Determination of antipyrine sulphoconjugates in isolated cultured rat hepatocytes.

1. An analytical method for the simultaneous determination of three major metabolites of antipyrine, namely, 3-hydroxymethylantipyrine (phase I), 4-hydroxyantipyrine sulphoconjugate (phase II) and norantipyrine sulphoconjugate (phase II) in cultured rat hepatocytes has been developed and applied to the study of induction or inhibition of these metabolic pathways following pretreatment of rats with several inducers or inhibitors. 2. Phenobarbital was the most effective inducer of the three pathways studied, particularly the formation of 4-hydroxyantipyrine sulphoconjugate; 3-methyl-cholanthrene (MC) increased the formation of the two sulphoconjugates, but decreased the formation of 3-hydroxymethylantipyrine. 3. Rifampicin inhibits antipyrine metabolism in rat. The three metabolic pathways studied were decreased by 30%. 4. SKF 525A markedly inhibited the three metabolic pathways; piperonyl butoxide and omeprazole decreased the three pathways but to a lesser extent. Cimetidine decreased the two sulphoconjugates pathways, but did not affect 3-hydroxymethylantipyrine formation.

Importance of plasminogen activator inhibitor type 1 (PAI-1) for preventing single chain urokinase plasminogen activator (scu-PA) conversion into two chain urokinase plasminogen activator (tcu-PA) in plasma in vitro.

We have studied the effects of PAI-1 on the conversion of scu-PA into tcu-PA in vitro in plasma containing or not a 125I-fibrin clot by determining tcu-PA activity on S2444. Two preparations of PAI-1 have been used, a fraction of medium conditioned with the monkey Vero cells (Vero-Prep), the antiurokinase activity of which is inhibited at 83% by anti PAI-1 IgG, or purified human PAI-1 from HT 1080 fibrosarcoma cells. Scu-PA purified from human kidney cells has been treated with diisopropylfluorophosphate before use. In plasma, conversion of scu-PA into the tc form is accelerated by addition of anti PAI-1 IgG. In plasma containing a clot, generation of tcu-PA, is considerably delayed after addition of the Vero-Prep or human PAI-1. Clot lysis is also decreased but to a lesser extent than it would be expected from the level of tcu-PA activity. Addition of anti PAI-1 antibodies shortens the lag phase before tcu-PA appears and moderatly accelerates clot lysis. These results demonstrate the importance of PAI-1 for the stability of scu-PA in plasma in vitro by delaying its conversion into tcu-PA.

Tumor necrosis factor (TNF) stimulates plasminogen activator inhibitor (PAI) production by endothelial cells and decreases blood fibrinolytic activity in the rat.

The effect of human recombinant tumor necrosis factor (TNF) was studied in vitro on human endothelial cells. TNF (1-1000 pg/ml) induced a dose-dependent increase in PAI level in the supernatant from 6 to 25 U/ml as estimated against urokinase. This effect was time-dependent. It was not suppressed by Polymyxin B thus excluding a possible contribution of an endotoxin contamination. Fibrinoenzymography performed after SDS-PAGE showed that this inhibitor neutralized urokinase and tissue plasminogen activator and gave rise to high molecular weight complexes. TNF (30 micrograms/kg) was also injected in rat. Blood fibrinolytic activity determined 4 hr later was decreased as estimated by the prolongation of the euglobulin clot lysis time from 37 to 188 min. Fibrinoenzymographic profile of the plasma was then characterized by a fainting of the tPA lysis band but the capacity of plasma to neutralize urokinase was not significantly modified. These results suggest that TNF could alter the fibrinolytic balance by stimulating PAI production at the endothelial level. This might be of importance in synergy with the TNF-induced procoagulant activity for promoting vascular occlusion of tumor capillaries.

Effect of polymyxin B and colimycin on induction of plasminogen antiactivator by lipopolysaccharide in human endothelial cell culture.

The effect of lipopolysaccharide (LPS) on the production of fibrinolytic inhibitor by human endothelial cells was determined because results of previous experiments have shown us that it is possible to stimulate this synthesis with muramyl dipeptide. Treatment of these cells with LPS resulted in a marked enhancement of fibrinolytic inhibitor, as estimated in a urokinase-induced fibrinolysis assay. A dose-response curve was obtained for LPS concentrations ranging from 10 to 1,000 ng/ml, thus demonstrating the great sensitivity of these cells. This inhibitor did not reduce plasmin activity and formed complexes with high- and low-molecular-weight urokinase as visualized by fibrin enzymography on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. The molecular weight of this inhibitor was estimated to be 54 to 58 kilodaltons. These findings led us to conclude that LPS stimulates formation of a plasminogen antiactivator. This LPS effect could be suppressed by polymyxin B and colimycin. The stimulatory effect of muramyl dipeptide required doses which were at least 1,000 times greater than those of LPS and was not decreased by polymyxin B. These results show the possibility of independent modulation of plasminogen antiactivator production at the endothelial level, which could be important in endotoxemia. Under these conditions colimycin might have an additional advantage for clinical use because of its ability to prevent fibrinolytic inhibition.

Stimulation of human endothelial cells by synthetic muramyl peptides: production of colony-stimulating activity (CSA).

The in vivo induction of colony-stimulating activity (CSA) by well-defined immunomodulatory synthetic muramyl peptides has been demonstrated recently in mice. In the present study, we tested the capacities of three muramyl peptides to induce CSA production in human endothelial cell (HEC) cultures. Two adjuvant-active peptides (MDP and Murabutide) induced CSA in the supernatant of cultured endothelial cells, whereas an adjuvant-inactive compound had no effect. This effect of MDP and Murabutide appeared to be time and concentration dependent and was not secondary to decreased production of inhibitors of colony formation. CSA secretion by stimulated HEC required de novo protein synthesis and did not result from the release of preformed active CSA. Maximal concentration appeared in the supernatant media within the first 24 h after addition of muramyl peptides, and a substantial second CSA secretion could be observed after a subsequent 24 h reexposure. This CAS was not dialyzable and promoted granulocyte-macrophage formation of nonadherent human marrow and unfractionated murine marrow. Our data demonstrate that the human endothelial cell is a target cell for MDP and Murabutide and suggest that in vivo endothelium might play an active role in muramyl peptide-induced modulation of hematopoiesis.

Increased synthesis of fibrinolytic inhibitor induced by muramyl dipeptide derivatives in human cultured endothelial cells.

A decreased fibrinolytic activity induced by bacterial products and some muramyl peptides has been previously demonstrated in macrophages preparations. Since vascular endothelial cells are important for the fibrinolytic balance, we have studied the effects of MDP derivatives on cultured endothelial cells. The supernatant of MDP and murabutide treated cell cultures exhibited an increased fibrinolytic inhibitory activity when tested with urokinase. The MDP(D-D)-treatment had no effect. This increased inhibitory activity was detectable in the supernatant after a 6 h treatment and was suppressed by the addition of puromycin to the cell cultures. Furthermore, the endothelial cell culture supernatant also reduced the lytic activity of the human plasma plasminogen activator induced by venostasis. This was enhanced by MDP treatment of the cultures. These in vitro results suggested that adjuvant-active muramyl peptides may regulate the fibrinolytic balance at the vessel wall level. This could be of possible significance in the transendothelial cell migration where the role of plasminogen activator(s) has been involved.