditions: control, parathyroid hormone. (PTH l U/ml), vasopressin, (AVP 10(-6)M), isoproterenol (10(-6)M), fluoride (5 X 10(-3)M). Highly significant and reproducible AC stimulations by these hormones were obtained for the following portions, respectively: DCTa, DCTg and CCTg with PTH; DCTl and CCTl with AVP; DCTg, CCTg and CCTl with isoproterenol. From these data, it is concluded that (a) the distal convoluted tubule can no longer be regarded as a single well-defined functional structure; (b) DCTa is actually a short CAL portion extending beyond MD, (c) DCTg and CCTg are two portions of a same functional segment; (d) similarly, DCTl belongs to the functional segment mainly constituted by CCTl; and, finally, (e) DCTb is the only functional segment which is entirely located in the distal convoluted tubule, i.e., included between the macula densa and the first branching with another tubule.
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PMID:Functional segmentation of the rabbit distal tubule by microdetermination of hormone-dependent adenylate cyclase activity. 94 Feb 69

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

Many biotransformation activities have absolute or modulated localization within the hepatic acinus. To investigate the intrahepatic acinar zonation of vitamin D3 (D3) metabolism, hepatic D3 extraction was investigated by antegrade or retrograde perfusion of normal livers and livers bearing selective periportal (PP) or perivenous (PV) destruction; D3 C-25 hydroxylation was studied after selective harvesting of PP or PV hepatocytes by digitonin-collagenase perfusion. Data indicate that hepatic D3 extraction is not regioselective and not perturbed by destruction of the proximal (PP) or distal (PV) part of the acinus, indicating that D3 extraction takes place in the most proximal hepatocytes being perfused. These observations suggest that, in vivo, D3 extraction will take place according to its concentration gradient within the hepatic acinus, thus resulting in a preferential PP extraction of the vitamin. D3 C-25 hydroxylation was higher in PP than in PV hepatocytes in the presence of 1.9 mM Ca2+, with 25-hydroxyvitamin D3 [25(OH)D3] formation of 34.6 +/- 3.9 and 24.4 +/- 1.1 fmol.h-1.(10(6) hepatocytes)-1, respectively (P less than 0.05). Modulators of extracellular [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)] or intracellular Ca2+ (parathyroid hormone, A23187), however, significantly influenced 25(OH)D3 formation with similar decreases in the PP (31%) and PV (26%) areas in the presence of EGTA but with increases in the presence of Ca2+ ionophore A23187 of 189 +/- 16% in PP and of 260 +/- 20% in PV hepatocytes, resulting in similar production in both regions of the acinus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:C-25 hydroxylation of vitamin D3 in periportal and perivenous region of hepatic acinus. 131 77

The present study was designed to further understand the role of PTH on the secretion of the neutral metalloproteinases, collagenase and gelatinase, from the rat osteosarcoma clonal cell line, ROS 17/2.8. Semiconfluent cells were treated with bovine parathyroid hormone, b-PTH-(1-34) at 100 nM-0.01 nM for 24-96 hours and pooled, concentrated media were analyzed by functional assay for collagenase (3H-methyl collagen) and gelatinase (3H-methyl gelatin). Collagenase activity significantly decreased (P less than 0.01) in the PTH conditioned media in a dose-dependent manner before (98-64%) and after (91-39%) reduction and alkylation. SDS-PAGE and fluorography apparently showed the most degradation to alpha A chains in collagen with controls, whereas this substrate remained intact with PTH (100 nM). PTH (100 nM) media also showed neutral gelatinase activity approximately 2% compared to control before and after reduction and alkylation (P less than 0.01). Significant amounts of an inhibitor to collagenase and gelatinase might have been secreted at 1 nM and 0.01 nM PTH, since collagenase and gelatinase activities were greater after reduction and alkylation. Reduction and alkylation likely destroyed these significant amounts of inhibitor. Polymorphonuclear leukocyte collagenase activity was also inhibited 80% by PTH conditioned media, but not by control. However, upon reduction and alkylation which destroyed inhibitor, the PTH treated media showed only a 14% inhibition against polymorphonuclear leukocyte collagenase (P less than 0.01). PTH appeared to downregulate neutral metalloproteinase activities through its effects on an inhibitor. This downregulation may represent a specific phenotypic response to PTH in ROS 17/2.8 cells.
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PMID:Parathyroid hormone regulation of matrix degrading enzymes in rat osteoblastic osteosarcoma 17/2.8 cells. 132 16

Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents. In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2- to 3-fold more potent than either PTH or 1,25(OH)2D3. Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines. 133 77

Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.
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PMID:A serum factor promotes collagenase synthesis by an osteoblastic cell line. 164 67

During continuous culture with serial passage, the human osteosarcoma cell line SaOS-2 showed a time-dependent decrease in skeletal alkaline phosphatase (ALP) activity. Because this was indicative of heterogeneity, subpopulations of SaOS-2 cells were isolated from replicate low-density cultures. The subpopulations were less heterogeneous and more stable (with respect to ALP) than the parent population. ALP specific activity in the subpopulations ranged from 0.05 to 2.3 U/mg protein, and cytochemical analyses indicated multiple steady-state levels of ALP activity per cell. The amount of ALP activity in SaOS-2 subpopulations was proportional to collagen production ([3H]proline incorporation into collagenase-digestible protein; r = .84, P less than .005), and to parathyroid hormone (PTH)-linked synthesis of cyclic adenosine monophosphate (cAMP) (r = .88, P less than .01). From these data, we inferred that ALP activity in SaOS-2 cells can provide a useful index of the osteoblastic phenotype, and that ALP activity, collagen production, and PTH-linked adenylate cyclase were coordinately regulated in these osteoblast-like osteosarcoma cells (ie, selection of subpopulations for ALP activity coselected for collagen synthesis and PTH-linked synthesis of cAMP). Further comparative studies showed that micromolar fluoride concentrations stimulated cell proliferation ([3H]thymidine incorporation into DNA) in low-ALP SaOS-2 subpopulations, but not in high-ALP cells (P less than .001), and that this differential sensitivity to fluoride was associated with an inverse correlation between fluoride-sensitive acid phosphatase and ALP activities (r = -.91, P less than .001).
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PMID:Skeletal alkaline phosphatase specific activity is an index of the osteoblastic phenotype in subpopulations of the human osteosarcoma cell line SaOS-2. 165 38


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