Sulfido-peptide leukotrienes in coronary heart disease - relationship with disease instability and myocardial ischaemia.

BACKGROUND: Urinary excretion of leukotriene (LT) E(4) is an index of LTC(4) biosynthesis and platelet-neutrophil interactions, which may occur in coronary heart disease and contribute to myocardial ischaemia. Enhanced LTC(4) biosynthesis may be a consequence of myocardial ischaemia or be linked to its pathogenetic substrate. METHODS AND RESULTS: Overnight urine collections were obtained from 17 patients with chronic stable angina, three patients with Prinzmetal's angina, 16 patients with non ST-elevation acute coronary syndromes (NSTE-ACS) and six patients with acute ST-elevation myocardial infarction (STEMI). LTE(4) excretion was measured by enzyme immunoassay after HPLC separation. Compared with healthy controls (51.1 +/- 21.3 pg mg(-1) creatinine, mean +/- SD, n = 11) and with non-coronary cardiac controls (36.6 +/- 9.8 pg mg(-1) creatinine, n = 9), LTE(4) excretion was unchanged in stable angina (40.5 +/- 25.8 pg mg(-1) creatinine), but significantly (P < 0.01) increased in NSTE-ACS (122.7 +/- 137.2 pg mg(-1) creatinine) and STEMI (213.4 +/- 172.4 pg mg(-1) creatinine). In these patients, LTE(4) excretion rapidly dropped after day 1, consistent with effective coronary reperfusion. In patients with NSTE-ACS, the increase in LTE(4) excretion was entirely restricted to patients with recent (< 48 h) spontaneous anginal episodes. Myocardial ischaemia elicited by a positive exercise stress test was not accompanied by any detectable increase in LTE(4) excretion, while a significant (P < 0.01) increase was detected after a single-vessel percutaneous coronary interventions (PCI) procedure (n = 10), as compared with diagnostic angiography (n = 9). CONCLUSIONS: In coronary heart disease, increased LTC(4) biosynthesis is restricted to ACS and not linked to myocardial ischaemia per se, but likely to the occurrence of plaque disruption.

The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis.

Evidence for increased oxidant stress has been reported in human atherosclerosis. However, no information is available about the importance of in situ oxidant stress in relation to plaque stability. This information is relevant because the morbidity and mortality of atherosclerosis are essentially the consequences of acute ischemic syndromes due to unstable plaques. We studied 30 carotid atherosclerotic plaques retrieved by endarterectomy from 18 asymptomatic (stable plaques) and 12 symptomatic patients (unstable plaques). Four normal arteries served as controls. After lipid extraction and ester hydrolysis, quantitation of different indices of oxidant stress were analyzed, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatetraenoic acids (EETs), ketoeicosatetraenoic acids (oxo-ETEs), and F2-isoprostanes using online reverse-phase high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS). All measurements were carried out in a strictly double-blind procedure. We found elevated levels of the different compounds in atherosclerotic plaques. Levels of HETEs were 24 times higher than EETs, oxo-ETEs, or F2-isoprostanes. Levels of HETEs, but not those of EETs, oxo-ETEs or F2-isoprostanes, were significantly elevated in plaques retrieved from symptomatic patients compared with those retrieved from asymptomatic patients (1, 738 +/- 274 vs. 1,002 +/- 107 pmol/ micromol lipid phosphorous, respectively; P < 0.01). One monooxygenated arachidonate species, 9-HETE, which cannot be derived from known enzymatic reactions, was the most abundant and significant compound observed in plaques, suggesting that nonenzymatic lipid peroxidation predominates in advanced atherosclerosis and may promote plaque instability.

Localization of distinct F2-isoprostanes in human atherosclerotic lesions.

F2-Isoprostanes are prostaglandin (PG) isomers formed in situ in cell membranes by peroxidation of arachidonic acid. 8-epi PGF2alpha and IPF2alpha-I are F2-isoprostanes produced in humans which circulate in plasma and are excreted in urine. Measurement of F2-isoprostanes may offer a sensitive, specific, and noninvasive method for measuring oxidant stress in clinical settings where reactive oxygen species are putatively involved. We determined whether isoprostanes were present in human atherosclerotic lesions, where lipid peroxidation is thought to occur in vivo. 8-epi PGF2alpha ranged from 1.310-3.450 pmol/micromol phospholipid in atherectomy specimens compared with 0.045-0.115 pmol/micromol phospholipid (P < 0.001) in vascular tissue devoid of atherosclerosis. Corresponding values of IPF2alpha-I were 5.6-13.8 vs. 0.16-0.44 pmol/micromol phospholipid (P < 0.001). Levels of the two isoprostanes in vascular tissue were highly correlated (r = 0.80, P < 0.0001). Immunohistochemical studies confirmed that foam cells adjacent to the lipid necrotic core of the plaque were markedly positive for 8-epi PGF2alpha. These cells were also reactive with anti-CD68, an epitope specific for human monocyte/macrophages. 8-epi PGF2alpha immunoreactivity was also detected in cells positive for anti-alpha-smooth muscle actin antibody, which specifically recognizes vascular smooth muscle cells. Our results indicate that 8-epi PGF2alpha and IPF2alpha-I, two distinct F2-isoprostanes and markers of oxidative stress in vivo, are present in human atherosclerotic plaque. Quantitation of these chemically stable products of lipid peroxidation in target tissues, as well as in biological fluids, may aid in the rational development of antioxidant drugs in humans.

Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition of both extracellular signal-regulated kinase and c-Jun kinase and upregulation of endothelin B receptors.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.

Interference of some flavonoids and non-steroidal anti-inflammatory drugs with oxidative metabolism of arachidonic acid by human platelets and neutrophils.

The effect of ten flavonoids was studied on the stimulation of washed human platelets by either arachidonic acid or thrombin. The oxygenated metabolites released were analyzed by radioimmunoassay, glass-capillary-column gas chromatography and high-pressure liquid chromatography. No effect was evidenced for naringenin, rutinose and phloridzin up to 1000 microM. Thromboxane B2 and 12-hydroxyeicosatetraenoic acid production was depressed simultaneously by all other compounds at different IC50. When tested for their effect on reversibility, however, cyclooxygenase and lipoxygenase inhibition was found to be different depending upon the flavonoid used. All compounds, except morin and rutin, inhibited platelet aggregation and [14C]serotonin release with parallel inhibition of thromboxane synthesis when tested on arachidonic acid-induced platelet-rich plasma stimulation. Some flavonoids inhibited the metabolism of human neutrophils stimulated by ionophore A23187 as assessed by high-performance liquid chromatography. Our results show that flavonoids interfere with the different oxidative metabolisms of arachidonic acid. No clearcut specificity could be found between one compound and one metabolic pathway.

The use of iodinated tracers for a sensitive radioimmunoassay of 13,14-dihydro-15-ketoprostaglandin Falpha.

Radioimmunoassay of 13,14-dihydro-15-ketoprostaglandin Falpha is reported using various 125I-labelled derivatives. The apparent association constants of the antiserum for iodinated tracers are higher than with the homologous hapten. In spite of this, the high specific activities of iodinated tracers (2000 Ci/mmol) allow a 3-fold increase in the sensitivity of the assay when compared with the tritiated derivative. Human plasma levels of 13,14-dihydro-15-ketoprostaglandin Falpha reported (25+/-6 pg/ml) are lower than those previously found by radioimmunoassay, and no sexual difference was found.

125I derivatives of prostaglandins. A novel approach in prostaglandin analysis by radioimmunoassay.

We report the production of radioactive iodinated (125 I) derivatives of prostaglandins E1, E2, F2alpha and their use in radioimmunological assays. Histamine or tyramine was coupled to the prostaglandins carboxyl group and the iodination was accomplished using the chloramine T method. The high specific radioactivity of these tracers and the resolution of the purification procedure allowed the detection of 0.5 pg of prostaglandins. A comparison with tritiated prostaglandin was made and showed a 10-fold gain in sensitivity. Furthermore in the case of the prostaglandin E1 system using 125I-labelled histamine or tyramine as tracer the cross reaction curves obtained were different from those obtained with [3H]prostaglandin E1; we suggest that the blocking of the carboxyl group alters the prostaglandin E1 structure, modifying its immunoreactivity.

Immunological studies of human constitutive cyclooxygenase (COX-1) using enzyme immunometric assay.

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.

Differential measurement of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays.

We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580-598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570-581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570-595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4 alpha-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.

Development of enzyme immunoassays for leukotrienes using acetylcholinesterase.

We have developed sensitive solid phase enzyme immunoassays (EIA) to analyze quantitatively leukotrienes (LTs) using acetylcholinesterase from Electrophorus electricus as a label for LTB4, LTC4 and LTE4. However, because of problems specific to LTs, we used different coupling procedures to prepare LTs conjugates necessary for the production of antibodies and for the preparation of enzymatic tracers. For the immunogens, all LTs were coupled to bovine serum albumin using glutaraldehyde (ethylene diamine was used to add an amino group to LTB4). Immunizations in rabbits were done following classical procedures. For the enzymatic tracers, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate was selected to conjugate the LTs via their amino groups to acetylcholinesterase. Titers of the different antisera ranged from 1:30,000 (LTE4), 1:40,000 (LTC4) to 1:50,000 (LTB4) and sensitivities (IC50) were 5.5 pg, 4.3 pg and 2.4 pg, respectively. Cross reactivities were also examined against other LTs. Sensitivities and specificities of the different systems were dependent on the conditions of incubation (temperature). Validation of the technique was done (i) after spiking known amounts of LTC4 in plasma and measuring the substance added after prior extraction and purification, (ii) by analyzing the supernatant of human neutrophils suspended in buffer or in plasma, (iii) by measuring LTE4 in urine. Due to the background provided by these complex matrixes, quantitation was performed after addition of [3H]LTs for recovery, protein precipitation, extraction by Sep-PakR and purification by HPLC. Measurement of LTs can be done in biological fluids with the same ease and advantages as other enzyme immunoassays that we have previously developed for eicosanoids analysis.

Radioimmunoassay of 11-dehydrothromboxane B2 in human plasma and urine.

Because of the discrepancy between the capacity of platelets to synthesize thromboxane B2 ex vivo and the actual synthetic rate in vivo, measurement of thromboxane B2 in plasma is highly influenced by sampling-related artifacts. We have developed and validated a radioimmunoassay for a major enzymatic derivative of thromboxane B2 with an extended plasma half-life, i.e., 11-dehydrothromboxane B2. The binding of the tracer is displaced by as low as 1 pg/ml of the homologous ligand, with a high degree of specificity for the open ring structure as well as for the omega side-chain. This method can detect changes in the plasma concentration and urinary excretion of 11-dehydrothromboxane B2 associated with stimulated short-term increases of thromboxane B2 secretion in the human circulation.

Time-dependent utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations.

The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

Selective stimulation of human platelet lipoxygenase product 12-hydroxy-5,8,10,14-eicosatetraenoic acid by chlorpromazine and 8-(n,n-diethylamino)-octyl-3,4,5-trimethoxybenzoate.

Stimulation of human platelets by thrombin and by the Ca2+ ionophore A23187 leads to a rapid Ca2+-dependent activation of phospholipases that release membrane-bound arachidonic acid for oxidation by a cyclooxygenase and lipoxygenase enzymes into so-called eicosanoids. Chlorpromazine and the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited the release of eicosanoids, as estimated by a quantitative glass capillary-gas chromatography analysis. TMB-8 was more efficient for thrombin- than for ionophore-induced eicosanoids liberation. Chlorpromazine, the more potent inhibitor, was active at the same concentration against either inducer. The reduction of oxidative metabolism by the cyclooxygenase pathway was more pronounced than reduction in the lipoxygenase pathway. When exogenous arachidonic acid was added to the platelets, both drugs stimulated selectively the production and the formation rate of 12-hydroxy-5,8,10,14-eicosatetraenoic acid by a factor of 2-2.5 in the absence of variation of cyclooxygenase products. Therefore, the stimulation of the lipoxygenase metabolite by the two drugs was obtained with both endogenous and exogenous arachidonic acid. This selective stimulation by drugs of a lipoxygenase product in the absence of inhibition of cyclooxygenase is the first reported of this type and suggests a differential control for the two oxidation enzymes. These findings emphasize the importance of a simultaneous quantitative analysis of both oxidation pathways.

Change in the physical state of platelet plasma membranes upon ionophore A23187 activation. A fluorescence polarization study.

Human platelets were isolated and fluorescence-labelled by 1,6-diphenylhexatriene. Diphenylhexatriene was essentially localized in the plasma membrane, as indicated by trinitrobenzenesulfonate-quenching experiments. A decrease of the fluorescence polarization of diphenylhexatriene was observed upon ionophore A23187 addition in the absence of aggregation. 0.3 microM ionophore allowed to reach the maximum rate of the decrease of fluorescence polarization; it also maximally stimulated the light transmission change, the serotonin release and the thromboxane B2 synthesis. The amplitude of the fluorescence polarization decrease was maximum at platelet concentrations between 4 X 10(7) and 7 X 10(7)/ml. The presence of Ca2+ in the medium increased the rate constant of the polarization change. Chlorpromazine (60 microM) completely inhibited this transition, but at 30 microM its inhibitory effect was reversed by Ca2+. The membrane events implied in platelet activation very likely lead to fluidization of the plasma membrane, perhaps by its fusion with the membranes of internal granules which are relatively depleted of cholesterol. Ca2+ plays a central role in the triggering of the observed effects at the membrane level.

Autocrine control of adipose cell differentiation by prostacyclin and PGF2 alpha.

The mitogenic-adipogenic effect exerted by arachidonic acid, which leads to terminal differentiation of Ob1771 mouse preadipocytes, has been shown to be (i) blocked by cyclooxygenase inhibitors, (ii) mimicked by a stable analogue of prostacyclin (carbaprostacyclin) and (iii) potentiated by PGF2 alpha. Since these prostanoids are known to be synthesized and secreted by preadipocytes, we have proposed that both prostacyclin as the key mediator and PGF2 alpha as a modulator control the expression of terminal events of adipose conversion by means of an autocrine mechanism (Gaillard, D. et al. and Negrel, R. et al. Biochem. J. (1989) 257, 389-397 and 399-405). In order to test this hypothesis, the release of prostacyclin, characterized under the form of its stable degradation product 6-keto-PGF1 alpha, and that of PGF2 alpha have been studied in the culture medium of Ob1771 cells. A striking increase in the release of 6-keto-PGF1 alpha and to a minor degree of PGF2 alpha was observed when cells were exposed to arachidonic acid as shown by using [3H]arachidonic acid prelabelled cells or by radio-immunoassays. Since antagonists of PGF2 alpha and PGI2 receptors were not available, specific antibodies directed against PGF2 alpha and 6 beta-PGI1, another stable analogue of prostacyclin, were added as neutralizing agents in the culture medium. These antibodies were able to counteract the mitogenic-adipogenic effect of arachidonic acid. Prostacyclin and PGF2 alpha thus appear as autocrine mediators in the process of adipose conversion.

Heme oxygenase-1 induction by nitric oxide in RAW 264.7 macrophages is upregulated by a cyclo-oxygenase-2 inhibitor.

Unstimulated RAW 264.7 macrophages express negligible heme oxygenase-1 (HO-1) protein but incubation with the nitric oxide (NO) donor spermine nonoate (SPNO) induced HO-1 and weakly cyclo-oxygenase-2 (COX-2) protein. This effect was potentiated by coincubation with the COX-2 selective inhibitor, SC58125. Cells incubated with SPNO showed a strong increase in HO-1 mRNA levels after 4 h with a significant potentiation in the presence of SC58125, which did not modify HO-1 mRNA stability. The induction of HO-1 by NO and its potentiation by anti-inflammatory agents may play a role in inflammatory and immune responses.

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.

Eicosanoid biosynthesis in patients with stable angina: beneficial effects of very low dose aspirin.

OBJECTIVE: We assessed the production of eicosanoids and the effects of very low dose aspirin in patients with stable angina under basal conditions and during rapid atrial pacing. BACKGROUND: Platelet activation occurs in acute ischemic syndromes but is still controversial in stable angina. Very low dose aspirin is known to be platelet selective and can be used to test the hypothesis of the platelet origin of increased thromboxane production in stable angina. METHODS: Urinary excretion of eicosanoids was measured in 42 patients, including 24 patients with and 18 patients without coronary artery disease. The effects of 50 mg/day of aspirin were measured at rest and during pacing-induced ischemia in 10 patients with stable angina and were compared with a similar group of patients not treated by aspirin. RESULTS: Excretion of 11-dehydro-thromboxane B2 was 2.6 times higher in patients with stable angina than in healthy subjects (mean [+/- SEM] 74.8 +/- 13.0 [24 patients] vs. 29.0 +/- 5.4 [18 patients] ng/mmol of creatinine, p < 0.01). Urinary prostacyclin metabolite levels did not differ between the two groups. Treatment for 8 days with 50 mg/day of aspirin inhibited platelet cyclooxygenase, as reflected by the 97% reduction of in vitro serum thromboxane production. This aspirin regimen normalized the level of urinary thromboxane metabolites in patients with angina (17.3 +/- 3.4 ng/mmol of creatinine [10 patients], p < 0.001 from baseline level before treatment) and did not change prostacyclin metabolite levels. Atrial pacing in patients with angina not treated with aspirin caused lactate and thromboxane release into the coronary sinus. In patients with very low dose aspirin therapy, pacing did not cause thromboxane release despite inducing myocardial ischemia. However, fractional lactate extraction decreased less sharply in patients with than without aspirin therapy. CONCLUSIONS: Thromboxane production is greatly increased in patients with stable angina. Very low dose aspirin administered to these patients reduces thromboxane synthesis to normal levels, preserves prostacyclin biosynthesis and prevents acute thromboxane release into the coronary circulation during pacing-induced ischemia. Our data suggest that platelets (not monocytes/macrophages) are activated in stable angina to produce thromboxane.

Total inhibition of phospholipase C and phosphatidylinositol 3-kinase by okadaic acid in thrombin-stimulated platelets.

The strong inhibition of thrombin-induced platelet functions induced by okadaic acid is not correlated with the partial modification of pleckstrin phosphorylation, which remains still phosphorylated two min after stimulation, indicating that protein kinase C is not affected by okadaic acid. We then investigated the effect of okadaic acid on platelet lipid metabolism. Our data indicate that inhibition indeed strongly affects phosphatidic acid as well as phosphatidylinositol 3,4-bisphosphate synthesis at low concentrations of okadaic acid, and phosphatidylinositol 4,5-bisphosphate at higher concentrations. Since thrombin-induced tyrosine phosphorylations were completely inhibited in the presence of okadaic acid, as a consequence, phosphatidylinositol 3-kinase was no longer detected in antiphosphotyrosine immunoprecipitates, thus explaining the absence of phosphatidylinositol, 3,4-bisphosphate synthesis. Finally, okadaic acid inhibited thrombin-induced fibrinogen binding, indicating that serine/threonine phosphatases may affect the inside-out signalling which regulates the alpha 11bb3 integrin, downstream protein kinase C activation.

Expression of cyclooxygenase-1 and -2 in ovine endometrium during the estrous cycle and early pregnancy.

In this study we investigated the expression of the two cyclooxygenases, cox-1 and -2, in sheep uterine tissues during the estrous cycle and early pregnancy. We identified the cox-2 isoform in the ovine uterus by Western blot and demonstrated that the two cyclooxygenases exhibited different patterns of expression. Cox-1 was expressed at steady state levels in the endometrium during the estrous cycle and comparable stages of pregnancy. In contrast, cox-2 was highly and transiently expressed from days 12-15 of the estrous cycle and declined thereafter to undetectable levels. Endometrium from early pregnant ewes showed a similar pattern of cox-2 expression, although there was a slower decrease beyond day 15. Immunohistochemical studies demonstrated that cox-1 was localized in both epithelial and stromal cells, whereas cox-2 was localized solely in the luminal epithelium and to a lesser extent in the superficial glands. Treatment of ovariectomized ewes with steroids indicated that expression of cox-1 remained at constant levels whatever the treatment. In contrast, endometrial cox-2 was highly induced by a 10-day progesterone treatment. Estradiol slightly increased cox-2 expression but only after progesterone priming. Collectively these results suggest that the developing ability of the uterus to synthesize PGs is due to the induction of cox-2.