phenylmercuric-acetate and Periodontitis

Matrix metalloproteinases (MMPs) are capable of cleaving almost all macromolecules of the extracellular connective tissue matrix and are thought to play a major role in tissue destructive inflammatory diseases such as periodontitis. The aim of this study was to determine the effects of siderophores, which are iron-chelating molecules produced by a variety of microorganisms, on the activity of MMP-2. Heat-denatured type I collagen (gelatin) was incubated with p-aminophenylmercuric acetate-activated MMP-2 and siderophores. Degradation of gelatin was monitored by SDS-PAGE and Coomassie blue staining. Ferrichrome, rhodotorulic acid, desferoxamine mesylate and 2,3-dihydroxybenzoic acid were found to inhibit the MMP-2 activity whereas beta-phenylpyruvic acid had no effect. The inhibition could be reversed by adding an excess calcium chloride or ferric chloride to the assay mixtures. Our study suggests that microbial siderophores may represent new-potential therapeutic molecules for the treatment of destructive inflammatory diseases involving excess MMP-2 activity, such as periodontitis.

Topics: Bacteria; Calcium Chloride; Chelating Agents; Chlorides; Chromium; Collagen; Connective Tissue; Deferoxamine; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Extracellular Matrix; Ferric Compounds; Ferrichrome; Gelatinases; Humans; Hydroxybenzoates; Indicators and Reagents; Iron Chelating Agents; Macromolecular Substances; Matrix Metalloproteinase 2; Metalloendopeptidases; Periodontitis; Phenylmercuric Acetate; Phenylpyruvic Acids; Piperazines; Rosaniline Dyes; Siderophores; Sodium Dodecyl Sulfate; Sulfhydryl Reagents

We previously reported that low-dose doxycycline (DOXY) therapy reduces host-derived collagenase activity in gingival tissue of adult periodontitis (AP) patients. However, it was not clear whether this in vivo effect was direct or indirect. In the present study, inflamed human gingival tissue, obtained from AP patients during periodontal surgery, was extracted and the extracts partially purified by (NH4)2SO4 precipitation. The extracts were then analyzed for collagenase activity using SDS-PAGE/fluorography/laser densitometry, and for gelatinase activity using type I gelatin zymography as well as a new quantitative assay using biotinylated type I gelatin as substrate. DOXY was added to the incubation mixture at a final concentration of 0-1000 microM. The concentration of DOXY required to inhibit 50% of the gingival tissue collagenase (IC50) was found to be 16-18 microM in the presence or absence of 1.2 mM APMA (an optimal organomercurial activator of latent procollagenases); this IC50 for DOXY was similar to that exhibited for collagenase or matrix metalloproteinase (MMP)-8 from polymorphonuclear leukocytes (PMNs) and from gingival crevicular fluid (GCF) of AP patients. Of interest, Porphyromonas gingivalis collagenase was also inhibited by similar DOXY levels (IC50 = 15 microM), however the collagenase activity observed in the gingival tissue extracts was found to be of mammalian not bacterial origin based on the production of the specific alpha A (3/4) and alpha B (1/4) collagen degradation fragments. In contrast, the inhibition of collagenase purified from culture media of human gingival fibroblasts (MMP-1) required much greater DOXY levels (IC50 = 280 microM). The predominant molecular forms of gelatinolytic activity presented in the AP patients gingival tissue extracts were found to closely correspond to the 92 kD PMN-type gelatinase (MMP-9) although small quantities of 72 kD fibroblast-type gelatinase (MMP-2), and some other low molecular weight gelatinases, were also detected. The IC50 of DOXY versus gingival tissue gelatinolytic activity was estimated at 30-50 microM measure using either type I gelatin zymography or the biotinylated type I gelatin assay. We conclude that MMPS in inflamed gingival tissue of AP patients, like those in GCF, originate primarily from infiltrating PMNs rather than resident gingival cells (fibroblasts and epithelial cells) or monocyte/macrophages, and that their pathologically-elevated tissue-degrading activities can be d

Topics: Adult; Cells, Cultured; Collagenases; Doxycycline; Electrophoresis, Polyacrylamide Gel; Female; Fibroblasts; Gelatinases; Gingiva; Gingival Crevicular Fluid; Humans; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 8; Matrix Metalloproteinase Inhibitors; Neutrophils; Periodontitis; Phenylmercuric Acetate; Porphyromonas gingivalis; Sodium Dodecyl Sulfate

Topics: Collagenases; Endopeptidases; Enzyme Activation; Humans; Mouth, Edentulous; Neutrophils; Periodontitis; Phenylmercuric Acetate; Saliva; Salivary Proteins and Peptides

Analysis of inflammatory exudate collected from sites of experimental periodontitis in cynomolgus monkeys has revealed the presence of collagenase and a 92-kDa gelatinase that comigrated after electrophoresis with the 92-kDa gelatinase released from polymorphonuclear leukocytes. Since neutralizing antibodies to fibroblast collagenase had no effect on the collagenase activity and bacterial collagenases could not be detected, polymorphonuclear leukocytes appear to be the major source of collagenolytic proteinases in inflammatory fluid from gingiva.

Topics: Animals; Bacteria; Fibroblasts; Gelatinases; Gingiva; Humans; Macaca fascicularis; Microbial Collagenase; Molecular Weight; Neutrophils; Pepsin A; Periodontitis; Phenylmercuric Acetate

In previous studies, elevations in the levels of active and latent collagenase in gingival crevicular fluid (GCF) have been correlated positively with periodontal disease activity. To provide a simple diagnostic approach for testing collagenolytic activity, the feasibility of using a 3.0 ml water mouthrinse to collect GCF simultaneously from all sites in the mouth was assessed. Patients with adult periodontitis (AP, n = 23) and local juvenile periodontitis (LJP, n = 7) were sampled before periodontal therapy and some (12 AP, 4 LJP) were also assessed longitudinally after scaling and root planing, administration of antibiotics, and following periodontal surgery. Healthy patients (n = 19) were used as controls. The levels of active collagenase, procollagenase, and collagenase inhibitor activity were determined by functional assays and quantitated after SDS-PAGE and fluorography. Gelatinase and progelatinase were assayed by enzymography on gelatin-substrate gels. Active collagenase levels were found to be significantly higher (14- to 20-fold) in AP and LJP patients compared to controls, whereas matrix metalloproteinase activity was not detected in mouthrinses from edentulous patients. Collagenase inhibitor levels were generally low in all groups of subjects tested. Following clinical treatment the levels of active collagenase and gelatinase were reduced; the reduction was significant for active collagenase after tetracycline treatment and scaling in LJP patients. Of the clinical indices recorded (gingival index, plaque index, and pocket depth) there were no significant correlations with enzyme activity but similar trends were observed between the changes in active collagenase and gingival index. In patients with untreated periodontal disease, collagenase occurred predominantly in the active form. N-ethylmaleimide (NEM) and p-aminophenylmercuric acetate (AMPA) were equally effective as activators of the latent collagenase, indicating that the collagenase was derived from PMNs, which were also the source of gelatinase. The results of these studies indicate that measurement of active collagenase and gelatinase in mouthrinse samples is potentially useful in the diagnosis and assessment of periodontal disease activity.

Topics: Adolescent; Adult; Aggressive Periodontitis; Collagen; Collagenases; Enzyme Precursors; Ethylmaleimide; Female; Gelatinases; Gingival Crevicular Fluid; Humans; Male; Microbial Collagenase; Mouthwashes; Neutrophils; Pepsin A; Periodontitis; Phenylmercuric Acetate

To study the regulation of collagen degradation in periodontium, human gingival homogenate was incubated at 36 degrees C and the release of hydroxyproline was assayed as a measure of collagenase activity. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and serum albumin inhibited the in vitro collagenolysis while p-aminophenylmercuric acetate, a sulfhydryl reagent, increased the degradation. When latent collagenase obtained from gingival fibroblast culture was added to the incubation a marked increase in the collagen degradation was found. This increase was prevented by phenylmethylsulfonyl fluoride. The data suggests that collagenase may exist in gingiva partly in a latent form and its activation may be brought about by 2 mechanisms. A serine proteinase present in tissue may activate collagenase by producing a limited cleavage, or the activation may occur through a reaction that involves the sulfhydryl groups of the collagenase molecule.

Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Collagen; Enzyme Activation; Fibroblasts; Gingiva; Humans; Microbial Collagenase; Periodontitis; Phenylmercuric Acetate