Platelet activation during thrombolysis and percutaneous transluminal coronary angioplasty in the acute phase of myocardial infarction

To clarify the mechanism of recanalization and reocclusion in thrombolysis and percutaneous transluminal coronary angioplasty (PTCA), the plasma concentrations of beta-thromboglobulin (beta-TG), thromboxane B2 (TXB2) and platelet aggregation adenosine diphosphate (ADP) (2 microM/ml, collagen 2 micrograms/ml) were assessed in 11 normal subjects and in 19 patients with acute myocardial infarction whose infarct-related vessels were recanalized by thrombolysis and/or PTCA. In patients with acute myocardial infarction, the plasma concentrations of beta-TG and TXB2 were significantly higher than those in normal subjects (beta-TG: 128 +/- 132 ng/ml vs 38 +/- 17 ng/ml, TXB2: 131 +/- 154 pg/ml vs 36 +/- 18 pg/ml). Collagen-induced platelet aggregation decreased significantly in patients with acute myocardial infarction; whereas, ADP-induced platelet aggregation showed no significant difference. Infarct-related vessels recanalized by thrombolysis (seven patients: group 1) and PTCA (seven patients: group 2) were patent on the follow-up angiograms. Infarct-related vessels were reoccluded in five patients immediately after PTCA or during the follow-up angiography (group 3). Beta-TG and TXB2 did not change before and after recanalization in groups 1 and 2, but increased significantly after recanalization in group 3 (beta-TG: 155 +/- 185 ng/ml----269 +/- 233 ng/ml, TXB2: 104 +/- 87 pg/ml----169 +/- 91 pg/ml). Platelet aggregation did not differ significantly among the three groups. We concluded that platelets are not activated during thrombolysis and/or PTCA in cases without reocclusion, while platelets are markedly activated during PTCA in cases with reocclusion. Thus, it is suggested that platelet activation plays an important role in the mechanism of reocclusion.     

Induction of functional lipoxin A4 receptors in HL-60 cells

The appearance of [11,12-3H]lipoxin A4 (LXA4) specific binding sites was examined with human acute promyelocytic leukemic cell line 60 (HL- 60) cells exposed to either retinoic acid, phorbol 12-myristate 13- acetate (PMA), or dimethyl sulfoxide (DMSO). All three agents induced a threefold to fivefold increase in the expression of specific [11,12- 3H]LXA4 binding. Similar results were obtained in parallel with [14,15- 3H]leukotriene (LT) B4. For both 3H-ligands, homologous displacement curves were similar and independent of the agent used to induce differentiation. Specific binding of [11,12-3H]LXA4 to differentiated HL-60 cells gave a kd = 0.6 +/- 0.3 nmol/L. The appearance of both [11,12-3H]LXA4 and [14,15-3H]LTB4-specific binding sites was inhibited by actinomycin D, and LXA4 binding was sensitive to protease treatment. Specific binding of [11,12-3H]LXA4 was not evident with human platelets, red blood cells (RBCs) or the cultured B-cell (Raji), T-cell (Jurkat) lines save human endothelial cells (kd = 11.0 +/- 0.3 nmol/L). The structural specificity of induced [11,12-3H]-LXA4 recognition sites was assessed with LXB4, LTC4, LTB4, and trihydroxyhepatanoic methyl ester. Only LTC4, at 3-log molar excess, competed for 3H-LXA4-specific binding with HL-60 cells and gave a 30% reduction. The leukotriene D4 receptor antagonist SKF 104353 was ineffective in blocking [11,12- 3H]LXA4-specific binding with HL-60 cells while it competed for specific [11,12-3H]LXA4 binding with endothelial cells where LTD4 binding appears to be virtually identical to that of LXA4 binding. In addition, the LTB4 receptor antagonist ONO 4057 was ineffective at competing for [11,12-3H]LXA4 binding. When phospholipase D activation was monitored in human polymorphonuclear leukocytes (PMN) and HL-60 cells, a correlation was shown between activation and specific 3H-LXA4 binding. LXA4-induced phospholipase D (PLD) activation gave a biphasic concentration-dependent response comprised of at least two components: one phase being islet-activating protein (IAP)-sensitive (LXA4 10(-9) mol/L peak activity) and the other was staurosporine-sensitive (LXA4 10(-7) mol/L peak activity). Results indicate that HL-60 cells exposed to differentiating agents express [11,12-3H]LXA4 recognition sites also present in PMN. In addition, specific LXA4 recognition sites of myeloid cells can be distinguished by competition binding with SKF 104353 and 3H-LXA4 cross-reactivity with putative LTD4 receptors present on human endothelial cells. Moreover, they provide evidence indicating that binding of LXA4 to its recognition sites confers functional responses.     

Cyclooxygenase- and lipoxygenase-derived eicosanoids in bronchoalveolar lavage fluid from patients with scleroderma lung disease: an imbalance between proinflammatory and antiinflammatory lipid mediators

OBJECTIVE: Eicosanoids play a key role in the regulation of inflammation and fibrosis. Recently we showed that levels of 5-lipoxygenase (5-LOX)-derived proinflammatory/profibrotic leukotrienes are elevated in bronchoalveolar lavage (BAL) fluid from patients with scleroderma lung disease (SLD). The present study was undertaken to investigate whether increased levels of leukotrienes are balanced by the antiinflammatory/antifibrotic cyclooxygenase (COX)- and 15-LOX-derived eicosanoids in the lungs of patients with SLD. METHODS: Levels of 5-LOX-derived leukotriene B(4) (LTB(4)), COX-derived prostaglandin E(2) (PGE(2)), and 15-LOX-derived 15-hydroxyeicosatetraenoic acid (15-HETE) and lipoxin A(4) (LXA(4)) in BAL fluid from systemic sclerosis (SSc) patients with SLD (n = 32) and without SLD (n = 16) and from healthy individuals (n = 12) were measured by enzyme-linked immunosorbent assay. RESULTS: Levels of LTB(4) (mean +/- SEM 248 +/- 29 pg/ml) and PGE(2) (51 +/- 10 pg/ml) in SSc patients with SLD were significantly higher compared with patients without SLD (LTB(4) 119 +/- 35 pg/ml, PGE(2) 17 +/- 3 pg/ml; P < 0.05 for both) and with healthy controls (85 +/- 12 pg/ml and 19 +/- 2 pg/ml, respectively; P < 0.05 for both). Accordingly, the mean +/- SEM PGE(2):LTB(4) ratio was similar in SSc patients with SLD (0.30 +/- 0.05), SSc patients without SLD (0.29 +/- 0.07), and controls (0.31 +/- 0.07). In contrast, levels of 15-HETE and LXA(4) in patients with SLD did not differ significantly from levels in patients without SLD or in controls. The ratio of LXA(4):LTB(4) in SLD patients (0.16 +/- 0.03) was significantly lower (P < 0.05) than that in patients without SLD (0.40 +/- 0.10) or controls (0.34 +/- 0.08). CONCLUSION: Increased production of LTB(4) in the lungs of patients with SLD is not balanced by an up-regulation of 15-LOX-derived antiinflammatory/antifibrotic eicosanoids such as 15-HETE or LXA(4). Targeting the 5-LOX/15-LOX balance may be of practical value in the treatment of SLD.     

Induced sputum eicosanoid concentrations in asthma

Further definition of the role of leukotrienes (LT) and prostaglandins (PG) in asthma would be helped by a noninvasive method for assessing airway production. The supernatant from sputum induced with hypertonic saline and dispersed using dithiotrietol has been successfully used to measure other molecular markers of airway inflammation and might be a useful method. We have measured induced sputum supernatant LTC(4)/D(4)/E(4) concentrations using enzyme immunoassay and PGE(2), PGD(2), TXB(2), and PGF(2alpha) using gas chromatography-negative ion chemical ionization-mass spectroscopy in 10 normal subjects and in 26 subjects with asthma of variable severity. Sputum cysteinyl-leukotrienes concentrations were significantly greater in subjects with asthma (median, 9.5 ng/ml) than in normal control subjects (6.4 ng/ml; p < 0.02) and greater in subjects with persistent asthma requiring inhaled corticosteroids (median, 11.4 ng/ml) or studied within 48 h of an acute severe exacerbation of asthma (13 ng/ml) than in subjects with episodic asthma treated with inhaled beta(2)-agonists only (7.2 ng/ml). There were no significant differences in the concentrations of other eicosanoids between groups, although there was a negative correlation between the percentage sputum eosinophil count and sputum PGE(2) concentration (r = -0.48; p < 0.01) in subjects with asthma. We conclude that induced sputum contains high concentrations of eicosanoids and that sputum LTC(4)/D(4)/E(4) concentrations are significantly greater in subjects with asthma than in normal subjects. The inverse relationship between eosinophilic airway inflammation and sputum PGE(2) concentration would be consistent, with the latter having an anti-inflammatory role.     

Inflammatory mediators in exhaled breath condensate of children with obstructive sleep apnea syndrome

BACKGROUND: Upper airway inflammation is now recognized in adults with obstructive sleep apnea (OSA) syndrome. However, the role played by eicosanoids such as leukotrienes and prostaglandins is unclear. OBJECTIVE: To investigate whether eicosanoids are measurable in exhaled breath condensate (EBC), and to determine whether differences in these inflammatory mediators emerge among children with and without sleep-disordered breathing (SDB). METHODS: EBC was collected from 50 consecutive snoring children undergoing overnight polysomnography for suspected SDB, and from 12 nonsnoring control subjects. Prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and cysteinyl leukotrienes (cys-LTs: leukotriene C4 [LTC4]/leukotriene D4 [LTD4]/leukotriene E4 [LTE4]) EBC levels were analyzed using enzyme-linked immunosorbent assay. RESULTS: LTB4 levels were elevated in children with an apnea-hypopnea index (AHI) > 5/h (SDB; 97.6 +/- 6.3 pg/mL) compared to children with an AHI < 5/h (mild SDB; 66.4 +/- 19.1 pg/mL; p < 0.01) and control subjects (27.8 +/- 3.7 pg/mL; p < 0.01). Similarly, cys-LT (LTC4/LTD4/LTE4) concentrations were also increased in SDB (45.1 +/- 10.6 pg/mL in SDB vs 27.6 +/- 8.3 pg/mL in mild SDB, and 15.7 +/- 7.6 pg/mL in control subjects; p < 0.01). In contrast, PGE2 concentrations were similar among the three groups. CONCLUSIONS: Inflammatory mediators such as leukotrienes and prostaglandins can be readily quantified in EBC collected from the upper airway of children. Disease severity-dependent increases in leukotriene concentrations (LTB4 and LTC4/LTD4/LTE4) emerge among children and may serve as a noninvasive tool in the clinical assessment of these children.     

Sudden death induced by intracoronary platelet aggregation

Sodium arachidonate (AA, 1.5 mg/kg) was injected into the coronary arteries in 16 rabbits. Arrhythmia, marked ST-T depression and apnea appeared in all cases, and 7 cases died within 10 min after the AA. Before the injection, the thromboxane B2 (TXB2) value was 1.2 +/- 0.2 ng/ml (mean +/- SE) and the 6-keto-PGF1 alpha value was 2.1 +/- 0.4 ng/ml. Three minutes after the AA, TXB2 values were 5.0 +/- 1.1 in the surviving cases and 17.9 +/- 6.5 ng/ml in the cases which died. 6-keto-PGF1 alpha values were 47.7 +/- 4.6 in the surviving cases and 248.5 +/- 69.3 ng/ml in the cases which died. There occurred no deaths in 7 cases pretreated with aspirin (ASA) and in 11 cases pretreated with OKY-046 and 1581, which are specific inhibitors of TXA2 synthetase. TXB2 values did not change in the ASA and OKY groups after the AA injection. 6-keto-PGF1 alpha values did not change in the ASA group and increased in the OKY group after the AA injection. Histological findings of the heart showed more remarkable ischemic changes in the non-pretreated group than in the ASA and OKY groups. These results suggest a role for TXA2 in sudden death. PGI2 production was extremely enhanced, suggesting the presence of a protective mechanism against thrombogenesis in vivo.     

Thromboxane A2 and peptidoleukotrienes contribute to the myocardial ischemia and contractile dysfunction in response to intracoronary infusion of complement C5a in pigs

Intracoronary infusions of activated complement C5a result in myocardial ischemia, contractile dysfunction, and leukocyte accumulation. The hypothesis was tested that the generation of the coronary vasoconstrictors, thromboxane A2 and the 5-lipoxygenase leukotrienes (LTC4 and LTD4), contributes to the C5a-induced decrease in coronary blood flow and contractile function. The left anterior descending coronary artery in anesthetized swine was cannulated and servo pump-perfused with arterial blood at constant pressure and measured flow. Regional subendocardial contractile function was assessed with sonomicrometry. The interventricular vein was cannulated for sampling of coronary venous blood for leukocyte count. The responses in left anterior descending coronary artery blood flow and percent segment shortening to intracoronary infusions of LTC4 (1 microgram), LTD4 (1 microgram), thromboxane agonist U46619 (7.5 micrograms), and C5a (500 ng) were assessed before and after 1) LTD4/LTE4 receptor blockade with leukotriene receptor blocker LY171883 (10 mg/kg i.v.) (n = 5), 2) thromboxane A2/prostaglandin H2 receptor blockade with thromboxane receptor blocker BM13505 (2 mg/kg i.v.) (n = 5), and 3) combined thromboxane and leukotriene receptor blockade (n = 5). In the absence of receptor blockade, intracoronary C5a decreased coronary flow (50-60%) and regional segment function (60-70%) compared with the preinfusion levels. This was accompanied by a fall in coronary venous blood leukocyte levels by 5-6 x 10(6) cells/ml in the absence of alterations in arterial blood leukocyte count. Intracoronary injections of LTD4, LTC4, or U46619 also resulted in prompt decreases in coronary blood flow (50-60%) and segment function (70-80%) from preinfusion levels. Leukotriene receptor blockade with LY171883 abolished these responses to LTD4 and LTC4. Administration of LY171883 also attenuated (p less than 0.05) the myocardial response to C5a; coronary flow and segment function decreased by approximately 28% from preinfusion levels. Thromboxane receptor blockade with BM13505 eliminated the response in coronary flow and segment function to intracoronary U46619. Similar to LY171883, administration of BM13505 blunted (p less than 0.05) the C5a-induced decreases in coronary flow and contractile function, which fell by approximately 20-25% from the preinfusion level. After the combined LTD4/LTE4 receptor and thromboxane A2/prostaglandin H2 receptor blockade, intracoronary C5a resulted in little change in both coronary blood flow and segment shortening. In contrast to the flow and function effects, the C5a-induced myocardial leukocyte extraction was not decreased by leukotriene and/or thromboxane receptor blockade.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterogeneity of leukotriene C4 production by eosinophils from asthmatic and from normal subjects

We evaluated the formation of leukotriene C4 (LTC4) by peripheral blood eosinophils of different densities obtained from asthmatic and normal subjects. When stimulated with 1 microgram/ml of the calcium ionophore A23187 for 15 min at 37 degrees C, eosinophils with densities greater than 1.093 g/ml from asthmatic and normal subjects released 19.1 +/- 4.2 ng LTC4/10(6) eosinophils and 23.9 +/- 5.0 ng LTC4/10(6) eosinophils, respectively. In contrast, lower density eosinophils (densities 1.093 g/ml or less) isolated from the asthmatic subjects released significantly less LTC4 than did eosinophils of similar densities from normal subjects (41.6 +/- 3.0 versus 79.0 +/- 6.7 ng LTC4/10(6) eosinophils, p less than 0.05). Differences could not be demonstrated between the two subject groups in LTC4 metabolism, time course of extracellular release of LTC4, or dose response to A23187, nor were interactions between eosinophils and neutrophils with regard to LTC4 release evident. Thus, hypodense eosinophils elaborate greater quantities of LTC4 than do eosinophils of normal density whether obtained from normal or asthmatic subjects. However, the finding that peripheral blood eosinophils from asthmatic subjects have decreased capacity for the synthesis of LTC4 compared with cells of similar densities isolated from normal subjects demonstrates that the capacity of eosinophils to produce LTC4 is regulated by factors that are not necessarily reflected in the cell density.     

The metabolism of peptide leukotrienes in the lung may affect their coronary vasoconstrictive effect in anesthetized dogs

The deteriorative effect of peptide leukotrienes (LTs) on the coronary circulation are well known. On the other hand, it has been recognized that some of these prostanoids are metabolized in the pulmonary vascular bed. To clarify the influence of the metabolism of LTs in the lung on their effects to the coronary circulation, we compared the changes of coronary circulation parameters by leukotriene D4 (LTD4) and C4 (LTC4) administered (5 mcg/kg) into the right atrium (RA) and the left atrium (LA) in 8 mongrel dogs. After the administration of LTD4 and LTC4, significant reduction of coronary blood flow (LTD4-RA 65.8 +/- 3.4%, LTD4-LA 54.8 +/- 2.3%, LTC4-RA 64.3 +/- 3.9%, LTC4-LA 66.9 +/- 3.7%) and elevation of coronary vascular resistance (LTD4-RA 130.5 +/- 8.2%, LTD4-LA 157.3 +/- 6.1%, LTC4-RA 138.7 +/- 8.0%, LTC4-LA 129.5 +/- 10.1%) were recognized. The effect of LTD4, administered into the left atrium was significantly greater than that administered into the right atrium (p less than 0.05). The difference between right and left atrial administration was not statistically significant, although a tendency for the effect to be greater with right atrial administration than left was recognized. The LTC4 concentration in aortic blood measured by radioimmunoassay was significantly lower in right atrial administration than left (p less than 0.05). These results suggested that LTD4 was metabolized and inactivated in the lung during its passage through the pulmonary circulation, and LTC4 was not only inactivated but also converted to LTD4 and activated in the pulmonary vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoxin A4 antagonizes cellular and in vivo actions of leukotriene D4 in rat glomerular mesangial cells: evidence for competition at a common receptor.

Lipoxin A4 (LXA4) was competitive with [3H]leukotriene D4 (LTD4) for specific binding to cultured rat glomerular mesangial cells. Half-maximal inhibition was obtained with 100 nM LXA4, compared with 10 nM for unlabeled LTD4. At 10 and 50 nM LXA4 induced low, but significant, increases in mesangial-cell inositol trisphosphate generation: 48% and 44% increases as compared to vehicle controls, respectively (compared with 146% and 106% increments obtained for equimolar LTD4), which were abolished in the presence of 100-fold concentrations of the LTD4 receptor antagonist, SKF 104353. In addition, exposure to 100 nM LXA4 prevented mesangial cell inositol trisphosphate generation induced by 10 nM LTD4. To test the in vivo relevance of these results, we established a dose-response curve for the reducing effects of intrarenal arterial LTD4 on glomerular filtration rate and renal plasma flow in anesthetized rats (LTD4 doses were 0.5, 7.0, 14.0, and 20.0 micrograms/kg per min) without or with LXA4 at 1 microgram/kg per min. Mean percent decreases in glomerular filtration rate/renal plasma flow during LTD4 administration were 27*/24, 25*/40*, 70*/65*, and 73*/70* at the above doses, respectively (*P less than 0.05 versus baseline). With LXA4, these values were as follows: 9/20*, 11/37*, 42*/51*, and 50*/68*, the latter value representing a shift in the LTD4/glomerular filtration rate dose-response curve. Thus, LXA4 competes for [3H]LTD4 binding to mesangial cells, its presence prevents LTD4-induced inositol trisphosphate generation, and its own stimulation of mesangial-cell inositol trisphosphate is blocked by an LTD4 receptor antagonist. In vivo, LXA4 antagonizes LTD4-induced falls in glomerular filtration rate but not renal plasma flow, implying prevention of LTD4-mediated reductions in the glomerular ultrafiltration coefficient, a consequence of mesangial-cell contraction. These results suggest that LTD4 and LXA4 interact at a common site on rat mesangial cells at which LXA4 provokes partial agonist responses and competitively antagonizes both the cellular and physiological actions of LTD4. Moreover, these results provide evidence for a potential counterregulatory interaction between leukotrienes and lipoxins that may be relevant during glomerular inflammation.     

Metabolism of leukotrienes by adult and fetal human lungs

The metabolism of leukotriene (LT) A4, B4, C4, D4, and E4 was studied using both bioassay and reversed phase high performance liquid chromatography (RP-HPLC) methods. The incubation of 20,000 g supernatants of homogenates of human adult lung with LTA4 and LTC4 for various periods of time produced substances of higher biologic activity than the controls (without incubation) when measured on strips of guinea pig lung parenchyma and ileum. RP-HPLC analyses of the incubation media revealed the formation of LTB4, LTC4, LTD4, and LTE4 from LTA4 and the formation of LTD4 and LTE4 from LTC4. LTD4 was converted to LTE4 whereas LTB4 and LTE4 were not catabolized to an appreciable extent during a 2-h incubation period. Supernatants (20,000 g) of human fetal lung homogenates also contain the enzymatic activities to transform LTC4 into LTD4 and LTE4; however, LTA4 was mainly converted to LTB4 and to products of the nonenzymatic hydrolysis of LTA4 such as the delta 6-trans-LTB4, delta 6 -trans-12-epi-LTB4 and the 5,6-dihyroxyeicosatetraenoic acids; much smaller quantities of the peptidoleukotrienes were formed than in adult lung homogenates.     

Eicosanoid levels in CSF of premature infants with posthemorrhagic hydrocephalus

The cerebrospinal fluid (CSF) of 11 premature infants suffering from posthemorrhagic hydrocephalus was examined by radioimmunoassay for prostaglandin (PG) E2, PGF2 alpha, PGD2, 6-keto PGF1 alpha, thromboxane B2 (TxB2) and peptidoleukotrienes (LTC4/LTD4). The LTs were detected in the CSF of more of these patients (70%) than any of the other eicosanoids, and usually in the highest concentration. Among the 11 posthemorrhagic patients CSF eicosanoid levels were highest when determined soon after injury. Moreover, the variety of eicosanoids present, as well as concentrations, in these infants decreased with time. The types of eicosanoids most evident in the CSF of patients who required shunting were TxB2 and LTs, being present together in 5 of 6 (83%) of these infants. In contrast, 1 of 5 (20%) of the patients who did not require this neurosurgical intervention contained both TxB2 and LTs, the remaining having only one or neither eicosanoid. The highest average concentration for each eicosanoid studied was (pg/ml): PGE2, 628; PGF2 alpha, 985; PGD2, 1410; 6-keto PGF1 alpha, 544; TxB2, 486 and LTs, 1229. This study is the first to demonstrate that the CSF of preterm infants may contain a wide variety of eicosanoids and indicates that these lipids are a manifestation of neurological assault.     

Ex-vivo regulation of endotoxin-induced tissue factor in whole blood by eicosanoids.

The influence of several eicosanoids of the lipoxygenase pathway was examined in an ex vivo system of human whole blood subjected to stimulation by lipopolysaccharide (LPS). Exogenously added leukotriene B4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4)] or 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) significantly (P<0.05) enhanced LPS-evoked expression of monocyte tissue factor (TF) activity in a concentration-dependent manner. 15(S)-HETE, on the other hand, exerted such activity only when added at certain concentrations, whereas 5(S)-HETE was devoid of any apparent activity. LPS-induced TF activity was inhibited by the lipoxygenase inhibitors nordihydroguaiaretic acid, CGS 23885 and ZM 230487, by 59, 32 and 88%, respectively. Furthermore, the production of LTB4 in LPS-stimulated whole blood was investigated, in the absence or presence of either tumor necrosis factor alpha (TNFalpha) or phorbol-12-myristate-13-acetate (PMA). LPS alone induced a moderate time-dependent and concentration-dependent release of LTB4, reaching the maximum concentration (1260 +/- 202 pg/ml) within 90 min at 5 ng/ml LPS. The prior and concurrent presence of PMA (5 ng/ml) or TNFalpha (10 ng/ml) further enhanced the LTB4 production approximately twofold (P < 0.05). TNFalpha added alone evoked approximately twice the LTB4 production seen when LPS (2200 +/- 243 versus 1260 +/- 203 pg/ml) was added alone. Considering these results, LPS and TNFalpha emerge as important agonists of LTB4 production in whole blood. LTB4 in turn appears to be of importance for the expression of TF in monocytes, potentially amplifying the thrombogenic potential of these cells.     

Cardiovascular effects of leukotrienes

Leukotrienes are among the most potent lipid mediators of inflammation. Leukotriene B4 is one of the most potent endogenously synthesized chemotactic substances. It is probable that LTB4 plays an important role in the initial phase of the inflammatory reaction. The peptidoleukotrienes LTC4 and LTD4 are potent broncho- and vasoconstrictors. Administration of LTC4 and LTD4 leads to an increase of blood pressure in most species. These effects are sometimes dependent on the liberation of vasoactive prostaglandins. In addition, peptidoleukotrienes enhance vascular permeability rapidly. Peptidoleukotrienes elicit coronary vasoconstriction which is the most likely reason for the negative inotropic effect of these compounds. Recently the synthesis of peptidoleukotrienes has been demonstrated in the heart under experimental conditions. While the kidney shows a relatively mild vasoconstrictor response to leukotrienes, the pulmonary vascular bed generally responds with a brisk increase of resistance. Pathophysiological roles of leukotrienes require further definition. A striking increase in leukotrienes synthesis has been observed following endotoxin shock.     

Angioplasty increases coronary sinus F2-isoprostane formation: evidence for in vivo oxidative stress during PTCA

OBJECTIVES: Isoprostanes, stable end-products of oxygen free radical mediated-lipid peroxidation, were measured in the coronary vessels during percutaneous transluminal coronary angioplasty (PTCA) to provide direct evidence for enhanced oxidative stress in a local milieu in vivo. BACKGROUND: Percutaneous transluminal coronary angioplasty is associated with complications such as myocardial stunning and accelerated restenosis, which at least in part are mediated by oxygen free radicals. Because isoprostanes are markers of oxidant stress and potent vasoactive compounds, the formation of which is not inhibited by aspirin treatment in vivo, it is possible that these mediators are increased locally during PTCA. METHODS: In 12 coronary artery disease patients who were given aspirin and ticlopidine, blood samples from coronary sinus were taken immediately before and immediately upon balloon deflation during PTCA. Isoprostane F2alpha-III, isoprostane F2alpha-VI, and TxB2 were quantified after extraction and chromatography using a stable dilution isotope gas chromatography/mass spectrometry assay. RESULTS: Coronary sinus and left main coronary artery levels of iPF2alpha-III and iPF2alpha-VI at baseline were (mean +/- SEM) 40 +/- 9 pg/ml and 115 +/- 10 pg/ml, respectively. The TxB2 levels were undetectable. Following PTCA, isoprostane levels markedly increased (mean +/- SEM): iPF2alpha-III, 125 +/- 12 pg/ml (p < 0.001); iPF2alpha-VI, 295 +/- 20 pg/ml (p < 0.001), whereas TxB2 levels remained undetectable. CONCLUSIONS: These results indicate that PTCA induces coronary sinus increase in F2-isoprostane formation, and they also provide direct evidence for enhanced oxidative stress in a local milieu in vivo. Thus, an increased F2-isoprostane formation could play a role in the pathogenesis of some PTCA-associated untoward events.     

Radioimmunoassay of the leukotrienes of slow reacting substance of anaphylaxis.

A rabbit immunized with a conjugate of leukotriene D4 (LTD4) and bovine albumin via the icosanoid carboxyl produced antibodies with comparable affinities for leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4) and their 11-trans stereoisomers. The antibodies bound 3H-labeled 11-trans-LTC4 and 11-trans-LTC4 with the same average association constant (Ka) of 2.8 x 10(9) M-1 at 37 degrees C and were present at a concentration of 0.32 microgram/ml of the immune rabbit plasma. When 9.5 microliter of anti-LTD4 and 108 pmol of 11-trans-[3H]LTC4 (40 Ci/mmol) were incubated in a volume of 300 microliter with LTC4, LTD4, LTE4, or their 11-trans stereoisomers, 50% inhibition of 11-trans-[3H]LTC4 binding was achieved at levels varying between 0.3 and 0.7 ng. As assessed with synthetic analogs of the natural leukotrienes, the antibodies recognized neither those changes within the 6-sulfidopeptide unit of LTD4 produced by deamination or modest peptide lengthening nor the specific stereochemistry of the delta 14-cis double bond. However, the antibodies did recognize the triene lipid domain and the position and spatial orientation of the glutathione or cysteinylglycine function. Binding of 11-trans-[3H]LTC4 by anti-LTD4 was not inhibited by glutathione, cystinylbisglycine, arachidonic acid, or 5-hydroxy-6,8,11,14-icosatetraenoic acid, and leukotriene B4 (LTB4) was about 1/1000th as active as LTC4, LTD4, or LTE4. Mouse lymphoma (WEHI-5) and rat basophil leukemia (RBL-1) cells, when stimulated with calcium ionophore A23187, each produced immunoreactive leukotrienes; and LTC4, LTD4, and LTE4 from RBL-1 cells were individually quantitated by radioimmunoassay after resolution by high-performance liquid chromatography.     

Increased leukotrienes in exhaled breath condensate in childhood asthma

Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4, and LTE4) are generated predominantly by mast cells and eosinophils and induce airway smooth muscle contraction, microvascular leakage, and mucous hypersecretion whereas leukotriene B4 (LTB4) is a potent chemoattractant of neutrophils. We measured cys-LTs and LTB4 in exhaled breath condensate from children aged 7-14 years including healthy nonatopic children (n = 11) and children with mild intermittent asthma (steroid naive, n = 11), mild persistent asthma (low-dose inhaled steroid treatment, n = 13), or moderate to severe persistent asthma (high-dose inhaled steroid treatment, n = 13). Exhaled LTB4 levels were increased in patients with mild and moderate to severe persistent asthma compared with patients with mild intermittent asthma (126.0 +/- 8.8 and 131.9 +/- 7.1 versus 52.7 +/- 3.8 pg/ml, p < 0.001 and p < 0.0001) and normal subjects (126.0 +/- 8.8 and 131.9 +/- 7.1 versus 47.9 +/- 4.1 pg/ml, p < 0.0001). Elevated exhaled cys-LT levels were found in patients with mild and moderate to severe persistent asthma compared with normal subjects (27.9 +/- 2.8 and 31.5 +/- 4.5 versus 18.5 +/- 0.5 pg/ml, p < 0.01 and p < 0.05). There was an inverse correlation between exhaled cys-LTs and LTB4 in patients with mild persistent asthma. We conclude that exhaled cys-LTs and LTB4 may be noninvasive markers of airway inflammation in pediatric asthma.     

Angiotensin II-induced contractions in human internal mammary artery: effects of cyclooxygenase and lipoxygenase inhibition

OBJECTIVE: This study investigated, in isolated human internal mammary artery, the involvement of the cyclooxygenase and the lipoxygenase pathways of arachidonic acid metabolism in the contraction induced by angiotensin II. METHODS: Rings of human internal mammary arteries were suspended in organ baths for recording of isometric tension. In addition, the release of eicosanoids in response to angiotensin II (0.3 microM) was measured by enzyme immunoassay. RESULTS: In human arterial rings without endothelial dependent relaxation in response to substance P or acetylcholine, the angiotensin II-induced contractions were significantly (P<0.05) reduced by 27% in the presence of GR32191 0.3 microM (thromboxane A(2) (TXA(2)) receptor antagonist) but remained unchanged in the presence of dazoxiben 100 microM (thromboxane synthase inhibitor). In addition, angiotensin II failed to modify TXB(2) and 6-keto-PGF(1alpha) production. These results suggest the contribution of a TXA(2)/PGH(2) agonist other than TXA(2) in angiotensin II-induced contractions. However, indomethacin increased (P<0.05) angiotensin II-mediated contractile response and cysteinyl leukotriene production, suggesting a redirection of arachidonic acid metabolism from the cyclooxygenase pathway to the lipoxygenase pathway. Indeed, the contractions induced by angiotensin II were inhibited (P<0.05) by phenidone 100 microM (cyclooxygenase and lipoxygenase inhibitor), baicalein 100 microM (5-, 12- and 15-lipoxygenases inhibitor), AA861 10 microM (5-lipoxygenase inhibitor) and MK571 1 microM (CysLT(1) receptor antagonist). Cysteinyl leukotrienes were released in response to angiotensin II (pg/mg dry weight tissue: 32+/-9 (basal, n=6) vs. 49+/-9 (angiotensin II 0.3 microM, n=6), P<0.05). LTD(4), and at a lesser degree LTC(4), induced contractions of internal mammary artery and MK571 1 microM abolished the contraction to LTD(4). CONCLUSIONS: This study suggests that the in vitro vasoconstrictor effects of angiotensin II in human internal mammary artery are enhanced at least in part by eicosanoids produced by the cyclooxygenase pathway, probably PGH(2), acting on TXA(2)/PGH(2) receptors, and by lipoxygenase-derived products, particularly cysteinyl leukotrienes acting on CysLT(1) receptors.     

Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4.

5-Lipoxygenase pathway-derived products of arachidonic acid released by human eosinophils activated in vitro have been measured by using radioimmunoassays specific for leukotriene B4 (LTB4) and for sulfidopeptide leukotrienes including leukotriene C4 (LTC4). Eosinophil-enriched leukocytes (mean, 85% eosinophils) from five hypereosinophilic donors activated with 5.0 microM ionophore A23187 for 15 min at 37 degrees C in the presence of 50 mM L-serine released 69 +/- 28 and 1.5 +/- 0.8 (mean +/- SEM) ng of LTC4 and LTB4, respectively, per 10(6) cells; ratios of LTC4 to LTB4 ranged from 16 to 149. Eosinophils stimulated with ionophore (2.5 microM) or phorbol myristate acetate (1 microgram per ml) metabolized exogenously added LTC4 to products that coeluted on reverse-phase high-performance liquid chromatography with synthetic S-diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 diastereoisomers, and this metabolic inactivation was inhibited by L-serine or catalase. Ionophore-activated eosinophils purified from three normal donors also preferentially generated LTC4 (38 +/- 3 ng per 10(6) cells) relative to LTB4 (6.0 +/- 3.1 ng per 10(6) cells), whereas neutrophils from the same donors released LTB4 (48 +/- 21 ng per 10(6) cells) in a greater than 7-fold excess to LTC4. The predominant production by human eosinophils of LTC4 with its potent smooth muscle spasmogenic and vasoactive properties may contribute to the pathobiology of allergic and other diseases associated with eosinophilia.