Matrix metalloproteinases (MMPs) are zinc-containing proteinases that participate in tissue remodeling under physiological and pathological conditions. To test the involvement of bacterial proteinases in tissue injury during bacterial infections, we investigated the activation potential of various bacterial proteinases against precursors of MMPs (proMMPs) purified from human neutrophils (proMMP-8 and -9) and from human fibrosarcoma cells (proMMP-1). Each proMMP was subjected to treatment with a series of bacterial proteinases at molar ratios of 0.01-0.1 (bacterial proteinase to proMMP), and activities of MMPs generated were determined. Among six different bacterial proteinases, thermolysin family enzymes (family M4) such as Pseudomonas aeruginosa elastase, Vibrio cholerae proteinase, and thermolysin strongly activated all three proMMPs via limited proteolysis to generate active forms of the MMPs. N-terminal sequence analysis of the active MMPs revealed that cleavage occurred at the Val 82 -Leu 83 and Thr 90 -Phe 91 bonds of proMMP-1 and proMMP-9, respectively, which are located near the N terminus of the catalytic domain of MMPs. In contrast, Serratia 56-kDa proteinase and Pseudomonas alkaline proteinase, both of which are classified as members of the serralysin subfamily of zinc metalloproteinases (family M10), and Serratia 73-kDa thiol proteinase did not evidence proteolytic processing or activation of proMMP-1, -8, and -9 under these experimental conditions. These results indicate that bacterial proteinases may play an important role in tissue destruction and disintegration of extracellular matrix at the site of infections.Collagen, one of the major structural components of the extracellular matrix, has a triple-helical structure and exhibits resistance to proteolytic cleavage by endogenous and exogenous proteinases (1) except for matrix metalloproteinases (MMPs) 1 such as human neutrophil collagenase (MMP-8). Bacterial proteinases have been suggested to mediate direct tissue destruction, resulting in impairment of host defense mechanisms in septic foci (2-4). Most bacterial proteinases, however, have weak degradative activity against collagen (1, 5). Thus, the mechanism of extracellular matrix destruction at the site of bacterial infection is poorly understood.MMPs, a family of zinc neutral endopeptidases, are secreted by a variety of cells as inactive precursors (proMMPs) and degrade a series of collagens (6). Two distinct proMMPs (proMMP-8 and neutrophil progelatinase, proMMP-9) are synthesized and secreted extracellularly from specific granules of human neutrophils after membrane stimulation (7, 8). Macrophages and fibroblasts produce interstitial procollagenase (proMMP-1) (6, 9) and 92-kDa progelatinase (proMMP-9), whose expressions vary constitutively or inducibly after stimulation with proinflammatory cytokines and lipopolysaccharide (10 -12). MMP-8 and -1 specifically cleave native triplehelical type I collagen into two major fragments, one-fourth and three-fourths the size of native collagen, respect...

BACKGROUND.Rapid tumor growth is caused by angiogenesis factors, growth fac-

Nitric oxide (NO), now almost synonymous with endothelium-derived relaxing factor (EDRF), reacts with superoxide anion radical (O2-) and forms a potentially toxic molecular species, peroxynitrite (ONOO-). Because xanthine oxidase (XO) seems to be a major O2- -producing enzyme in the vascular system, it is important to clarify the mechanism of XO regulation of NO/EDRF. We first characterized the inhibition of XO in vitro by three types of pyrazolopyrimidine derivatives. Kinetic studies indicated that 4-amino-6-hydroxpyrazolo[3,4-d]pyrimidine (AHPP) and allopurinol competitively inhibited the conversion of xanthine to uric acid catalyzed by XO, with apparent Ki values of 0.17 +/- 0.02 and 0.50 +/- 0.03 micro M respectively; alloxanthine inhibited this conversion in a noncompetitive manner with an apparent Ki value of 3.54 +/- 1.12 microM. O2- generation in the xanthine/XO system assayed by lucigenin-dependent chemiluminescence was suppressed most strongly by AHPP in a dose-dependent fashion; allopurinol itself appears to reduce the enzyme by transfer of an electron to O2, thus generating O(2-). AHPP significantly augmented EDRF-mediated relaxation of aortic rings from both rabbits and spontaneously hypertensive rats (SHR) in a dose-dependent manner, whereas allopurinol did not affect the relaxation and only marginal potentiation of the vasorelaxation was observed with alloxanthine. Finally, iv injection of AHPP (50.4 mg/kg; 100 micromol/300 g rat) reduced the blood pressure of SHR rats to 70% of the initial pressure; this pressure is almost the blood pressure of normal rats. Allopurinol (100 micromol/300 g rat; iv) showed transient decrease in blood pressure and moderate reduction of hypertension of SHR (10%) was observed with iv injection of alloxanthine (100 mumol/300 g rat). On the basis of these results, it seems that XO regulates EDRF/NO via production of O2-.

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