The pulmonary pharmacology of [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide] (2NTX-99), an anti-atherotrombotic compound with therapeutic potential in pathological conditions that target lung vasculature.

The pharmacological activity of 2NTX-99 ([4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide]) was investigated in vitro in the intact, rat pulmonary vasculature and in guinea pig airways. Rat lungs were perfused at constant flow and changes in vascular tone recorded. Challenge with the TXA2 analogue 9,11-dideoxy-9α11α-methanoepoxy ProstaglandinF2 (U46619, 0.5 µM) increased vessel tone (32.48±1.5 vs 13.13±0.56 mmHg; n=12). 2NTX-99 (0.1-100 µM; n=5), caused a concentration-dependent relaxation, prevented by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 µM, n=4), an inhibitor of soluble guanylate cyclase. Acetylcholine (0.1-10 µM; n=3) and a reference NO-donor, isosorbide-5-mononitrate (5-100 µM; n=4), were ineffective. Intraluminal perfusion of washed human platelets (2 × 108 cells/ml) increased intravascular pressure after challenge with arachidonic acid (AA, 2 µM; n=5), an increase abolished by acetylsalicylic acid and significantly reduced by 2NTX-99 (40 µM; n=5). TXB2 in the lung perfusate was detected after platelet activation, 2NTX-99 inhibited TXA2 synthesis (6.45±0.6 and 1.10±0.2 ng/ml, respectively). 2NTX-99 did not alter central or peripheral airway responsiveness to Histamine (0.001-300 µM; n=6), U46619 (0.001-3 µM, n=3) or LTD4 (1 pM-1 µM; n=6). 2NTX-99 vasodilates the pulmonary vasculature via the release of nitric oxide (NO) and reduces intraluminal, AA-induced, TXA2 formation. The combined activity of 2NTX-99 as an NO-donor and a TXA2-synthesis inhibitor provides strong support for its potential therapeutic use in pathologies of the pulmonary vascular bed (e.g. pulmonary hypertension).

Antagonism of thromboxane receptors by diclofenac and lumiracoxib.

BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) are analgesic and anti-inflammatory by virtue of inhibition of the cyclooxygenase (COX) reaction that initiates biosynthesis of prostaglandins. Findings in a pulmonary pharmacology project gave rise to the hypothesis that certain members of the NSAID class might also be antagonists of the thromboxane (TP) receptor. EXPERIMENTAL APPROACH: Functional responses due to activation of the TP receptor were studied in isolated airway and vascular smooth muscle preparations from guinea pigs and rats as well as in human platelets. Receptor binding and activation of the TP receptor was studied in HEK293 cells. KEY RESULTS: Diclofenac concentration-dependently and selectively inhibited the contraction responses to TP receptor agonists such as prostaglandin D2 and U-46619 in the tested smooth muscle preparations and the aggregation of human platelets. The competitive antagonism of the TP receptor was confirmed by binding studies and at the level of signal transduction. The selective COX-2 inhibitor lumiracoxib shared this activity profile, whereas a number of standard NSAIDs and other selective COX-2 inhibitors did not. CONCLUSIONS AND IMPLICATIONS: Diclofenac and lumiracoxib, in addition to being COX unselective and highly COX-2 selective inhibitors, respectively, displayed a previously unknown pharmacological activity, namely TP receptor antagonism. Development of COX-2 selective inhibitors with dual activity as potent TP antagonists may lead to coxibs with improved cardiovascular safety, as the TP receptor mediates cardiovascular effects of thromboxane A2 and isoprostanes.

Activation of cerebral endothelium is required for mononuclear cell recruitment in a novel in vitro model of brain inflammation.

Brain inflammation is a common event in the pathogenesis of several neurological diseases. It is unknown whether leukocyte/endothelium interactions are sufficient to promote homing of blood-borne cells into the brain compartment. The role of mononuclear cells and endothelium was analyzed in a new experimental model, the isolated guinea-pig brain maintained in vitro by arterial perfusion. This preparation allows one to investigate early steps of brain inflammation that are impracticable in vivo. We demonstrate by confocal microscopy analysis that in vitro co-perfusion of pro-inflammatory agents and pre-activated fluorescent mononuclear cells induced endothelial expression of selectins and intracellular adhesion molecule-1 in correspondence of arrested mononuclear cells, and correlates with a moderate increase in blood-brain barrier permeability. Separate perfusion of pro-inflammatory agents and mononuclear cells induced neither mononuclear cell adhesion nor adhesion molecule expression. We demonstrate that co-activation of mononuclear cells and cerebral endothelium is an essential requirement for cell arrest and adhesion in the early stages of experimental cerebral inflammation.

The Cycloxygenase-2 inhibitor SC58236 is neuroprotective in an in vivo model of focal ischemia in the rat.

Focal ischemia was induced in the fronto-parietal region of rat brain, by injection of Rose Bengal, followed by light activation. Focal ischemia was accompanied by formation of PGD(2) peaking 60-90 min post irradiation and declining thereafter. Increased Cycloxygenase-2 (COX-2) expression was also observed. Control ischemic rats showed distinct morphological alterations with necrosis of neurons, glial cells and blood vessels, surrounded by a halo with pyknotic cells with cytoplasm swelling and vacuolization. Compound SC58236, a selective COX-2 inhibitor, dose-dependently prevented, ischemia-induced eicosanoid formation (area under the curve (AUC) of controls: 3.11 +/- 0.87; AUC of 20 mg/kg SC58236: 0.39 +/- 0.24), and caused significant reduction of damaged area (30.7 and 18.9% at SC58236 20 and 6.6 mg/kg), suggesting that selective inhibitors of COX-2 are neuroprotective.

Effect of endogenous and exogenous prostaglandin E(2) on interleukin-1 beta-induced cyclooxygenase-2 expression in human airway smooth-muscle cells.

We studied the effect of endogenous and exogenous prostaglandin E(2) (PGE(2)), a metabolite of arachidonic acid through the cyclooxygenase (COX) pathway, on interleukin (IL)-1 beta-induced COX-2 expression, using primary cultures of human bronchial smooth-muscle cells (HBSMC). Treatment with exogenous PGE(2) resulted in enhanced expression of IL-1 beta-induced COX-2 protein and messenger RNA (mRNA) as compared with the effect of the cytokine per se. Inhibition of PGE(2) production with a nonselective COX inhibitor (flurbiprofen, 10 microM) resulted in a significant reduction in IL-1 beta- induced COX-2 expression, supporting a role of endogenous COX metabolites in the modulation of COX-2 expression. None of the experimental conditions used in the study affected the expression of constitutive cyclooxygenase (COX-1). Treatment with cycloheximide to inhibit translation, and with dexamethasone or actinomycin D to inhibit transcription, linked the effect of PGE(2) to the transcriptional level of COX-2 mRNA rather than to a potential effect on protein and/or mRNA stabilization. PGE(2) increased adenylate cyclase activity in a concentration dependent manner, and forskolin, a direct activator of adenylate cyclase, caused a marked increase in IL-1 beta-dependent COX-2, suggesting the existence of a causal relationship between the two events. The same results were observed with salbutamol, a bronchodilator that acts by increasing cyclic adenosine monophosphate. The effect of PGE(2) on COX-2 expression may contribute to the hypothesized antiinflammatory role of PGE(2) in human airways, providing a self-amplifying loop leading to increased biosynthesis of PGE(2) during an inflammatory event.

Tramadol anti-inflammatory activity is not related to a direct inhibitory action on prostaglandin endoperoxide synthases.

The analgesic drug tramadol has been shown to relieve pain in inflammatory conditions, to inhibit the development of experimental inflammation, and to reduce prostaglandin (PG)E(2)concentrations in the inflammatory exudate. In this study, we evaluated the putative activity of tramadol to suppress prostaglandin endoperoxide synthase-1 (PGHS-1), and prostaglandin endoperoxide synthase-2 (PGHS-2) activities in human whole blood in vitro. Platelet thromboxane (Tx)B(2)production and monocyte PGE(2)production in LPS- stimulated blood were measured in samples incubated with different concentrations (300 ng/ml, 3 microg/ml, 30 microg/ml) of tramadol or its enantiomers. Neither tramadol nor the enantiomers inhibited the formation of arachidonic acid metabolites. Our results indicate that the anti-inflammatory effect of tramadol demonstrated in some models is not related to a direct inhibitory effect on the formation of prostanoids.

Monoclonal anti-CD18 antibody prevents transcellular biosynthesis of cysteinyl leukotrienes in vitro and in vivo and protects against leukotriene-dependent increase in coronary vascular resistance and myocardial stiffness.

BACKGROUND: Cysteinyl leukotrienes (cys-LT) can constrict small and large vessels and increase vascular permeability. Formation of cys-LT arising from polymorphonuclear leukocytes (PMNL) and endothelial cell cooperation (transcellular synthesis) led to the hypothesis that PMNL-endothelial cell adhesion may represent a key step toward the formation of vasoactive cys-LT. METHODS AND RESULTS: We studied the effect of pretreatment with a monoclonal antibody directed against the CD18 subunit of PMNL beta(2)-integrin on the synthesis of cys-LT in a PMNL-perfused isolated rabbit heart in vitro and in a model of permanent ligature of the left descending coronary artery in the rabbit in vivo. Challenge of PMNL-perfused rabbit hearts with formyl-met-leu-phe (0.3 micromol/L) caused synthesis of cys-LT and increase in coronary perfusion pressure that were prevented by the anti-CD18 antibody. Similar results were obtained with the use of A-23187 (0.5 micromol/L) as a challenge. Persistence of PMNL-associated myeloperoxidase activity in the perfusion buffer was observed in the presence of the anti-CD18 antibody, indicating decreased PMNL infiltration. Coronary artery ligature in vivo increased urinary excretion of leukotriene E(4), supporting the activation of the 5-lipoxygenase pathway during experimental acute myocardial infarction. Pretreatment with the anti-CD18 antibody (1 mg/kg) prevented the increase in leukotriene E(4) excretion. CONCLUSIONS: These data support the importance of adhesion in promoting cys-LT formation, originating from PMNL-endothelial cell cooperation, and contributing to myocardial stiffness and increased coronary resistance.

Inhibition of prostanoid synthesis protects against neuronal damage induced by focal ischemia in rat brain.

Changes in prostanoids concentration and effects of the non-specific COX inhibitor indomethacin on prostanoids levels and extension of tissue damage were studied following focal ischemia induction in the fronto-parietal region of rat brain. Ischemia was induced in animals bearing a transcerebral microdialysis probe by injection of Rose Bengal, a photosensitive dye, followed by light activation. Prostanoid levels were determined in the dialysate using immunoenzymatic techniques. PGD2 levels rose significantly up to 237+/-22 pg/ml compared to a basal level measured before ischemia induction which was below the detection limit. TXB2 changes were smaller and had a different time course. Treatment with indomethacin abolished the ischemia-induced PGD2 release and reduced the extent of injury to the area by 43+/-3.7%. These results suggest that prostanoid release may play an important role in neurodegenerative processes and that cyclooxygenase inhibitors may contribute to protect against cerebral tissue damage.

Simultaneous investigation of the neuronal and vascular compartments in the guinea pig brain isolated in vitro.

We describe a new method for studying the interactions between vascular tone changes and neuronal activity in the arterially perfused isolated brain of the adult guinea pig maintained in vitro. Electrophysiological recordings were performed in the piriform and entorhinal cortices with the entire arterial bed preserved or after vascular restriction to the territories of median and posterior cerebral arteries of one hemisphere. The changes in vascular tone were measured by means of a pressure transducer. The arterial pressure was 53.77+/-12.74 mmHg in control conditions at 30 degreesC. Intraluminal application of vasoactive drugs, such as the tromboxane A2 receptor agonist U46619 (0.1 microM) and 5-HT (3 microM), induced an increase in the resistance to perfusion pressure that was prevented by the selective antagonists. The preservation of the endothelial function was verified by inducing the release of endogenous endothelial relaxant factor after intraluminal application of 1 microM acetylcholine. The study of the reciprocal interactions between neuronal activity and vascular tone modifications demonstrated that evoked responses in the piriform and entorhinal cortices were not modulated by rapid changes of the vascular tone. A sustained and elevated plateau of vasoconstriction maintained for several minutes determined a cortical spreading depression. Epileptiform discharges induced in limbic cortices by GABAa receptor blockade were consistently associated with a vasodilation (8.26+/-2.8 mmHg). The results demonstrate that the in vitro isolated guinea pig brain preparation can be exploited for studying simultaneously neuronal activity and cerebrovascular motility.

Nitric oxide modulation of transcellular biosynthesis of cys-leukotrienes in rabbit leukocyte-perfused heart.

1. We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys-LT) generated upon neutrophil-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. 2. Hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) purified human neutrophils (PMNL), and challenged with 0.5 microM A-23187 for 30 min. Coronary perfusion pressure (CPP) and left-ventricular end-diastolic pressure (LVEDP) were monitored. Cys-LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL, adhesion to the coronary endothelium. 3. Basal CPP and LVEDP values averaged 45 +/- 1.4 mmHg and 5 +/- 0.1 mmHg, respectively; A-23187 triggered an increase in CPP (134 +/- 9 mmHg, at 30 min) which was significantly attenuated by pretreatment with L-arginine, 100 microM (90 +/- 3 mmHg, at 30 min). Pretreatment with NG-monomethyl-L-arginine, 10 microM (L-NMMA), induced a marked increase in CPP (290 +/- 40 mmHg, at 20 min) and in LVEDP (47 +/- 16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by L-arginine, 100 microM, (CPP 115 +/- 10 mmHg, LVEDP 6 +/- 1.1 mmHg, at 30 min). 4. The increase in CPP was accompanied by the release of cys-LT in the circulating buffer, which was reduced significantly by L-arginine. Pretreatment with L-NMMA, caused a marked rise in cys-LT concentrations which was prevented by L-arginine. 5. Neither L-arginine nor L-NMMA affected directly the A-23187-induced arachidonic acid (AA) metabolism in isolated PMNL alone. 6. Pretreatment with L-NMMA caused a prompt drop in myeloperoxidase (MPO), activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by L-arginine. 7. This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys-LT by preventing PMNL adhesion to the coronary intima.

Endothelial cells as target for antiphospholipid antibodies. Human polyclonal and monoclonal anti-beta 2-glycoprotein I antibodies react in vitro with endothelial cells through adherent beta 2-glycoprotein I and induce endothelial activation.

OBJECTIVE: To investigate the ability of human anti-beta 2-glycoprotein I (anti-beta 2 GPI) antibodies to recognize the cofactor adherent on endothelial cells (EC) and to modulate endothelial functions. METHODS: Six human affinity-purified polyclonal anti-beta 2 GPI IgG and 2 IgM monoclonal antibodies (MAb) were obtained from patients with the antiphospholipid syndrome. The antibodies were tested for their ability to 1) bind to endothelial monolayers through the adherent beta 2 GPI and 2) modulate endothelial adhesion molecule expression and interleukin-6 (IL-6) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) secretion. RESULTS: The affinity-purified IgG and the MAb with anti-beta 2 GPI activity, but not the respective controls, displayed EC binding, which declined on cells incubated in serum-free medium and was restored in a dose-dependent manner by exogenous human beta 2 GPI. After EC binding, both polyclonal and monoclonal antibodies up-regulated adhesion molecule expression. Anti-beta 2 GPI MAb also significantly increased IL-6 and 6-keto-PGF1 alpha secretion. CONCLUSION: These findings support the hypothesis that anti-beta 2 GPI antibodies bind and activate EC through the adherent cofactor beta 2 GPI, likely leading to a procoagulant state.

The polymorphonuclear leukocyte: a cell tuned for transcellular biosynthesis of cys-leukotrienes.

Sulfidopeptide leukotrienes (cysLT) are potent vasoactive mediators that can constrict coronary vessels and alter caliber of the microcirculation. They can be formed "in situ" via a peculiar type of cell communication termed "transcellular biosynthesis" whereby donor cells (polymorphonuclear leukocytes, PMNL) feed acceptor cells (endothelial cells, EC) the unstable epoxide intermediate leukotriene A4 for further metabolism to cysLT. We have investigated the relative amount of leukotriene A4 that is synthesized by PMNL and made available for transcellular biosynthesis. This has been accomplished by measuring the relative amounts of enzymatic vs non-enzymatic leukotriene A4-derived metabolites after challenge with the Ca(2+)-ionophore A23187, using PMNL suspensions at different concentrations. Non-enzymatic leukotriene A4-derived metabolites were used as a quantitative index of the amount of leukotriene A4 released into the extracellular milieu. In human, as well as in bovine PMNL, the relative amounts of non-enzymatic vs enzymatic leukotriene A4-derived metabolites increased with decreasing cell concentrations. By diminishing possible cell-cell interactions via increased dilution, it is calculated that approx. 60% of leukotriene A4 synthesized is released from the PMNL. These data provide evidence that, in PMNL, transfer of leukotriene A4 to neighbouring acceptor cells is taking place as a predominant mechanisms of cell communication.

Vasoconstriction to polymorphonuclear leukocytes in the isolated, perfused rabbit heart: inhibition by prostacyclin mimetics.

Perfusion of the isolated rabbit heart with 5 x 10(6) human polymorphonuclear leukocytes (PMNL), under recirculating conditions (50 ml) and challenge with A-23187 (0.5 mu M) increased coronary perfusion pressure (CPP) sixfold, accompanied by increased levels of sulfidopeptide leukotrienes (CY-SLT), which had previously shown to correlate linearly with the increase in CPP. Pretreatment (20 min) of isolated rabbit hearts with the prostacyclin (PGI(2)) analogue iloprost (3 nM) resulted in significant protection against the increase in CPP and in almost complete inhibition of 5-lipoxygenase (5-LO) product synthesis. Similarly, pretreatment of isolated rabbit heart with defibrotide (200 mu g/ml), a polydeoxyribonucleotide derivative known to inhibit PMNL activation and enhance PGI(2) production by heart endothelial cells, produced significant protection against the increase in CPP and almost complete inhibition of 5-LO product synthesis. Neither iloprost nor defibrotide affected the A-23187-induced arachidonic acid (AA) metabolism in isolated PMNL alone. Inhibition of rabbit cyclooxygenase by intravenous (i.v.) administration of lysine-acetylsalicylate (60 mg/kg) 2 h before the animals were killed significantly reduced the protection provided by defibrotide, with a parallel fivefold increase in sulfidopeptide LT levels, returning to values in the range observed in control hearts. Control of endogenous modulators of leukocyte-vascular wall interactions such as PGI(2) results in significant changes in sulfidopeptide LT production in an organ model of transcellular metabolism of LT A(4), suggesting a novel mechanism of action for cardioprotective drugs in myocardial ischemia.

Morphological and functional changes of coronary vasculature caused by transcellular biosynthesis of sulfidopeptide leukotrienes in isolated heart of rabbit.

Morphological and functional modifications occurring in Langendorff rabbit heart preparations perfused with purified human leukocytes (PMNL), as an organ model of sulfidopeptide-leukotrienes (sLT) transcellular biosynthesis, were studied. Coronary perfusion pressure (CPP), monitored as an index of coronary vasospasm, increased by 295% after challenge with the Ca(2+)-ionophore A-23187 (0.5 micromol/L) for 30', accompanied by a significant formation of sLT. Increase in CPP was prevented by PMNL pretreatment with the 5-lipoxygenase inhibitor MK-886 (1 micromol/L) or by heart pretreatment with LTD4-receptor antagonist SKF 104353, indicating a pivotal role of PMNL-derived 5-lipoxygenase (5-LO) products in the observed functional modifications. Similar effects were obtained using granulocyte macrophage-colony stimulating factor-primed PMNL challenged with the tripeptide n-formyl-methionyl-leucyl-phenylalanine. Scanning electron microscopy (SEM) of coronary arteries showed craters on the vessel luminal surface, PMNL adhering to endothelial cells (EC), increased number of microvilli on EC, presence of nonviable, desquamating, fusiform EC. SEM and transmission electron microscopy of myocardial microvessels, showed presence of perivascular and intermuscle edema, presence of activated PMNL and decreased number of patent microvessels. These morphological alterations were significantly blunted by MK-886 or SKF 104353. These data provide evidence of close interaction between PMNL and myocardial EC, resulting in enhanced sLT formation via transcellular biosynthesis, originating from transfer of PMNL-derived LTA4 to EC. These potent proinflammatory autacoids are responsible for coronary vasospasm and the morphological alternations observed.

Myocardial protection by the leukotriene synthesis inhibitor BAY X1005: importance of transcellular biosynthesis of cysteinyl-leukotrienes.

Perfusion of the isolated rabbit heart with 5 x 10(6) human polymorphonuclear leukocytes, under recirculating conditions (50 ml), and challenge with A-23187 (0.5 microM) caused an increase in coronary perfusion pressure (from a prechallenge value of 46 +/- 1.1 to 176.2 +/- 29.7 mm Hg, 30 min after challenge, n = 6-4), which was linearly correlated (P < .006) with formation of cysteinyl leukotrienes (29.7 +/- 7.3 pmol/ml, 30 min after challenge). Pretreatment with the leukotriene synthesis inhibitor BAY X1005 (1 microM) (n = 6) resulted in significant protection against the increase in coronary perfusion pressure (76.7 +/- 12.8 mm Hg, 30 min after challenge) and in almost complete inhibition of sulfidopeptide leukotriene synthesis (3.2 +/- 1.7 pmol/ml, 30 min after challenge). In in vivo experiments, ligation of the left anterior descending coronary artery in the rabbit (n = 10) resulted in acute myocardial infarction marked by a mortality rate of 60% compared with sham-operated animals (n = 10). Intravenous treatment of the rabbits with BAY X1005 (10 mg/kg/h, for 2 h) (n = 10) markedly reduced the mortality rate (20%), protected the rabbits against the marked electrocardiogram derangement and abolished the significant increase in plasma creatine phosphokinase activity and cardiac tissue myeloperoxidase activity induced by coronary artery ligation. BAY X1005 exerts a significant cardioprotection and suggests that specific leukotriene synthesis inhibitors may lead to innovative therapy in myocardial ischemia.

Formation of sulphidopeptide-leukotrienes by cell-cell interaction causes coronary vasoconstriction in isolated, cell-perfused heart of rabbit.

1. We have studied the transcellular biosynthesis of bioactive leukotrienes (LTs), generated upon blood cell-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. Rabbit isolated hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) cells of unpurified (buffy coat) or purified human neutrophils (PMNL), and challenged with 0.5 microM A23187 for 30 min. Coronary perfusion pressure (CPP), heart rate (HR), left ventricular end-diastolic pressure (LVEDP) and left ventricular pressure (LVP) were monitored continuously. Leukotriene formation was measured by specific enzyme-immunoassay and confirmed by reversed phase h.p.l.c. and u.v. spectral analysis. 2. Basal CPP values averaged 44 +/- 1.4 mmHg; A23187 triggered a marked increase in CPP both in the presence of buffy coat cells (+100% above basal) and PMNL (+270% above basal); the latter change in CPP was accompanied by a rise in LVEDP (+138% above basal). 3. The increase in CPP was preceded by a statistically significant rise in iLTC4-D4 concentration in the circulating buffer. Pretreatment with two structurally unrelated LTD4 receptor antagonists, LY171883 and SKF104353 (10 microM), fully prevented the increase in CPP and LVEDP. A similar protection was also observed when the rabbit heart was perfused with PMNL that had been pretreated with MK886 (1 microM), a potent inhibitor of leukotriene biosynthesis. 4. The increased coronary tone was accompanied by a marked release of lactate dehydrogenase (LDH), a marker of ischaemic damage; pretreatment of the heart with the LTD4 receptor antagonists as well as of the PMNL with MK886 resulted in a complete suppression of LDH activity release. 5. Positive identification of LTC4-D4 in the perfusates was obtained and a significant correlation observed between the CPP values and iLTC4-D4 concentrations.6. This study suggests that challenge of PMNL present within the coronary vasculature, causes a LTD4-dependent coronary vasoconstriction, favoured by an efficient uptake of PMNL-derived LTA4 by endothelial cells. The activation of the 5-lipoxygenase pathway in the context of tight interactions between blood cells and coronary vasculature, is suggested to have an important outcome in the alterations of coronary flow and cardiac contractility.