Cartilage cyclic nucleotide phosphodiesterase: inhibition by anti-inflammatory agents

Soluble 3′,5′-nucleotide phosphodiesterase (PDE) activity is described in chicken epiphyseal and articular cartilage. Kinetic studies of these enzymes demonstrate a high and low K_m for the substrates, adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Epiphyseal and articular PDE activities are inhibited by those anti-inflammatory agents which are potent inhibitors of the enzyme, prostaglandin synthetase (PS). Specificity of this inhibition is indicated by the activity of these agents against the low K_m enzyme. Other anti-inflammatory agents with significantly less potency as PS inhibitors or with no activity against prostaglandin synthetase are found to be either inactive or relatively less potent as inhibitors of cartilage PDE activity. A variety of other anti-inflammatory or anti-rheumatic agents, which are not known to affect prostaglandin synthetase activity, are poor inhibitors of cartilage PDE activity. These data provide insight into the mechanism of action of certain anti-inflammatory agents and into the relationships between prostaglandins and inflammatory reactions.     

The effect of anti-inflammatory agents on human synovial fibroblast prostaglandin synthetase

Human synovial fibroblast prostaglandin synthetase activity is inhibited by many different non-steroidal anti-inflammatory agents. Aspirin, indomethacin and phenylbutazone significantly inhibit both PGE₁, PGE₂ and PGF_1α and PGF_2α synthesis; whereas penicillamine and aurothioglucose are more potent inhibitors of the F prostaglandins. Histidine and antimalarials do not inhibit, to a significant degree, human synovial prostaglandin synthetase activity. Hydrocortisone has no direct effect on prostaglandin synthetase activity. No changes in synthetase activity are observed when synovial cells are incubated with hydrocortisone, and the prostaglandin synthetase system subsequently isolated and assayed. The proposed inhibitory effects of hydrocortisone on prostaglandin production by synovium may be the result of an alteration of enzyme substrate or cofactor concentration rather than a direct effect on prostaglandin synthetase.     

Effect of docosahexaenoic acid (DHA) on arachidonic acid metabolism and platelet function

Studies from our laboratory have suggested a role for ferrous iron in the metabolism of arachidonic acid and demonstrated that inhibitors of prostaglandin synthesis exert their effect by complexing with the heme group of cyclooxygenase. Docosahexaenoic acid (DHA) is a potent competitive inhibitor of arachidonic acid metabolism by sheep vesicular gland prostaglandin synthetase. In this study we have evaluated the effect of exogenously added DHA on platelet function and arachidonic acid metabolism. DHA at 150 microM concentration inhibited aggregation of platelets to 450 microM arachidonic acid. At this concentration DHA also inhibited the second wave of the platelet response to the action of agonists such as epinephrine, adenosine diphosphate and thrombin. Inhibition induced by this fatty acid could be overcome by the agonists at higher concentrations. DHA inhibited the conversion of labeled arachidonic acid to thromboxane by intact, washed platelet suspensions. However, platelets in plasma incubated first with DHA then washed and stirred with labeled arachidonate generated as much thromboxane as control platelets. These results suggest that the polyenoic acids, if released in sufficient quantities in the vicinity of cyclooxygenase, could effectively compete for the heme site and inhibit the conversion of arachidonic acid.     

Stimulation of the cyclooxygenase activity of prostaglandin H synthase by salicylate-derived quinhydrones

Two novel salicylate-derived quinhydrones were evaluated for their effect on the kinetics of cyclooxygenase activity of sheep seminal vesicle prostaglandin H synthase. These quinhydrones, which form semiquinone radicals in solution, were designed to resemble oxidized intermediates of salicylic acid metabolism. Although initially investigated for their potential role in irreversible inactivation of cyclooxygenase, these derivatives were found to give three-fold stimulation of this activity. In the absence of arachidonic acid, preincubation of the enzyme with these quinhydrones did not lead to inactivation of the cyclooxygenase activity. These compounds thus resemble the phenolic antioxidants in their effects on the cyclooxygenase activity of the synthase.     

Triacsin C: a differential inhibitor of arachidonoyl-CoA synthetase and nonspecific long chain acyl-CoA synthetase

Triacsins A, B, C, and D are newly discovered compounds isolated from the culture filtrate of streptomyces which are known to inhibit nonspecific long chain acyl-CoA synthetase (EC 6.2.1.3.). These inhibitors have not been previously studied with regard to their effects on arachidonoyl-CoA synthetase, an enzyme which specifically utilizes arachidonate and other icosanoid precursor fatty acids. To explore this question, we used triacsin C, a potent inhibitor of the nonspecific acyl-CoA synthetase. Triacsin C was found to inhibit the action of arachidonoyl-CoA synthetase and the nonspecific enzyme in sonicates of HSDM1C1 mouse fibrosarcoma cells. Importantly, however, the triacsin concentration and length of pre-incubation with the enzymes could be adjusted to almost completely inhibit (greater than 80%) the nonspecific long chain acyl CoA-synthetase, with less than 20% inhibition of arachidonoyl-CoA synthetase. Using intact cultured cells exposed to 1 ug/ml triacsin for up to 15 minutes, we unexpectedly observed preferential inhibition of arachidonoyl-CoA synthetase activity. In intact cell studies, arachidonoyl-CoA synthetase was inhibited greater than 90%, with 55-60% inhibition of the nonspecific acyl-CoA synthetase. As additional evidence of its inhibition of acyl-CoA synthetase enzymes in intact cells, triacsin C inhibited both fatty acid uptake into cells and icosanoid production, metabolic processes which in certain cell types appear to be dependent on acyl-CoA synthetase activity. Thus, triacsin C is a novel inhibitor which can alter the fatty metabolism of intact cells. This compound can be of significant value in determining the specific cellular functions of the two acyl-CoA synthetase enzymes.     

Structure-activity relationships for inhibition of prostaglandin cyclooxygenase by phenolic compounds

Sixty-three phenolic compounds were examined for their ability to inhibit sheep vesicular gland prostaglandin cyclooxygenase. Examination of structure-activity relationships for these compounds indicated that inhibition was increased by ring substituents which were electron donating and by substituents which were hydrophobic. Inhibition was decreased by steric masking of the phenolic hydroxyl. The most potent inhibitors possessed a two aromatic ring structure connected by a short bridge. In these inhibitors, one ring was apolar, the other ring contained a phenolic hydroxyl ortho to the bridge, and the bridge contained a Lewis base such that the compounds could form bidentate metal chelates. Compounds with [I]₅₀ values of less than 10 uM included 2,4,6-trimethyl phenol, 7.2 uM; 2-(2-hydroxyphenyl)benzothiazole, 7.0 uM; 2-benzyloxyphenol, 5.2 uM; and 2-hydroxybenzophenone, 3.8 uM.Inhibition of arachidonate induced platelet aggregation was examined for three of the more potent inhibitors. 2-Benzyloxyphenol and 2,4,6-trimethylphenol were more potent than indomethacin when assayed using a 2 min preincubation of inhibitor with platelets.     

Regulation of glial cyclooxygenase by bradykinin

The aim of the present study was to investigate the short-term effect of bradykinin on the two cyclooxygenase species in neonatal rat glial cells. In spite of the fact that cyclooxygenase protein levels were not altered, an increase in cyclooxygenase activity was observed. Use of cyclooxygenase-1 inhibitors and paracetamol resulted in complete elimination of the bradykinin-induced prostaglandin E(2) synthesis and of cyclooxygenase enzyme activity. Cyclooxygenase-2 inhibitors only partially inhibited enzyme activity and prostaglandin production. Our data suggest that cyclooxygenase-1 is probably the major contributor to short-term modulation of glial prostaglandin E2 synthesis by bradykinin.     

Design, synthesis and evaluation of potent thymidylate synthase X inhibitors

Three synthesized series of compounds based on a thiazolidine core allowed identification of potent inhibitors of thymidylate synthase X. The evaluation of the catalytic activity of the enzyme in the presence of these molecules revealed two distinct classes of compounds that inhibit ThyX with submicromolar concentrations, which could lead, after optimization, to effective inhibitors with potential biomedical interest.     

On the mode of action and biochemical properties of anti-inflammatory drugs-II.

The inhibition of prostaglandin (PG) synthetase by nonsteroidal anti-inflammatory drugs (NSAID) is not well understood. Co-factors (glutathione and hydroquinone) are needed for maximum enzymatic activity in vitro, and we suggest that NSAID might inhibit PG synthetase partly by interfering with co-factor induced stimulation of the enzyme. This hypothesis was tested by: A) Examining the effect of glutathione, noradrenaline and hydroquinone on bull seminal vesicle (BSV) PG synthetase in vitro. The stimulatory effects were concentration-dependent. B) Three structurally distinct NSAID, indomethacin, aspirin and paracetamol, inhibited the stimulation by each co-factor in a concentration-related manner. Drug effectiveness also depended on the concentration of co-factor.     

Anticancer Properties of Distinct Antimalarial Drug Classes

We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC₅₀s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC₅₀s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings.     

Stereoisomeric relationships among anti-inflammatory activity, inhibition of platelet aggregation, and inhibition of prostaglandin synthetase

This study compares the affects of a new non-steroidal anti-inflammatory drug, d,1-6-chloro-alpha-methyl-carbazole-2-acetic acid, its enantiomers, and indomethacin on platelet aggregation, prostaglandin synthetase, adjuvant arthritis, gastric ulceration and arachidonic acid induced diarrhea. In the adjuvant arthritic rat, doses producing anti-inflammatory activity were similar for all compounds with the exception of the I-isomer which was much less active. On the other hand, indomethacin was 10 to 25 times more potent with regard to inhibition of platelet aggregation, inhibition of prostaglandin synthetase, inhibition of arachidonic acid induced diarrhea, and induction of gastric ulceration than the racemate and its isomers. Such divergence of potencies suggests that the racemate, unlike indomethacin, would have no affect on platelet aggregation and, hence, produce no prolongation of bleeding time at doses possessing anti-inflammatory activity. The data also suggest that the racemate and d-isomer have greater specificity toward anti-arthritic activity and are less ulcerogenic than indomethacin. The d-isomer apparently is the more active component of the racemate in all the systems tested since: (a) the d-isomer has 2 to 3 times the inhibitory potency of the racemate and (b) the I-isomer, at high dosages or high concentrations had considerably less affect. Comparison of potencies relative to inhibition of platelet aggregation and of prostaglandin synthetase, are quite close; therefore, mechanistically, the anti-aggregatory affects of these drugs, or lack thereof, may be related to inhibition of prostaglandin synthetase.     

Stereoisomeric relationships among anti-inflammatory activity, inhibition of platelet aggregation, and inhibition of prostaglandin synthetase

This study compares the affects of a new non-steroidal anti-inflammatory drug, d,l-6-chloro-alpha-methyl-carbazole-2-acetic acid, its enantiomers, and indomethacin on platelet aggregation, prostaglandin synthetase, adjuvant arthritis, gastric ulceration and arachidonic acid induced diarrhea. In the adjuvant arthritic rat, doses producing anti-inflammatory activity were similar for all compounds with the exception of the l-isomer which was much less active. On the other hand, indomethacin was 10 to 25 times more potent with regard to inhibition of platelet aggregation, inhibition of prostaglandin synthetase, inhibition of arachidonic acid induced diarrhea, and induction of gastric ulceration than the racemate and its isomers. Such divergence of potencies suggests that the racemate, unlike indomethacin, would have no affect on platelet aggregation and, hence, produce no prolongation of bleeding time at doses possessing anti-inflammatory activity. The data also suggest that the racemate and d-isomer have greater specificity toward anti-arthritic activity and are less ulcerogenic than indomethacin. The d-isomer apparently is the more active component of the racemate in all the systems tested since: (a) the d-isomer has 2 to 3 times the inhibitory potency of the racemate and (b) the l-isomer, at high dosages or high concentrations had considerably less affect. Comparison of potencies relative to inhibition of platelet aggregation and of prostaglandin synthetase, are quite close; therefore, mechanistically, the anti-aggregatory affects of these drugs, or lack thereof, may be related to inhibition of prostaglandin synthetase.     

Aromatic hydroxamic acids and hydrazides as inhibitors of the peroxidase activity of prostaglandin H2 synthase-2

The cyclooxygenase activity of the bifunctional enzyme prostaglandin H(2) synthase-2 (PGHS-2) is the target of non-steroidal anti-inflammatory drugs. Inhibition of the peroxidase activity of PGHS has been less studied. Using Soret absorption changes, the binding of aromatic hydroxamic acids to the peroxidase site of PGHS-2 was examined to investigate the structural determinants of inhibition. Typical of mammalian peroxidases, the K(d) for benzhydroxamic acid (42mM) is much greater than that for salicylhydroxamic acid (475microM). Binding of the hydroxamic acid tepoxalin (25microM) resulted in only minor Soret changes. However, tepoxalin is an efficient reducing cosubstrate, indicating that it is an alternative electron donor rather than an inhibitor of the peroxidase activity. Aromatic hydrazides are metabolically activated inhibitors of peroxidases. 2-Naphthoichydrazide (2-NZH) caused the time- and concentration-dependent inhibition of both PGHS-2 peroxidase and cyclooxygenase activities. H(2)O(2) was required for the inactivation of both PGHS-2 activities and indomethacin (which binds at the cyclooxygenase site) did not affect the peroxidase inhibitory potency of 2-NZH. A series of aromatic hydrazides were found to be potent inhibitors of PGHS-2 peroxidase activity with IC(50) values in the 6-100microM range for 13 of the 18 hydrazides examined. Selective inhibition of PGHS-2 over myeloperoxidase and horseradish peroxidase isozyme C was increased by certain ring substitutions. In particular, a chloro group para to the hydrazide moiety increased the PGHS-2 selectivity relative to both myeloperoxidase and horseradish peroxidase isozyme C.     

Effects of suprofen and other prostaglandin synthstase inhibitors in a new animal model for myometrial hyperactivity

An model for studying factors related to dysmenorrhea and for evaluating drugs for their inhibitory effects on uterine contractility induced by arachidonic acid and prostaglandins has been developed. Intravenous administration of arachidonic acid and PGF₂α to guinea pigs during the late stage of the estrous cycle, induced dose related uterine contractions and an elevation in uterine basal pressure similar that seen in patients with dysmenorrhea. Pretreatment with prostaglandin synthetase inhibitors inhibited the response to arachidonic acid. The order of relative potency was suprofen (1) > indomethacin (0.65) > naproxen (0.52) > ibuprofen (0.43) > aspirin (0.31). The effectiveness of maximal response for suprofen was significantly greater than that of the other compounds tested. Simultaneous administration of suprofen with PGF₂α also blocked induction of uterine contractions, suggesting the possibility that suprofen also antagonizes PGF₂α receptor binding. Bradykinin also induced uterine constractions, an effect blocked by pretretment with suprofen. Finally, histochemical studies demonstrated stimulation of uterine catecholamine levels (norepinephrine) by arachidonic acid, PGF₂α and bradykinin. These effects were blocked by suprofen.These data suggest that suprofen, an analgesic prostaglandin synthetase inhibitor, may be of use in the clinical tretment of the uterine contractions associated with primary dysmenorrhea.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Influence of arachidonate metabolism on enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages

We examined whether arachidonate metabolism exerted any influence on the enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages. Enhancement of the intracellular transglutaminase activity was observed on stimulation of macrophages with normal sheep red blood cells (SRBC) or immunoglobulin G (IgG)-coated SRBC, and was inhibited by inhibitors of phospholipase A2 and cyclooxygenase. Moreover, prostaglandin E2 (PGE2), a main product of the cyclooxygenase pathway, leukotriene B4 (LTB4), a product of 5-lipoxygenase, and arachidonic acid also could directly induce high levels of intracellular transglutaminase activity without stimulation of macrophages by SRBC or IgG-coated SRBC, but leukotriene C4, prostaglandin D2, and prostacyclin were unable to induce high activity of the enzyme. Enhancement of transglutaminase activity induced by LTB4 was inhibited by cyclooxygenase inhibitor, but the enzyme activity induce by PGE2 was not inhibited. Furthermore, the quantity of PGE2 released into the culture medium of macrophages stimulated with SRBC or IgG-coated SRBC correlated well with the activity of intracellular transglutaminase in macrophages. Moreover, enhancement of transglutaminase activity by treatment of macrophages with SRBC or IgG-coated SRBC was partially suppressed by sodium benzoate, which is a scavenger of hydroxy radical. These findings suggest that arachidonate metabolism, in particular the cyclooxygenase pathway, plays an important role in the enhancement of intracellular transglutaminase activity.     

Inhibition of [3H]captopril binding by peptide analog angiotensin converting enzyme inhibitors

Ketomethylene containing peptide analogs, modeled after a snake venom pentapeptide, have been shown to be potent angiotensin converting enzyme inhibitors. Although the most potent compounds are up to five times more potent than captopril in inhibiting angiotensin converting enzyme activity, they are relatively weak inhibitors of [3H]captopril binding to membrane bound angiotensin converting enzyme. This indicates that inhibition of [3H]captopril binding and enzymatic activity is due to binding to distinct sites. These results suggest that the inhibitors bind to an additional site on the enzyme distinct from the captopril binding site.     

Is prostaglandin a mediator for the inhibitory action of histamine,hydrocortisone, and isoproterenol?

We examined the inhibitory actions of prostaglandin E₂, histamine, isoproterenol, hydrocortisone, and interferon on lymphocyte mitogenesis. There was a high degree of intercorrelation between the amount of inhibition caused by prostaglandin E₂, histamine, isoproterenol, and hydrocortisone, but not interferon, in any given subject; that is if lymphocytes from a given subject were highly sensitive to inhibition by one of those agents, they were also sensitive to the other agents. The inhibitory actions of histamine, isoproterenol, or hydrocortisone could be partially blocked by adding prostaglandin synthetase inhibitors to the mitogen cultures. Preincubation of the lymphocytes for 18 hr prior to the addition of mitogens and inhibitors resulted in a loss in sensitivity to the inhibitors other than interferon. Removal of glass-adherent cells (the prostaglandin-producing cells) prior to culture lessened the inhibition caused by histamine and isoproterenol. The above data would suggest that these inhibitors may act via prostaglandin; however, all of these compounds actually decrease prostaglandin production in cultures of peripheral blood mononuclear cells. The implications of these findings are discussed.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

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.