Leukotrienes as mediators in tissue trauma.

A significant increase in the production of cysteinyl leukotrienes was observed after mechanical or thermal trauma in the anesthesized rat. The amount of biliary N-acetyl-leukotriene E4, which represents a suitable indicator for blood plasma leukotrienes, was used as a measure of leukotriene generation. Cysteinyl leukotrienes were rapidly eliminated from blood plasma into bile where N-acetyl-leukotriene E4 was the major metabolite. Leukotrienes were at a much lower concentration in blood plasma than in bile and differed in the pattern of metabolites. The detected amounts of leukotrienes were sufficient to induce known phenomena associated with trauma, such as tissue edema and circulatory and respiratory dysfunction. Increased leukotriene generation appears to play an important role in the pathophysiology of tissue trauma.

Endogenous 12(S)-HETE production by tumor cells and its role in metastasis.

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

Cell adhesion-dependent differences in endogenous protein phosphorylation on the surface of various cell lines.

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [gamma-32P]ATP and [gamma-32P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.

Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme.

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.

Production of peptide leukotrienes in endotoxin shock.

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 mg/kg) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase-HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC4 and LTD4 in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC4 and LTD4 which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin-induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.

Substituted 4,6-diaminoquinolines as inhibitors of C5a receptor binding.

The anaphylatoxin C5a is implicated in a number of inflammatory diseases. It is a highly cationic protein with 13 of 74 amino acids being either arginine or lysine. A search focusing on positively charged molecules, particularly amine-containing functionalities, led to the discovery of substituted 4,6-diaminoquinolines 1 [N,N'-bis(4-amino-2-methyl-6-quinolyl)urea] and 7 [6-N-(2-chlorocinnamoyl)-4,6-diamino-2-methylquinoline] as inhibitors of C5a receptor binding. These two compounds inhibited the binding of radiolabeled C5a to its receptor isolated from human neutrophils with IC50's = 3.3 and 12 micrograms/mL, respectively. Our efforts to enhance their potencies by chemical modification revealed a narrow profile of potency for effective C5a receptor binding inhibition.

Synthesis and antiinflammatory/analgesic activities of 11H-dibenzo[b, e,][1,4]dioxepinacetic acids.

A new class of tricyclic arylacetic acids was synthesized and evaluated as antiinflammatory/analgesic agents as well as inhibitors of prostaglandin synthetase. 11H-Dibenzo[b,e][1,4]dioxepin-2-, -3, -7, and -8-acetic and alpha-methylacetic acids and their derivatives were prepared by cyclization of diaryl ether precursors or by condensation of catechol and an aryl dihalide. The most potent compound in the carrageenan foot edema assay was alpha-methyl-11H-dibenzo[b,e][1,4]dioxepin-8-acetic acid (1 mg/kg = 43% inhibition). The most potent enzyme inhibitors were the 2-acetic acid and the alpha-methyl-7-acetic acid (IC50 = 0.1 microM). Some of these compounds were also found to be highly ulcerogenic.

Glycolipids as host resistance stimulators.

6-(5-Cholesten-3 beta-yloxy)hexyl 1-thio-beta-D-mannopyranoside (L-644,257) enhances natural host resistance in cyclophosphamide-treated mice against Pseudomonas aeruginosa in a dose-dependent manner. It is active sc, im, and ip but not orally. L-644,257 is substantially more protective against P. aeruginosa than its alpha anomer. The beta-L-fucose glycolipid is more effective when given im and ip than sc. The lactose and beta-D-glucose glycolipids were only marginally effective to nonprotective. The 17 beta-steroidal side chain of L-644,257 can be modified without substantial loss of protective activity.

Orally active beta-lactam inhibitors of human leukocyte elastase. 2. Effect of C-4 substitution.

The effect of changing the C-4 substituent of 3,3-diethyl-1-[(benzylamino)carbonyl]-2-azetidinone on inhibition of HLE and in a model of HLE-induced lung damage in hamsters was explored. Substituents at this position do not appear to interact strongly with HLE with the most potent compounds having k(obs)/[I] = 6900 M-1 s-1. However, substituents at this position had a marked effect on in vivo activity. The greatest oral activity in the lung hemorrhage assay was achieved with C-4 aryl carboxylic acid ethers (60-85% inhibition at 30 mg/kg po). Based upon the established mechanism of inhibition by these compounds, the C-4 substituent would be released, and therefore, the pharmacological potential of these C-4 substituents was of considerable concern. Fortunately, compounds containing 4-hydroxybenzoic acid and 4-hydroxyphenylacetic acid ethers at C-4 were among the most active analogs. These phenolic acids are also found as urinary metabolites in healthy humans. Other heteroaryls at C-4 were also orally active in this model despite relatively modest enzyme activity.

Orally active beta-lactam inhibitors of human leukocyte elastase-1. Activity of 3,3-diethyl-2-azetidinones.

A thorough analysis of the mechanism of inhibition of human leukocyte elastase (HLE) by a monocyclic beta-lactam and the mechanism of beta-lactam hydrolysis led to the preparation of potent and highly stable inhibitors of HLE. This work led to the identification of 4-[(4-carboxyphenyl)-oxy]-3,3-diethyl-1- [[(phenylmethyl)amino]carbonyl]-2-azetidinone (2) as the first orally active inhibitor of human leukocyte elastase (HLE). Analogs of 2 with different substituents on the urea N were synthesized and evaluated for their activity in vitro against HLE as well as in vivo in a hamster lung hemorrhage model. Compounds with a methyl or a methoxy group in the para position of the benzene ring were very potent in both assays. The results are discussed on the basis of the proposed model for the binding of this class of inhibitors to HLE and a possible mechanism of inhibition is presented.

Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

Requirement for epidermal growth factor receptor tyrosine kinase and for 12-lipoxygenase activity in the expression of 12-lipoxygenase in human epidermoid carcinoma cells.

We studied the dependency of basal 12-lipoxygenase (12-LOX; arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) expression and activity on functional protein tyrosine kinase of the epidermal growth factor receptor (EGF-R) and on 12-LOX activity in human A431 epidermoid carcinoma cells. Treatment of cells with inhibitors of high specificity for EGF-R tyrosine kinase, namely PD 153035 and 4,5-dianilinophthalimide (DAPH1), decreased cellular 12-LOX at mRNA, protein, and activity levels in a time- and dose-dependent manner, with PD 153035 being effective at concentrations below 1 microM. After 24-hr incubation with 10 microM PD 153035 or DAPH1, 12-LOX activity dropped to 14% (39%), and 12-LOX protein to 25% (24%) of control level. Inhibition of 12-LOX activity by the compound N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP) also resulted in a substantial decrease in 12-LOX protein expression. 12-LOX mRNA levels were diminished or undetectable by reverse transcription-polymerase chain reaction after cell treatment with these inhibitors. Our results suggest that basal 12-LOX expression in A431 tumor cells largely depends on functional EGF-R tyrosine kinase, and that 12-LOX activity is required in the EGF-elicited intracellular signaling maintaining the expression of 12-LOX.

Leukotriene C4 metabolism by hepatoma cells and liver.

The metabolism of the glutathionyl leukotriene LTC4 in the mercapturic acid pathway was studied in suspensions of AS-30D hepatoma cells and hepatocytes, as well as in vivo in the bile duct-cannulated rat and in primates. 1. Isolated hepatocytes actively took up cysteinyl leukotrienes and metabolized LTC4 not only to LTD4 and LTE4 but also to N-acetyl-LTE4 and to metabolites more polar than LTC4. 2. AS-30D hepatoma cells are deficient in the transport system for the uptake of cysteinyl leukotrienes. Peptide cleavage of LTC4 to LTD4 and LTE4 was catalyzed by ectoenzymes of these cells. Inactivation of gamma-glutamyltransferase by acivicin and inhibition of LTD4 dipeptidase by penicillamine largely prevented further catabolism of LTC4 and LTD4, respectively. 3. [3H]LTC4 injected i.v. into rats was rapidly eliminated from the circulating blood, taken up by the liver, and excreted into bile where 77% of the administered radioactivity was recovered within 1 hr. The biliary LTC4 metabolites included LTD4, N-acetyl-LTE4, and metabolites more polar than LTC4. 4. Inhibition of [3H]LTC4 metabolism in vivo by i.v. penicillamine shifted the pattern of biliary cysteinyl leukotrienes; an extended half-life of [3H]LTD4 was associated with a retarded formation of N-acetyl-LTE4 and of polar metabolites. 5. Endogenous cysteinyl leukotrienes elicited by trauma were measured after HPLC separation by radioimmunologic analysis in plasma and bile of rats. The biliary concentration of these leukotrienes was up to 100 times as great as in plasma. N-Acetyl-LTE4 was the predominant endogenous metabolite in rat bile. 6. In the monkey Macaca fascicularis, cysteinyl leukotrienes were predominantly eliminated from blood via the liver into bile; renal excretion amounted to about 50% of the hepatobiliary elimination. Absorption of cysteinyl leukotrienes from the intestine resulted in enterohepatic circulation of these mediators. 7. Metabolites of [3H]LTC4 injected i.v. in the monkey were analyzed in bile and urine. In addition to polar metabolites and a small percentage of [3H]LTD4, [3H]LTE4 was a predominant metabolite particularly in bile. LTE4 was also the major endogenous cysteinyl leukotriene detected by radioimmunologic analysis in monkey bile. 8. LTE4 was the predominant endogenous cysteinyl leukotriene measured in human bile in patients suffering from acute pancreatitis. The detected amounts of LTE4 may be sufficient to induce known phenomena associated with acute pancreatitis including the shock-like reaction.

12-lipoxygenase expression in human melanoma cell lines.

12-lipoxygenase (12-LOX) expression and function in the regulation of the metastatic phenotype was demonstrated in several murine melanoma lines before. Here we have provided novel evidences that, though at a low level (in max. 15% of the cell population), human melanoma lines (HT168, M1, HT199, HT18 and WM35) express the platelet-type isoform of 12-LOX both at mRNA and protein levels. 12-LOX expression was demonstrated in cultured tumor cells and in skin tumor xenografts. Comparison of the expression of 12-LOX in skin primary tumors and its lung metastases indicated a stable expression. The low level of 12-LOX expression in human melanoma cell lines suggests that other lipoxygenase(s) could also be responsible for the metabolism of arachidonic acid to 12-HETE breakdown products.

Aminoterminal propeptide of type III procollagen: a marker of hepatic fibrosis after bile duct obstruction in the monkey.

BACKGROUND/AIMS: In an experimental study in monkeys, liver fibrosis development after segmental bile duct obstruction was investigated and correlated with the aminoterminal propeptide of type III procollagen (PIIINP). MATERIALS AND METHODS: Segmental bile duct obstruction was produced by ligation and section of the left hepatic bile duct in all monkeys. Fibrosis induction was examined by intravenous leukotriene C4 (LTC4, 5 nmol/kg) application, endogenous LT-production stimulated by endotoxin (LPS,salmonella abortus equi, 50 ng/kg), fibrosis inhibition by dexamethasone (1 mg/kg) intramuscularly and subsequent endogenous LT-production stimulation by LPS (50 ng/kg). Ligated and unligated liver lobe biopsies were taken 3, 7 and 12 weeks after ligation. All portal areas were measured morphometrically. PIIINP was measured by a specific radioimmunoassay each week and correlated with the morphometric results. RESULTS: Bile duct obstruction leads to secondary sclerosing cholangitis with bile duct vanishing and subsequent biliary cirrhosis combined with perivenous sclerosis and cavernous transformation of the terminal vein. The collagen concentration increased in the nonligated lobe from mean +/-SEM 1.05 +/- 0.03% to 1.53 +/- 0.19% only after LTC4 and with no difference in the other groups. In the ligated lobe collagen concentration increased significantly in all groups continuously from 1.05 +/- 0.03% up to: controls 6.1 +/- 0.9%, dexamethasone 5.9 +/- 0.8%, LPS 8.2 +/- 0.8%, LTC4 9.075 +/- 1.4%. PIIINP concentration rose within 6 weeks in the controls with hepatic bile duct obstruction from 34.43 +/- 15 ng/ml up to 57 +/- 13.27 ng/ml, after dexamethasone to 48.5 +/- 18.23 ng/ml, after LPS to 57 +/- 13.27 ng/ml, after LTC4 to 80.25 +/- 16.04 ng/ml. After 12 weeks, PIIINP decreased in the controls resp. after dexamethasone to 41.25 +/- 6.94 ng/ml resp. 33.5 +/- 7.72 ng/ml and increased after LPS resp. LTC4 up to 64.25 +/- 17.07 ng/ml resp.104 +/- 22.46 ng/ ml. The correlation of collagen deposition and PIIINP was in the controls r = 0.83, after dexamethasone r = 0.71, after LPS r = 0.83 after LTC4 r = 0.91. CONCLUSION: PIIINP determination after segmental bile duct obstruction correlates with collagen deposition and allows evaluation of hepatic fibrosis activity.