A versatile assay for gelatinases using succinylated gelatin.

A spectrophotometric assay using succinylated gelatin as substrate is described for measuring the catalytic activity of gelatinases. The assay is based on measurement of primary amines exposed as a result of hydrolysis of the substrate by gelatinases. Comparison of hydrolysis by matrix metalloproteinase (MMP) 1, 2, 3, 7, 9 indicated that succinylated gelatin was primarily digested by MMP-2 and -9. The assay is rapid (<60 min), specific, suitable for measuring gelatinolytic activity of enzymes and high volume screening of MMP-2 and -9 inhibitors. Sensitivity of the assay is comparable to that of gelatin zymography, under similar experimental conditions. Thus, the assay combines ease and rapidity of assays based on synthetic peptide substrates with specificity of the gelatin zymography technique.

Hypercholesterolemia causes mechanical weakening of rabbit atheroma : local collagen loss as a prerequisite of plaque rupture.

Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0. 5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (P<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.

Hydroxylamine analogs of 2,6-di-t-butylphenols: dual inhibitors of cyclooxygenase and 5-lipoxygenase or selective 5-lipoxygenase inhibitors.

The preparation of hydroxylamine analogs of 2,6-di-tert-butylphenols (DTBP) and the inhibition of cyclooxygenase (CO) and 5-lipoxygenase (5-LO) by these compounds is discussed.

Stimulus-dependent effects of CI-986 on arachidonic acid metabolism by human neutrophils.

CI-986 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3, 4-thiadizole-2(3H)-thione, choline salt) was evaluated for its effect on arachidonic acid metabolism by human neutrophils in response to different stimuli. Leukotriene B4 (LTB4) release in response to calcium ionophore A23187 was 15 to 35 fold greater than the responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP) or serum-opsonized zymosan (SOZ), respectively, while the thromboxane B2 (TXB2) release response was similar for the three stimuli tested. CI-986 inhibited the release of LTB4 and TXB2 in response to A23187 with IC50s of 63.4 and 1.6 microM, respectively. In comparison, the compound inhibited SOZ-stimulated LTB4 release with an IC50 of 11.2 microM, while having no effect on TXB2 at concentrations up to 100 microM. Conversely, CI-986 inhibited FMLP-stimulated LTB4 release by 42% at 100 microM, while inhibiting TXB2 release with an IC50 of 0.13 microM. These results demonstrate a stimulus-dependent inhibitory effect of CI-986 on human neutrophil eicosanoid metabolism.

Selective regulation of human neutrophil functions by the cell activation inhibitor CI-959.

The cell activation inhibitor CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-ylbenzo[ b]thiophene-2- carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI-959 inhibited spontaneous migration and chemotaxis toward N-formyl-methionyl-L-leucyl-L-phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 microM, respectively. CI-959 also inhibited superoxide anion generation in response to C5a, fMLP, serum-opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 microM, respectively. In comparison, CI-959 inhibited myeloperoxidase microM, respectively. In comparison, CI-959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and < 1.0 microM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 microM. At concentrations up to 100 microM, CI-959 had no effect on the respiratory burst or degranulation in response to L-alpha-1,2-dioctanoylglycerol (DiC8) or phorbol 12-myristate 13-acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 microM, respectively), while having less activity against the A23187-stimulated response (IC50 > 100 microM). These results demonstrate that CI-959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to inophore-mediated calcium influx, only oxygen radical production was inhibited by CI-959. CI-959 was further evaluated for its effects on neutrophil stimulus-response coupling. At 100 microM, CI-959 had no effect on human neutrophil phospholipase C or protein kinase C. CI-959 inhibited fMLP-stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 microM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 microM). These results indicate that CI-959 may exert its stimulus- and response-specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium-regulated signalling mechanisms.

1,3,4-Oxadiazole, 1,3,4-thiadiazole, and 1,2,4-triazole analogs of the fenamates: in vitro inhibition of cyclooxygenase and 5-lipoxygenase activities.

N-Arylanthranilic acids, known generically as the fenamates, are nonsteroidal antiinflammatory drugs (NSAIDs) that block the metabolism of arachidonic acid by the enzyme cyclooxygenase (CO). Substitution of the carboxylic acid functionality of several fenamates with acidic heterocycles provided dual inhibitors of CO and 5-lipoxygenase (5-LO) activities when tested in an intact rat basophilic leukemia (RBL-1) cell line. Compound 5b (IC50 = 0.77 microM (5-LO), 0.27 microM (CO)) which contains an 1,3,4-oxadiazole-2-thione replacement and 10b (IC50 = 0.87 microM (5-LO), 0.85 microM (CO)) which contains a 1,3,4-thiadiazole-2-thione are the most potent inhibitors of 5-LO and CO activities from these series. Both of these heterocyclic analogs of flufenamic acid are also active in carageenin-induced rat footpad edema (CFE), a model of acute inflammation. When dosed orally the ID50s for 5b and 10b in CFE are 8.5 and 4.7 mg/kg, respectively.

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-949.

The allergic mediator release inhibitor Cl-949 [5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H -indole-2-carboxamide, L-arginine salt] was evaluated for its effects on human neutrophil functions. Cl-949 (100 microM) inhibited spontaneous migration and chemotaxis toward f-met-leu-phe (FMLP) by 49.1% and 45.8%, respectively. At the same concentration, Cl-949 inhibited the phagocytosis of serum-opsonized zymosan (SOZ) by 39.0%. Cl-949 inhibited leukotriene B4 and thromboxane B2 release in response to SOZ with IC50s of 2.0 microM and 3.3 microM, while inhibiting the response to FMLP with IC50s of 1.7 and 2.0 microM. Cl-949 also inhibited myeloperoxidase release from primary lysosomal granules in response to the following stimuli with the respective IC50s (microM): C5a (40.3); FMLP (34.4): SOZ (21.4); concanavalin A (Con A) 3.9); and calcium ionophore A23187 (91.2). In contrast, Cl-949 inhibited lysozyme release from secondary granules in response to SOZ and Con A with IC50s of 99.3 and 56.1 microM, while inhibiting the response to C5a, FMLP, and A23187 by 41.2%, 52.4%, and 10.0%, respectively, at 100 microM. Cl-949 (100 microM) had no inhibitory effect against lysozyme release in response to L-alpha-1,2 dioctanoylglycerol (DiC8), or phorbol 12-myristate 13-acetate (PMA). Cl-949 inhibited superoxide anion generation stimulated by FMLP and Con A with IC50s of 33.9 and 25.8 microM, while inhibiting the response to C5a, SOZ, and A23187 by 36.6%, 24.8%, and 14.1% and having no effect on the response to DiC8 or PMA at 100 microM. These results demonstrate preferential inhibition of arachidonic acid metabolism and degranulation of primary lysosomal granules by Cl-949 with selectivity for stimuli which promote intracellular calcium mobilization or calcium influx.

Differential inhibition of human neutrophil functions. Role of cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase.

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been characterized in various tissues and cells according to their substrate specificity, intracellular location, and calmodulin dependence. The purpose of this study was to evaluate the possible involvement of different molecular forms of phosphodiesterase in regulating the respiratory burst and lysosomal enzyme release responses of human neutrophils. Treatment with the selective cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase inhibitors Ro 20-1724 or rolipram, or the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), resulted in inhibition of respiratory burst stimulated by the chemoattractants formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (IC50 values: 0.71-17 microM) and complement fragment C5a (IC50 values: 61-93 microM), but did not inhibit phagocytosis-stimulated respiratory burst (less than 10% inhibition at 100 microM). Selective inhibitors of calmodulin-dependent phosphodiesterase (ICI 74,917), calmodulin-insensitive, cyclic GMP-specific phosphodiesterase (M & B 22,948), cyclic GMP-stimulated phosphodiesterase (AR-L 57), or cyclic AMP-specific, cyclic GMP-inhibited phosphodiesterase (amrinone and cilostamide) exhibited little or no inhibitory effect on FMLP- or phagocytosis-stimulated respiratory burst (0-42% inhibition at 100 microM). Regulation of neutrophil activation by phosphodiesterase was also response specific, as Ro 20-1724, rolipram and IBMX were less potent inhibitors of FMLP-induced lysosomal enzyme release (0-14% inhibition at 100 microM). Analysis of human neutrophil preparations confirmed the existence of a cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase, which was associated with the particulate fraction of the cell. These results demonstrate a role for the cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase in the regulation of human neutrophil functions, which appears to be both stimulus specific and response specific.

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-949: mechanism of inhibitory activity.

CI-949 [5-methyl-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H- indole-2- carboxamide, L-arginine salt] inhibits human neutrophil activation in response to stimuli which promote calcium mobilization or calcium influx. This report further examines the effect of CI-949 on phosphoinositide-dependent stimulus-response coupling. At 100 microM, CI-949 had no inhibitory effect on human neutrophil phospholipase C or protein kinase C. In contrast, CI-949 inhibited FMLP-stimulated intracellular calcium mobilization with an IC50 of 8.4 microM. The compound was also a potent calmodulin antagonist, inhibiting calmodulin-dependent phosphodiesterase activity with an IC50 of 31.0 microM. The calmodulin antagonist activity of CI-949 was confirmed by fluorescence spectroscopy. These results demonstrate that CI-949 may function through inhibition of calcium- and calmodulin-dependent signal transduction processes.