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D J Crutchley

Publications and source records attributed to D J Crutchley.

At least 19 recordsLinked to original sources
PubMed1995

Copper-induced tissue factor expression in human monocytic THP-1 cells and its inhibition by antioxidants.

BACKGROUND: Transition metals such as copper are known to initiate free radical formation and lipid peroxidation. Recent reports suggest that intracellular reactive oxygen intermediates can induce the transcription of a number of important genes. The present study examines the effects of copper and iron on the ability of monocytic cells to synthesize and express tissue factor, the potent procoagulant factor. METHODS AND RESULTS: Exposure of human monocytic THP-1 cells to 5 to 10 mumol/L Cu2+ led to cell damage and the expression of tissue factor activity to levels up to 70 times higher than control, as measured by a single-stage plasma coagulation assay. These effects were seen only in the presence of a lipophilic chelating agent, 8-hydroxyquinoline, suggesting that intracellular transport of Cu2+ was required. The effects of Cu2+ were mimicked by ceruloplasmin but not by Fe3+ or hemin. The induction of tissue factor activity by Cu2+ was slow in onset (6 hours) but sustained (24 hours) and was accompanied by increased tissue factor mRNA levels, measured by reverse transcription/polymerase chain reaction after annealing with oligomer primers. Increases in tissue factor protein, measured by a specific immunoassay, also occurred but were smaller than those in activity. Cu2+, therefore, appears to act at both the transcriptional and posttranslational levels. The effects of Cu2+ were inhibited by a number of lipophilic antioxidants, including probucol, vitamin E, butylated hydroxytoluene, and a 21-aminosteroid, U74389G. CONCLUSIONS: Exposure of monocytes to oxidizing conditions may lead to the expression of high levels of tissue factor activity, with accompanying risk for disseminated intravascular coagulation, and this may be inhibited by lipophilic antioxidants.

Antioxidants↗
PubMed1995

K+ channel blockers inhibit tissue factor expression by human monocytic cells.

Human monocytes express the important procoagulant protein, tissue factor (TF), after stimulation by a variety of agents, including bacterial lipopolysaccharide (LPS). Monocyte TF expression may contribute to intravascular coagulation in a number of disease states. The present studies show that monocytic cell TF expression can be inhibited by several agents known to block cellular K+ channels. Exposure of human peripheral blood to 100 ng/mL LPS for 2 hours led to pronounced TF procoagulant activity associated with the mononuclear cell fraction. This was inhibited by 4-aminopyridine (2 mmol/L), tetraethylammonium chloride (10 mmol/L), and apamin (1 mumol/L). In contrast, charybdotoxin (100 nmol/L) was inactive. More detailed studies were carried out in cultured human monocytic tumor THP-1 cells. These cells exhibited low but detectable levels of TF mRNA, measured by reverse transcription and polymerase chain reaction; cell surface procoagulant activity, measured by a plasma clotting assay; and cell homogenate TF antigen, measured by immunoassay. Exposure of THP-1 cells to 1 microgram/mL LPS led to threefold to fivefold increases in all three parameters. Basal and LPS-induced levels of all three parameters were reduced in a dose-dependent manner by 4-aminopyridine (I50, 1 mmol/L) and tetraethylammonium chloride (I50, 20 mmol/L) but not by apamin or charybdotoxin. Expression of TF activity was also inhibited by glibenclamide, an inhibitor of ATP-dependent K+ channels (I50, 25 mumol/L). These results suggest that facilitation of TF synthesis may be an important role for K+ channels in monocytes.

Apamin↗
PubMed1994

Effects of prostacyclin analogs on the synthesis of tissue factor, tumor necrosis factor-alpha and interleukin-1 beta in human monocytic THP-1 cells.

Previous studies have shown that prostacyclin analogs can inhibit the expression of tissue factor (TF) procoagulant activity by human monocytes. The present studies have investigated this phenomenon further, by using a plasma coagulation assay to measure cellular TF activity, an immunoassay to measure TF antigen and reverse transcription/polymerase chain reaction with appropriate oligomer primers to measure TF mRNA. Iloprost and cicaprost inhibited lipopolysaccharide-induced increases in TF activity, antigen and mRNA (50% inhibition, 2-8 nM), with no apparent effect on TF mRNA stability. These agents therefore act at or before the level of transcription of the TF gene. The analogs were more potent inhibitors of tumor necrosis factor-alpha synthesis (50% inhibition at 334 +/- 40 pM cicaprost or 846 +/- 182 pM iloprost) and extraordinarily potent when combined with a phosphodiesterase inhibitor (50% inhibition at 101 +/- 31 pM iloprost in the presence of 20 microM isobutylmethylxanthine). Iloprost and cicaprost were less potent in inhibiting the synthesis of interleukin-1 beta (50% inhibition, 50-100 nM). Cicaprost inhibited lipopolysaccharide-induced increases in mRNA levels for TF, tumor necrosis factor-alpha and interleukin-1 beta; differential potency was again observed. We conclude that these three important monocyte functions can be down-regulated by prostacyclin analogs, and with differential sensitivity. Furthermore, the extreme sensitivity of tumor necrosis factor-alpha synthesis to inhibition suggests that such inhibition may be a major physiological function of prostacyclin itself.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Effects of prostacyclin analogues on human endothelial cell tissue factor expression.

Prostacyclin analogues have been reported to inhibit the expression of tissue factor procoagulant activity in human monocytes, primarily by elevating intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP). The present studies have investigated whether prostacyclins can also inhibit tissue factor expression in endothelial cells. Iloprost, carbacyclin, and ciprostene had no effect on human umbilical vein endothelial tissue factor activity induced by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), or interleukin-1 beta (IL-1 beta). Iloprost failed to elevate intracellular levels of cAMP, even when combined with a phosphodiesterase inhibitor. In contrast, forskolin increased endothelial cAMP and inhibited tissue factor expression. Conditioned medium from LPS-challenged monocytic THP-1 cells, which contained both TNF-alpha and IL-1 beta, induced endothelial cell procoagulant activity to levels 20-fold higher than those achieved in response to LPS alone. Iloprost abolished LPS-induced TNF-alpha secretion by THP-1 cells and inhibited IL-1 beta secretion by 45%. In keeping with this, iloprost reduced levels of TNF-alpha and IL-1 beta mRNA in LPS-challenged cells. Treatment of THP-1 cells with iloprost strongly inhibited the ability of conditioned medium to induce endothelial tissue factor expression, an effect that was mimicked by treating the medium with blocking antibodies to the cytokines. We conclude that although prostacyclin analogues do not directly suppress endothelial tissue factor expression due to their failure to elevate cAMP, they may do so indirectly by inhibiting the amplification produced by monocyte-derived cytokines.

Cells, Cultured↗
PubMed1992

Prostacyclin analogues inhibit tissue factor expression in the human monocytic cell line THP-1 via a cyclic AMP-dependent mechanism.

Increased expression of tissue factor procoagulant by peripheral blood monocytes has been implicated in a number of thrombotic disorders. The present studies were undertaken to determine whether stable analogues of prostacyclin, a potent endothelium-derived platelet inhibitor and vasodilator, could inhibit tissue factor expression by human monocytic cells. Exposure of monocytic tumor THP-1 cells to 100 ng/ml endotoxin, 2 units/ml interleukin-1 beta, or 5 ng/ml tumor necrosis factor-alpha for 4 hours led to increased tissue factor procoagulant activity. Preincubation for 30 minutes with iloprost, ciprostene, and carbacyclin led to a dose-dependent inhibition of tissue factor expression induced by all three challenging agents. Iloprost was the most potent: 50% inhibition occurred at 5 nM, a concentration close to the reported dissociation constant for iloprost binding to the platelet prostacyclin receptor. An orally active analogue, cicaprost, was equally effective against endotoxin-induced tissue factor expression. Carbacyclin and ciprostene were 100 times less potent. Iloprost prevented the endotoxin-induced expression of tissue factor antigen on the surface of THP-1 cells, as determined by flow cytometry. Iloprost (500 pM-50 nM) increased intracellular levels of cyclic AMP. This effect was potentiated by isobutylmethylxanthine, an inhibitor of phosphodiesterase. The inhibitory effects of iloprost on tissue factor expression were also potentiated by isobutylmethylxanthine and mimicked by forskolin and dibutyryl cyclic AMP but not dibutyryl cyclic GMP. These results suggest that prostacyclin may play a role in downregulating tissue factor expression in monocytes, at least in part via elevation of intracellular levels of cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1991

The stable prostacyclin analog, iloprost, and prostaglandin E1 inhibit monocyte procoagulant activity in vitro.

Exposure of human peripheral blood to 100 ng/mL of bacterial endotoxin for 2 hours resulted in a 20-fold increase in monocyte procoagulant activity. The activity was functionally identified as tissue factor, because it was not expressed in plasma deficient in factor VII and was specifically inhibited by a monoclonal antibody directed against human tissue factor. When the stable prostacyclin analog, iloprost, was added to blood 30 minutes before endotoxin, a dose-dependent inhibition of monocyte procoagulant activity occurred, with an I50 of 20 nmol/L. Prostaglandin E1 (PGE1) produced similar effects, with an I50 of 150 nmol/L. Exposure of THP-1 monocytic cells to 100 ng/mL endotoxin resulted in a threefold increase in procoagulant activity after 2 hours and a 20-fold increase after 6 hours. A 30-minute pretreatment with iloprost or PGE1 again inhibited development of procoagulant activity, with I50 values of 5 nmol/L and 150 nmol/L, respectively. Treatment of THP-1 cells with iloprost 2 hours after exposure to endotoxin significantly inhibited further increases in procoagulant activity. Iloprost was less potent under these conditions, 30% inhibition being obtained at 100 nmol/L and 70% at 1 mumol/L. These results suggest that prostacyclin may be a physiologic modulator of monocyte tissue factor expression; in addition, its stable analog, iloprost, may have clinical potential for the treatment of thrombotic disorders in which elevated monocyte procoagulant activity plays a role.

Alprostadil↗
PubMed1987

Endotoxin-induced secretion of an active plasminogen activator inhibitor from bovine pulmonary arterial and aortic endothelial cells.

Treatment of bovine pulmonary arterial and aortic endothelial cells for 24 h with 10-20 ng/ml endotoxin appeared to suppress urokinase secretion by over 90%. Immunodepletion of urokinase from conditioned medium revealed high levels of a fully active plasminogen activator inhibitor (PAI). In contrast, medium from aortic cells not treated with endotoxin expressed low levels of a latent PAI. The endotoxin-induced PAI from both cell types was not immunoprecipitated by incubation with IgG directed against the endothelial-type inhibitor, PAI-1, or antiserum directed against the placental-type inhibitor, PAI-2. Endotoxin-induced PAI was inactivated by 0.2-1.0 M of 2-mercaptoethanol, but unaffected by treatment with up to 500 microM of chloramine-T. We conclude that the exposure of bovine endothelial cells to endotoxin causes the secretion of a fully active PAI with properties different from those reported for PAI-1.

Animals↗
PubMed1987

Monovalent cation dependence of tissue plasminogen activator synthesis by HeLa cells.

HeLa cells synthesize and secrete increased levels of tissue plasminogen activator (tPA) when incubated for 18 h with 10-20 nM phorbol myristate acetate. This response was inhibited by a number of conditions which affect intracellular Na+ and K+ concentrations. Removing extracellular Na+, while maintaining isotonicity with choline+, reduced the secretion of both functional and antigenic tPA in a linear fashion. A series of cardiac glycosides and related compounds strongly inhibited tPA secretion with the following rank order of potency: digitoxin = ouabain greater than digoxin greater than digitoxigenin greater than digoxigenin greater than digitoxose greater than digitonin. These compounds also inhibited cellular Na+/K+-ATPase activity over an identical concentration range. Two compounds which selectively increase cellular permeability to K+, valinomycin, and nigericin, strongly inhibited tPA secretion, with IC50 values of approximately 50 nM. In contrast, monensin, which selectively increases cellular permeability to Na+, was much less active. Valinomycin, but not nigericin, also inhibited cellular Na+/K+-ATPase activity. Phorbol myristate acetate, 5-20 nM, increased Na+/K+-ATPase activity up to 2-fold and tPA secretion up to 15-fold. We conclude that the secretion of tPA by HeLa cells treated with phorbol myristate acetate proceeds via a mechanism which requires extracellular Na+ and a functional Na+/K+-ATPase ("sodium pump") enzyme.

Cations, Monovalent↗
PubMed1986

Calcium regulation of tissue plasminogen activator and prostaglandin biosynthesis in HeLa cells.

Phorbol 12-myristate 13-acetate, 1-20 nM, induced the synthesis in HeLa cells of a 65 200 Mr tissue-type plasminogen activator, and of prostaglandin E2. Omission of Ca2+ from the incubation medium inhibited the induction of plasminogen activator synthesis by 40-60% and abolished the induction of prostaglandin E2 synthesis. Maximal plasminogen activator synthesis could be maintained at extracellular Ca2+ concentrations of approx. 0.1 mM, while maximal prostaglandin synthesis required at least 0.45-0.9 mM Ca2+. The induction of each factor was inhibited by 10-100 microM 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular C2+ mobilization. Prostaglandin synthesis, but not plasminogen activator synthesis, was also inhibited by 10-100 microM verapamil and nifedipine, which inhibit intracellular Ca2+ uptake via the so-called 'slow-channels' and by 0.5-10 microM trifluoperazine, an inhibitor of calmodulin. Neither plasminogen activator synthesis nor prostaglandin synthesis were stimulated by 5-50 microM 1-oleoyl-2-acetylglycerol or 1-250 microM 1,2-dioctanoylglycerol, alone and in combination with 50 nM-1 microM ionophore A23187. These results indicate that the synthesis of plasminogen activator and prostaglandins in HeLa cells is Ca2+-dependent, and that the Ca2+ requirements for each process are not identical. Thus, Ca2+ regulation of the production of tissue plasminogen activator and prostaglandin E2 occurs at multiple points in their biosynthetic pathways.

Calcimycin↗
PubMed1986

Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells.

We have examined the effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner (threshold dose, 0.1 ng/ml; maximal dose, 10-100 ng/ml). The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP) abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56 degrees C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of 125I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa. The complex could be detected by chromatography on Sephadex G-100, but not by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These findings suggest that low doses of endotoxin suppress fibrinolytic activity in endothelial cells by stimulating the production or expression of a fast-acting, relatively labile inhibitor of plasminogen activator.

Animals↗
PubMed1985

Hydroxyeicosatetraenoic acids and other unsaturated fatty acids inhibit endotoxin-induced thromboplastin activity in human monocytes.

Human peripheral blood monocytes possessed increased thromboplastin activity when exposed to 10 micrograms/ml of bacterial endotoxin for 2 h. The effects of endotoxin were strongly inhibited by 10-20 microM of several unsaturated fatty acids: arachidonic, dihomo-gamma-linolenic, linoleic, linolenic and oleic acids. Two saturated fatty acids, arachidic and stearic acids, produced minimal inhibition at 20 microM. Three lipoxygenase-derived hydroxyfatty acids were also inhibitory, with IC50 values of 10 microM (5- and 12-hydroxyeicosatetraenoic acids) and 20 microM (15-hydroxyeicosatetraenoic acid). Leukotriene B4, 1 nM to 1 microM, was inactive. None of the fatty acids affected the ability of standard thromboplastin to shorten the clotting time of normal plasma or affected the activity of endotoxin as measured by a Limulus clotting assay.

Arachidonic Acid↗
PubMed1985

The pulmonary endothelial surface.

The understanding of endothelial metabolic properties has increased dramatically in recent years. Endothelial cells (ECs) process hormones, drugs, and many blood-borne substances by means of enzymes and transport processes. In turn, some hormones, blood cells, and cellular products interact with ECs via specific receptors on the luminal surface. Functional complexity is exemplified by the metabolism of the adenine nucleotides. ATP, ADP, and AMP are metabolized by enzymes of the endothelial surface to release adenosine, which may be immediately taken up into endothelium and reincorporated intracellularly into nucleotides. Equally complex is the metabolism of the kinins and angiotensins by ECs. Bradykinin is inactivated whereas angiotensin I is converted to angiotensin II. Bradykinin not thus degraded can act on endothelial receptors and stimulate the release of prostacyclin (PGI2). Thus bradykinin can amplify the release of another vasodilator, PGI2, and can stimulate the release of a powerful antiaggregatory agent (PGI2). Many of these complex metabolic reactions occur at the endothelial surface, a structure that is itself complex. ECs possess endothelial projections and caveolae as well as a fuzzy coat, or glycocalyx. Functions of the endothelial glycocalyx are not well understood, but the glycocalyx can now be visualized: it may act as a molecular sieve and provide a substratum for the initiation and progression of immunologic reactions.

Adenosine Diphosphate↗
PubMed1985

Glucocorticoid modulation of prostacyclin production in cultured bovine pulmonary endothelial cells.

The present study has examined the ability of glucocorticoids to modify the production of prostacyclin by endothelial cells derived from bovine pulmonary arteries. Binding studies with [3H]dexamethasone indicated that these cells possessed high-affinity binding sites for glucocorticoids (Kd approximately 4 nM). The cells released prostacyclin, measured serologically as its hydrolysis product 6-keto-prostaglandin F1 alpha. The release was strongly stimulated by a 5-min incubation with 50 nM bradykinin, 2 microM calcium ionophore A23187 or 1 to 5 microM arachidonic acid. Dexamethasone, 1 to 20 nM, suppressed prostacyclin release in response to bradykinin and ionophore in a dose-dependent manner. The suppressive effects required 24 hr for full expression. Maximal inhibition of bradykinin-induced prostacyclin release was approximately 65% and that of ionophore-induced release was approximately 35%. Total inhibition was not observed. Hydrocortisone at 20 nM inhibited bradykinin-induced release of prostacyclin by approximately 45% but had no effect on ionophore-induced release. Neither glucocorticoid inhibited prostacyclin release in response to arachidonic acid. Triton X-100 extracts and conditioned media from cells treated with 20 nM dexamethasone failed to modify prostacyclin release when added to fresh endothelial cells. We conclude that dexamethasone, in concentrations likely to produce virtually complete binding site occupancy, inhibits agonist-induced release of prostacyclin from bovine pulmonary endothelial cells. Inhibition appears to occur at the level of intracellular arachidonic acid release. However, we have been unable to obtain definitive evidence for the production of a macromolecular phospholipase inhibitor by endothelial cells in response to these low concentrations of steroid.

Animals↗
PubMed1984

Effects of inhibitors of arachidonic acid metabolism on thromboplastin activity in human monocytes.

Human isolated monocytes possess low levels of procoagulant activity, which was stimulated 10-30 fold by brief (2 hr) exposure to 10 micrograms/ml endotoxin. This activity was expressed in normal or factor XII-deficient plasma, but lost in plasma deficient in factors X or VII, indicating that it was due to thromboplastin. The stimulation of monocyte thromboplastin by endotoxin was inhibited in a dose-dependent manner by two phospholipase A2 inhibitors, 4-bromophenacyl bromide and quinacrine, and by two lipoxygenase inhibitors, eicosatetraynoic acid and nordihydroguaiaretic acid. Two cyclooxygenase inhibitors, aspirin and indomethacin, prevented endotoxin-induced increases in thromboxane B2 production but had no effect on thromboplastin production. These results suggest that a component in the sequence of lipid deacylation, arachidonic acid release, and metabolism via lipoxygenase may mediate the stimulation of monocyte thromboplastin activity by endotoxin.

Arachidonate Lipoxygenases↗
PubMed1983

Serum-dependent induction of plasminogen activator in human fibroblasts by catecholamines and comparison with the effects of prostaglandin E1.

Human foreskin fibroblasts produce the protease plasminogen activator, as shown by the ability of cell extracts to lyse 125I-labelled fibrin in the presence of plasminogen. Cellular plasminogen activator was stimulated up to 3-fold by 0.01-10 microM epinephrine, norepinephrine, or isoproterenol. Increases in plasminogen activator were slow in onset (24 h) and long-lived (greater than 48 h), and were abolished by 10 micrograms/ml of cycloheximide or 1 microgram/ml of actinomycin D, suggesting de novo synthesis of the protease. Stimulation of plasminogen activator by catecholamines was inhibited by 10 microM propranolol but not by 10 microM phentolamine, suggesting the involvement of beta-adrenergic receptors. Catecholamines stimulated plasminogen activator only in the presence of fetal bovine serum; under serum-free conditions they were inhibitory. Serum did not appear to alter the uptake and metabolism of epinephrine during incubation with fibroblasts. The ability of fetal bovine serum to support the induction of plasminogen activator by either 1 microM epinephrine or 3 microM prostaglandin E1 was maintained following dialysis but lost on heating (70 degrees C, 10 min) or acidification (pH 2.5). Human and calf sera supported the stimulatory effects of prostaglandin E1 but not of epinephrine. These results indicate that serum may influence the synthesis of plasminogen activator in cultured cells by modifying their response to vasoactive hormones.

Alprostadil↗
PubMed1983

Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Studies with lower homologs and calcium antagonists.

Bovine pulmonary artery endothelial cells, in serum-free culture medium, release small quantities of prostacyclin and thromboxane A2 (3-10 and 0.1-0.3 ng/ml; measured as immunoreactive 6-ketoprostaglandin F1 alpha and thromboxane B2, respectively). The release of these substances is stimulated by up to 20-fold during a 3 min incubation with the vasodilator, bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). Endothelial cells incubated with [3H]arachidonic acid for 24 h and then exposed to bradykinin for 3 min release 3H into the medium, approximately 65% of which co-chromatographs with 6-ketoprostaglandin F1 alpha and 3% with thromboxane B2. The effects of bradykinin are dose-related and are often discernible when the hormone is used at concentrations believed to occur physiologically (10 pg/ml; approximately 10 pM). Furthermore, the bradykinin molecule must be intact: none of its lower homologs affects the release of prostacyclin, thromboxane A2, or 3H unless used at concentrations (1 microM or higher) unlikely to be achieved in vivo. The release appears to involve calcium uptake and calmodulin: it is abolished by EGTA (5 mM) and inhibited by the 'slow channel' calcium antagonists, verapamil and nifedipine (10-100 microM), and by the calmodulin inhibitor, trifluoperazine (3-30 microM). Our findings suggest that bradykinin exerts some of its hormonal effects by acting on specific receptors possessed by vascular endothelial cells; receptor activation is associated with calcium transport, arachidonate mobilization, and a selective synthesis of prostacyclin, a vasodilator in its own right.

Animals↗
PubMed1983

Induction of plasminogen activator by 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. Suppression by inhibitors of fatty acid lipoxygenase.

HeLa cells incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and rat basophilic leukemia (RBL-1) cells incubated with calcium ionophore, showed increased levels of the protease plasminogen activator. These treatments have previously been shown to stimulate the cellular metabolism of arachidonic acid. The induction of plasminogen activator in both cell types was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, two compounds known to inhibit arachidonate metabolism via lipoxygenases. In contrast, indomethacin, which selectively inhibits arachidonate metabolism via cyclooxygenase, was inactive. The levels of four enzyme markers in HeLa cells were unchanged by treatment with TPA plus the lipoxygenase inhibitors, indicating that the inhibitors did not exert their effects on plasminogen activator via general cell toxicity. HeLa cells preincubated with [3H]arachidonate and subsequently challenged with TPA produced small amounts of material with the chromatographic mobilities and resistance to indomethacin expected of hydroxylated fatty acids derived via lipoxygenase. RBL-1 cells have been shown previously to produce leukotrienes and other lipoxygenase metabolites when treated with calcium ionophore. Plasminogen activator in HeLa cells was stimulated by up to 2.5-fold by incubation with 0.5-2 micrograms/ml 5-hydroxyeicosatetraenoic acid. Our results suggest that the induction of plasminogen activator in HeLa and RBL-1 cells is not mediated by prostaglandins or thromboxanes, but may be mediated or modulated by arachidonate metabolites derived via a lipoxygenase pathway.

5,8,11,14-Eicosatetraynoic Acid↗