Endogenous AA metabolites and their possible role in tracheal smooth muscle tone in guinea pigs

The effects of endogenous arachidonic acid (AA) metabolites on inherent tone and histamine-induced constriction were studied in guinea pig tracheal smooth muscle. Inhibitors of either cyclooxygenase (indomethacin) or lipoxygenase (AA 861) significantly diminished the inherent tone of the muscle. Antagonists of prostaglandins (SC 19220) or leukotrienes (FPL 55712) also diminished the inherent tone, whereas an inhibitor of thromboxane synthase (OKY 046) had no significant effect. These results show that the metabolites of the lipoxygenase pathway as well as prostaglandins also participate in the maintenance of inherent tone. To reexamine the previously reported augmentation of histamine constriction induced by the inhibitors and the antagonists, we compared the active tension of the muscle measured from the maximum relaxed level as the base line to eliminate the fluctuation of inherent tone. Such comparison revealed that the inhibitors and the antagonists have no augmentative effect on either the maximum response to histamine or the concentration required to produce 50% of maximum active tension and that there is functional synergism between the exogenously added histamine and the endogenously produced AA metabolites. Therefore the zero active tension is useful as a base line to compare the contractile response of a drug-treated preparation with that of a nontreated preparation.     

Role of prostaglandins and leukotrienes in the synergistic effect of oxytocin and corticotropin-releasing hormone (CRH) on the contraction force in human gestational myometrium.

We have recently demonstrated that corticotropin releasing hormone (CRH) potentiates the contractile response to oxytocin of human gestational myometrium, using a high flow microsuperfusion system and electrical field stimulation. We now report this potentiation to be equivalent to that of 1 nM prostaglandin F2 alpha (PGF2 alpha), while 10 nM PGF2 alpha did not potentiate the response to oxytocin. Prostaglandin E2 (PGE2) also showed no augmentation of the contraction force of the myometrium in response to oxytocin. The CRH potentiated response was inhibited by the lipoxygenase and cyclooxygenase inhibitor BW755C (1 microM) and by indomethacin (0.1 microM), but not by the lipoxygenase inhibitor BW4C (1 microM). Measurements of prostaglandins in the superfusate showed no significant trends. It is concluded that the potentiation of contraction force to oxytocin by CRH is dependent on prostaglandins, probably PGF2 alpha and that leukotrienes, generated via the lipoxygenase pathway are not involved.     

Involvement of lipoxygenase products in myometrial contractions

Studies with an preparation of guinea pig uterus suggest the possible involvement of the lipoxygenase pathway of arachidonic acid (AA) metabolism in myometrial contractions. Female guinea pigs were sensitized to ovalbumin (OA) on day one of their estrous cycle. On day 14, these pigs were anesthetized and the uterus was cannulated for measuring contractions. OA challenge, with histamine antagonism (methpyrilene) resulted in utering contractions which significantly raised myometrial tonus, presumably due to AA metabolites. Pretreatment with high doses of indomethacin resulted. in only 60% inhibition of the OA induced contraction, suggesting the remaining contraction was due to something other than cyclooxygenase products. In the presence of indomethacin and methapyrilene, the addition of AA to increase available substrate caused increased myometrial tone following antigen challenge. This increase in uterine tone was inhibited in a dose dependent fashion by FPL-55712 demonstrating that leukotrienes can contract the uterus and that antigen challenge may provide a means for studying leukotriene involvement in uterine pathophysiology.     

Involvement of lipoxygenase products in myometrial contractions

Studies with an in situ preparation of guinea pig uterus suggest the possible involvement of the lipoxygenase pathway of arachidonic acid (AA) metabolism in myometrial contractions. Female guinea pigs were sensitized to ovalbumin (OA) on day one of their estrous cycle. On day 14, these pigs were anesthetized and the uterus was cannulated for measuring contractions. OA challenge, with histamine antagonism (methapyrilene) resulted in uterine contractions which significantly raised myometrial tonus, presumably due to AA metabolites. Pretreatment with high doses of indomethacin resulted in only 60% inhibition of the OA induced contraction, suggesting the remaining contraction was due to something other than cyclooxygenase products. In the presence of indomethacin and methapyrilene, the addition of AA to increase available substrate caused increased myometrial tone following antigen challenge. This increase in uterine tone was inhibited in a dose dependent fashion by FPL-55712 demonstrating that leukotrienes can contract the uterus and that antigen challenge may provide a means for studying leukotriene involvement in uterine pathophysiology.     

Metabolism of arachidonic acid in sheep uterus: in vitro studies

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins - PGF2alpha and prostacyclin derivative 6-keto PGF1alpha. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.     

Paeonol alleviates primary dysmenorrhea in mice via activating CB2R in the uterus

Background and purposePrimary dysmenorrhea is the most common gynaecologic problem in menstruating women and is characterized by spasmodic uterine contraction and pain symptoms associated with inflammatory disturbances. Paeonol is an active phytochemical component that has shown anti-inflammatory and analgesic effects in several animal models. The aim of this study was to explore whether paeonol is effective against dysmenorrhea and to investigate the potential mechanism of cannabinoid receptor signalling.Experimental approachDysmenorrhea was established by injecting oestradiol benzoate into female mice. The effects of paeonol on writhing time and latency, uterine pathology and inflammatory mediators were explored. Isolated uterine smooth muscle was used to evaluate the direct effect of paeonol on uterine contraction.Key resultsThe oral administration of paeonol reduced dysmenorrhea pain and PGE2 and TNF-α expression in the uterine tissues of mice, and paeonol was found to be distributed in lesions of the uterus. Paeonol almost completely inhibited oxytocin-, high potassium- and Ca²⁺-induced contractions in isolated uteri. Antagonists of CB2R (AM630) and the MAPK pathway (U0126), but not of CB1R (AM251), reversed the inhibitory effect of paeonol on uterine contraction. Paeonol significantly blocked L-type Ca²⁺ channels and calcium influx in uterine smooth muscle cells via CB2R. Molecular docking results showed that paeonol fits well with the binding site of CB2R.Conclusions and implicationsPaeonol partially acts through CB2R to restrain calcium influx and uterine contraction to alleviate dysmenorrhea in mice. These results suggest that paeonol has therapeutic potential for the treatment of dysmenorrhea.     

Significance of myocardial eicosanoid production

Summary The precise role of eicosanoids in the development of myocardial injury during ischemia and reperfusion is still a matter of debate. Enhanced local production of these bioactive compounds appears to be a common response to tissue injury. Most likely, the cardiac tissue has the capacity to generate prostaglandins, thromboxanes as well as leukotrienes. Prostacyclin (PGI,) is the major eicosanoid produced by the jeopardized myocardium. In addition, at sites of tissue injury activation of platelets and infiltrating leukocytes results in the formation of considerable amounts of thromboxanes and leukotrienes. The production of eicosanoids requires prior release of arachidonic acid (AA) from phospholipids. Both ischemia and reperfusion are associated with a rise in the tissue level of AA. The absence of a proportional relationship between the tissue level of AA and the amounts of PGI, produced suggests that the sites of AA accumulation and PGI₂ formation are different. It is conceivable that AA accumulation is mainly confined to myocytes, whereas the capacity to synthesize PGI, mainly resides in vascular cells. Both beneficial and detrimental effects of eicosanoids on cardiac tissue have been described. Prostaglandins act as vasodilators. Besides, some of the prostaglandins, especially PGI,, are thought to possess cyto-protective properties. Thromboxanes and leukotrienes may impede blood supply by increasing smooth muscle tone. Besides, leukotrienes augment vascular permeability. Experimental studies, designed to evaluate the effect of pharmacological agents, like PGI₂-analogues and lipoxygenase and cyclo-oxygenase inhibitors, indicat that eicosanoids influence the outcome of myocardial injury. However, the delineation of the physiological significance of the locally produced eicosanoids is complicated by such factors as the wide variety of AA derivatives produced and the dose-dependency of their effects.     

Cyclooxygenase enzymes and prostaglandins in reproductive tract physiology and pathology

Prostaglandins, thromboxanes (TX) and leukotrienes, collectively referred to as eicosanoids, are cyclooxygenase (COX) metabolites of arachidonic acid (AA). Prostaglandins, have been recognised for many years as key molecules in regulating reproductive tract physiology and pathology. Numerous recent studies in in vitro model systems and knockout mouse models have demonstrated specific functional roles for the respective cyclooxygenase enzymes, prostaglandins and prostanoid receptors. Here we review the findings obtained in several of these studies with emphasis on the roles played by cyclooxygenase enzymes and prostaglandins, specifically prostaglandin E2 (PGE2) and F2alpha in reproductive tract physiology and pathology.     

On the role of arachidonic acid metabolites in mast-cell mediated mitogenesis in the rat

We investigated whether the mitogenic response induced by local mast-cell secretion in the rat mesentery was affected by suppression of phospholipase A2, lipoxygenase, or cyclooxygenase in arachidonic acid metabolism. Enzyme inhibitor was given in a single intravenous dose 5 min before intraperitoneal injection of the mast-cell secretagogue 48/80. Mepacrine, a phospholipase A2 inhibitor, suppressed the generation of both leukotrienes (SRS) and prostaglandins (PG), whereas the lipoxygenase inhibitor BW 755C reduced the generation of SRS, and the cyclooxygenase inhibitor indomethacin significantly suppressed the generation of PG. None of the enzyme inhibitors affected the basal mesenteric histamine content or histamine release in the mesentery after exposure to 48/80, and none of them affected mast-cell-mediated mitogenesis in the mesentery as judged by specific DNA activity and mitosis counting. The stimulation of DNA synthesis and mitosis initiated by secreting mast cells is apparently not mediated or modulated by synthesis of leukotrienes, prostaglandins, or other known arachidonic acid metabolites.     

Role of Ca(2+)-independent phospholipase A(2) and cyclooxygenase/lipoxygenase pathways in the nitric oxide production by murine macrophages stimulated by lipopolysaccharides.

There is evidence of molecular cross talk between inflammatory mediators such as nitric oxide (NO) and prostaglandins (PG), which may regulate tissue homeostasis and contribute to pathophysiological processes. Here we examine the role of endogenous arachidonic acid (AA) and its AA metabolites in the regulation of NO release by lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7. Our results suggest that bromoenol lactone-sensitive phospholipase A(2) is involved in AA release and the subsequent PG and leukotriene (LT) production. The cyclooxygenase inhibitor, indomethacin, and lipoxygenase inhibitors such as baicalein and zileuton blocked the dose-dependent PGE(2) or LTB(4) and nitrite (NO(2)(-)) production induced by LPS. Furthermore, the effects of indomethacin were reverted by exogenous PGE(2) and forskolin, whereas AH23848B, an EP(4) PGE(2) subtype receptor antagonist, decreased NO(2)(-) release. On the other hand, the effect of baicalein on NO(-)(2) production was reverted by exogenous LTB(4) and the fibrate WY 14,643, a natural and a synthetic peroxisome proliferator-activated receptor alpha (PPAR alpha), respectively. Thus, PGE(2) via EP(4) receptor/cAMP and LTB(4) via PPAR alpha may be involved in the control of NO synthesis by LPS in macrophage RAW 264.7 cultures.     

Inflammation: a novel mechanism for the transport of extracellular nucleotide-induced arachidonic acid by S100A8/A9 for transcellular metabolism

Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide-mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5-lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5-lipoxygenase-mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide-induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B(4)(LTB(4)), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.     

Effects of antigen on tracheal circulation and smooth muscle in sheep of different ages

The effects of Ascaris suum antigen on tracheal circulation and tracheal smooth muscle tone were compared in two groups of sheep: the first group was 1 yr old (14 sheep) and the second 5 yr old (8 sheep). Cranial tracheal arteries of anesthetized and paralyzed sheep were perfused at constant flow with monitoring of perfusion pressure. Tracheal smooth muscle tone was assessed by measuring changes in the external diameter of the cranial trachea. Close-arterial injection of antigen (1-20 micrograms) in young sheep produced dose-dependent vasodilation (6.1-15.5% fall in perfusion pressure) and smooth muscle contraction (0.06-0.28 mm reduction in tracheal diam). In old sheep, antigen (1-20 micrograms) produced vasoconstriction (4.1-16.8%) but no smooth muscle response. The smooth muscle contraction in young sheep was blocked by mepyramine (2 mg/kg iv) suggesting mediation by release of histamine. The vasodilation in young sheep and the vasoconstriction in old sheep were reduced by indomethacin (5 mg/kg iv), and the residual response was further reduced by FPL 55712 (2 mg/kg iv), suggesting mediation by both cyclooxygenase products and leukotrienes. Thus antigen given in the tracheal vasculature releases a mixture of inflammatory mediators. This mixture of mediators or their actions on the tracheal vasculature and smooth muscle may depend on the age of the sheep.     

Antagonism by ETYA of the effects of leukotrienes on ileum and lung parenchymal strips independent of effects on arachidonic acid metabolism

5,8,11,14-Eicosatetraynoic acid (ETYA), a compound which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism, antagonized the contraction of segments of guinea-pig ileal longitudinal muscle produced by SRS-A (IC₅₀ = 2.73 μM). This activity was unaffected by pretreatment of the tissues with 10 μM indomethacin. Phenidone, another mixed cyclooxgenese-lipoxygenese inhibitor, was inactive. FPL-55712, an SRS-A antagonist, was a very potent inhibitor (IC₅₀ = 0.011 μM).BW755C and NDGA nonselectively inhibited the contractions of the guinea-pig ileal longitudinal muscle induced by SRS-A or histamine.ETYA antagonized the contraction of the guinea-pig ileal strip produced by 6 nM synthetic LTC₄ (IC₅₀ = 9.3 μM). FPL-55712 demonstrated an IC₅₀ of 0.3 μM in a similar series of experiments. ETYA, 1, 3 or 10 μM did not inhibit the contractions elicited by 0.5 μM of histamine.This was not a tissue-selective effect since 100 μM ETYA antagonized the LTC₄-induced contraction of the guinea-pig lung parenchymal strip preparation.These data demonstrate that ETYA antagonized the contractile effect of the leukotrienes on tissues from the gastrointestinal tract and lung. Furthermore, the inability of indomethacin or phenidone to inhibit the contractile response suggests that antagonism by ETYA may occur by a mechanism independent of cyclooxygenase and lipoxygenase enzymes.     

Influence of platelet-activating factor on leukotriene D4-induced contractions of the guinea pig parenchymal strip

Platelet-activating factor (PAF) and sulphidopeptide leukotrienes, such as leukotriene D4 (LTD4), are potent constrictors that are probably released simultaneously in a variety of inflammatory respiratory events. The purpose of the present study was to determine whether LTD4-induced contractions of guinea pig parenchymal lung strips (GPPS) are modified in the presence of PAF. The contractile responses of isolated GPPS to cumulative doses of LTD4, acetylcholine, histamine, and potassium chloride in the presence of PAF (0.1 nM, 0.1 microM) were compared with parallel controls. There was no significant alteration of the response to acetylcholine and potassium chloride and the PAF-induced inhibition of the response to histamine, although significant, was not concentration dependent. In contrast, PAF in a concentration range from 0.1 nM to 1.0 microM caused a marked, concentration-dependent reduction of LTD4-induced contractions. Pretreatment with the PAF receptor antagonist, BN52021, prevented the attenuation of LTD4-induced contraction by PAF. The attenuation of LTD4-induced contraction by PAF was also prevented by pretreatment with indomethacin or with the thromboxane synthase inhibitor U63,557A, but not by pretreatment with the lipoxygenase inhibitors BW755c or nordihydroguaiaretic acid. Thus inhibition of LTD4-induced GPPS contraction by PAF is receptor dependent and probably secondary to thromboxane generation. The respiratory smooth muscle response to leukotrienes may be modified significantly by concomitant PAF release.     

Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A2 induce starfish oocyte maturation

In starfish oocyte maturation (meiosis reinitiation) is induced by the natural hormone 1-methyladenine (1-Me-Ade). This paper shows that arachidonic acid (AA) induces oocyte maturation at concentrations above 0.5 microM. This maturation shares many characteristics with 1-MeAde-induced maturation: same kinetics, same required contact time, same stimulations of protein phosphorylation and sodium influx. Although calcium facilitates the AA-induced but not the 1-MeAde-induced maturation, AA, like 1-MeAde, does not stimulate the uptake of calcium. Calcium does not facilitate the uptake of AA by oocytes. Out of 36 different fatty acids (saturated and unsaturated), only eicosatetraenoic (AA) and eicosapentaenoic acids were found to mimic 1-MeAde. Calcium-dependent phospholipases A2 from bee venom and Naja venom also induce maturation (0.1-1 unit/ml) when added externally to the oocytes. Phospholipase A2 inhibitors (quinacrine, bromophenacylbromide) block maturation; inhibition is reversed by increasing the 1-MeAde concentration and only occurs during the hormone-dependent period. AA is usually metabolized through oxidation by cyclooxygenase or lipoxygenase. Cyclooxygenase inhibitors (acetylsalicylic acid, indomethacin, tolazoline) do not block maturation; prostaglandins E2, D2, F2 alpha, I2, and thromboxane B2 do not induce meiosis reinitiation. On the other hand, lipoxygenase inhibitors (quercetin, butylated hydroxytoluene, and eicosatetraynoic acid) block 1-MeAde-induced maturation; although leukotrienes (A4, B4, C4, D4, E4) have no effects on oocytes, two other lipoxygenase products, 12- and 15-hydroxyeicosatetraenoic acids (and their corresponding hydroperoxy-) induce oocyte maturation (around 1 microM). The possible mode of action of the fatty acids inducing oocyte maturation is discussed.     

Cyclooxygenase inhibitors increase canine tracheal muscle response to parasympathetic stimuli in situ

To determine whether cyclooxygenase inhibitors alter parasympathetic control of airway smooth muscle in situ, we pretreated anesthetized dogs with intravenous indomethacin, meclofenamate, or normal saline and measured the isometric contraction of tracheal muscle in response to electrical stimulation of the vagus nerves. Indomethacin and meclofenamate increase the response of airway smooth muscle to parasympathetic stimulation. In subsequent experiments to determine the site of action of cyclooxygenase inhibitors, we found that indomethacin does not alter the response of tracheal muscle to intra-arterial acetylcholine (a muscarinic agonist) but does augment the response to intra-arterial dimethylpiperaziniumiodide (a nicotinic agonist). Moreover, the response to parasympathetic stimulation after pretreatment with a combination of indomethacin and BW755C (a combined cyclooxygenase-lipoxygenase inhibitor) does not differ significantly from the response after indomethacin or meclofenamate alone. We conclude that cyclooxygenase inhibitors increase the sensitivity of the contractile response of tracheal smooth muscle to parasympathetic stimulation, that they exert their effect on the postganglionic parasympathetic neuron, and that their effect is prejunctional. The effect appears secondary to a decrease in cyclooxygenase products rather than to an increase in lipoxygenase products. These findings suggest that endogenous cyclooxygenase products may modulate parasympathetic control of airway smooth muscle in vivo. They may relate to the mechanisms that underlie airway hyperresponsiveness, by which mediators of inflammation modulate airway responsiveness and by which nonsteroidal anti-inflammatory drugs induce severe bronchoconstrictor responses in some persons who have asthma.     

H(2)O(2) opens BK(Ca) channels via the PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle

H(2)O(2) is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H(2)O(2) opens the large-conductance, calcium- and voltage-activated (BK(Ca)) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H(2)O(2) on coronary smooth muscle involves the phospholipase A(2) (PLA(2))/arachidonic acid (AA) signaling cascades. H(2)O(2) stimulation of BK(Ca) channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA(2). Furthermore, H(2)O(2) stimulated release of [(3)H]AA from coronary myocytes, and exogenous AA mimicked the effect of H(2)O(2) on BK(Ca) channels. Inhibitors of protein kinase C activity attenuated the effect of H(2)O(2) on BK(Ca) channels, [(3)H]AA release, or intact coronary arteries. In addition, the effect of H(2)O(2) or AA on BK(Ca) channels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H(2)O(2) relaxes coronary arteries by stimulating BK(Ca) channels via the PLA(2)/AA signaling cascade and that lipoxygenase metabolites mediate this response.     

Cyclooxygenase and lipoxygenase-like activity in Drosophila melanogaster

To determine the possible activity of cyclooxygenase and lipoxygenase like enzymes in Drosophila melanogaster, we have investigated whether fly homogenates can biosynthesize prostaglandins and HETEs. Incubation of fly extracts with AA yields a mixture of 15- 12- 9- and 8-HETE as detected by selected ion monitoring GC-MS. Also the combination of HPLC-RIA using a PGE antibody shows the presence of endogenous PGE2 immunoreactivity in the extracts (405 pg/g in males and 165 pg/g in females). We have also detected the presence of lipoxygenase like immunoreactivity in the reproductive male system by using immunocytochemical techniques in whole body sections of the fly as well as reactivity in the digestive system of both males and females. Finally, we have not been able to detect endogenous AA in the fly by GC-MS methods. However, estimates by GC-MS of the total body fatty acids indicate substantial amounts of potential AA precursors.     

Inhibition of arachidonic acid-induced contraction of guinea pig lung strips

The effects of several enzyme inhibitors on arachidonic acid-induced contractions of guinea pig lung strips were studied. Varying concentrations of indomethacin, an inhibitor of cyclooxygenase, produced only a limited effect on contraction of tissue strips. By contrast, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and phenidone, which inhibit either lipoxygenase, or both lipoxygenase and cyclooxygenase, caused a dose-related antgonism of the arachidonic acid-induced contraction. The effects of these latter agents were similar to that of FPL 55712. Results indicate that the products of cyclooxygenase are predominantly involved in the early phase and the products of lipoxygenase are predominantly related to the late phase of arachidonic acid-induced contraction.     

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 μg/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.