Sorbus domestica leaves are a traditionally used herbal medicine recommended for the treatment of oxidative stress-related diseases. Dry leaf extracts (standardized by LC-MS/MS and LC-PDA) and nine model activity markers (polyphenols), were tested in scavenging assays towards six in vivo-relevant oxidants (O₂^•-, OH^•, NO^•, H₂O₂, ONOO^-, HClO). Ascorbic acid (AA) and Trolox (TX) were used as positive standards. The most active extracts were the diethyl ether and ethyl acetate fractions with activities in the range of 3.61-20.03 µmol AA equivalents/mg, depending on the assay. Among the model compounds, flavonoids were especially effective in OH^• scavenging, while flavan-3-ols were superior in O₂^•- quenching. The most active constituents were quercetin, (-)-epicatechin, procyanidins B2 and C1 (3.94-24.16 µmol AA/mg), but considering their content in the extracts, isoquercitrin, (-)-epicatechin and chlorogenic acid were indicated as having the greatest influence on extract activity. The analysis of the synergistic effects between those three compounds in an O₂^•- scavenging assay demonstrated that the combination of chlorogenic acid and isoquercitrin exerts the greatest influence. The results indicate that the extracts possess a strong and broad spectrum of antioxidant capacity and that their complex composition plays a key role, with various constituents acting complementarily and synergistically.

The effect of a selective LTD4 receptor antagonist SK & F104353 was studied in septic pigs anesthetized with isoflurane. Yorkshire pigs (25.2 +/- 2.3 kg) were instrumented and monitored for cardiac output (CO), mean arterial pressure (MAP), systemic vascular resistance (SVR), mean pulmonary arterial pressure (MPAP), pulmonary vascular resistance (PVR), renal artery blood flow (RABF), renal vascular resistance (RVR), arterial PO2, and extravascular lung water (EVLW). Blood samples were also collected for platelet, white blood cell and hematocrit determinations and plasma was assayed for thromboxane (TX) B2. Sepsis was induced by infusion of Pseudomonas aeruginosa (3 x 10(8) CFU%kg/h) for 2 h. Cardiovascular and hematologic data were determined at 30 min intervals for 4 h. Groups were infused with either SK & F104353 (3 mg/kg/h; n = 5) or drug vehicle (n = 6) beginning 15 min prior to infusion with the P. aeruginosa. In the vehicle group beginning at -90 min after sepsis induction, there was a 30 +/- 7% decrease of CO, a 27 +/- 5.0% decrease of MAP, and a 44 +/- 7% decrease of RABF, whereas, MPAP increased to 147 +/- 37% and plasma TXB2 increased from less than 200 pg/ml to 3,049 +/- 367 pg/ml (P less than 0.05). The EVLW and hematocrit increased (P less than 0.05), and the arterial PO2, white blood cell count, and platelet count decreased with the severity of the sepsis. In pigs pretreated with SK & F104353 the MAP and RABF were transiently improved (P less than 0.05), and the decrease in arterial PO2 was delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels of thromboxane (TX) B2 (the stable metabolite of TXA2) were quantified by radioimmunoassay in periovulatory ovine follicles. Follicles were obtained before, and at 8, 16, 24 and 32 h after the onset of the preovulatory surge in secretion of luteinizing hormone (the ovulatory event culminates at approximately 24 h). Unruptured follicles were segregated into tissue and fluid components. A portion of the wall of each follicle was processed for examination by high resolution light and transmission electron microscopy. Concentrations of TXB2 in homogenates of the follicular wall, and in fluid of follicles that had not yet ruptured, were dramatically elevated at 24 h. Aggregates of platelets (the suspected source of TXA2) were adhered to endothelial cells at this time; in some cases intra and extravascular clotting was apparent. It is suggested that platelets and(or) TXA2 might contribute to periovulatory processes in the ewe.

Escherichia coli endotoxin (1 mg/kg) infusion over 30 min into anesthetized artificially ventilated dogs caused a biphasic response: an early phase of pulmonary hypertension and a late phase of increased lung vascular permeability. During an early phase, PG F2 alpha, Tx A2 (as Tx B2) and prostacyclin (as 6-keto-PG F1 alpha) concentrations increased in plasma or right duct lymph of dogs. During a late phase, the concentrations of PG F2 alpha and Tx A2 decreased to near the base-line values, while the concentration of prostacyclin remained elevated. Administrations of PG synthetase inhibitors 45 min prior to endotoxin inhibited the increase in concentration of prostacyclin following the infusion of endotoxin and potentiated the increase in lung vascular permeability at the beginning of the late phase. Continuous infusion of prostacyclin (20 ng/kg/min) starting one hour before endotoxin for 5 hour periods prevented the increase in lung vascular permeability induced by endotoxin. Based on these results, we could conclude that endogenous prostacyclin might play an important role in preserving cell integrity of lungs and counteract the deleterious effects of endotoxin.

Cyclo-oxygenase metabolites are important regulators of pulmonary vascular and airway tone and may act to regulate ventilation-perfusion (VA/Q) relationships. Hypoxemia that follows aspiration of gastric acid is associated with increased venous admixture, and plasma levels of thromboxane (TX) B2 and 6-keto-PGF2 alpha are increased after experimental acid-induced acute lung injury. The present study was designed to determine the effects of cyclo-oxygenase metabolites on VA/Q relationships in canine acid aspiration. Eighteen anesthetized dogs received 0.2 mL/kg 0.1 N HCl intratracheally; six were pretreated with ibuprofen (IBU), a cyclo-oxygenase inhibitor, 12.5 mg/kg IV, and six other dogs received OKY-046 (OKY), a TX synthetase inhibitor, 0.5 mg/kg IV. The remaining six animals (ACID) served as controls. Continuous distributions of ventilation and perfusion were evaluated with the multiple inert gas elimination technique. Within 30 minutes, acid injury resulted in significant (p < 0.05) decreases in PaO2 from baseline values by 44.7 +/- 5.4 and 47.6 +/- 4.8 mm Hg in the ACID and OKY groups, respectively. Although decreased, the change in PaO2 of 21.0 +/- 4.8 mm Hg in IBU animals was significantly (p < 0.05) attenuated in comparison with the other groups. Ibuprofen increased pulmonary vascular resistance, attenuated perfusion to shunt and low VA/Q areas, and reduced ventilation to unperfused areas for the first 2 hours after acid injury (all p < 0.05), whereas OKY exacerbated hypoxemia and VA/Q inequality.(ABSTRACT TRUNCATED AT 250 WORDS)

A method of simultaneous analysis of prostaglandins (PGs) and thromboxane (TX) B2 in cerebrospinal fluid (CSF) with GC-MS-SIM was established. Deuterated PGs and TXB2 were used as internal standards: tetra-deuterated PGE2 (d4-PGE2) for PGE2, PGE1 and PGD2; d5-PGF2alpha for PGF2alpha and 9alpha,11beta-PGF2 and 8-epi PGF2alpha; d4-TXB2 for TXB2; and d4-6-keto PGF1alpha for 6-keto PGF1alpha. The PGs and TXB2 were derivatized to the methyl ester of the methoxim dimethyisopropylsilyl (DMiPSi) ether form or the methyl ester of the DMiPSi ether form with simultaneous preparation. Samples were extracted with octadecyl silica gel and purified in two steps with silisic acid gel chromatography between derivatization steps. The calibration curve of each PG and TXB2 was linear from 10 pg to 10 ng with the isotope dilution method. The levels of the seven types of PG and of TXB2 were assayed simultaneously in the cerebrospinal fluid (CSF) from patients with aseptic meningitis. The CSF pattern of the PG and TXB2 concentrations in mumps meningitis differed from those in other types of aseptic meningitis and in disease controls.

We investigated the role of neurogenic inflammation and the subsequent mechanisms in cigarette smoke-induced airway hyperresponsiveness in guinea pigs. Exposure to cigarette smoke was carried out at tidal volume for 3 min. Airway responsiveness to histamine was determined before and after smoke exposure followed by bronchoalveolar lavage (BAL). Plasma extravasation was evaluated by measuring the extravasation of Evans blue dye in the airway. Cigarette smoke produced significant airway hyperresponsiveness and plasma extravasation, with an influx of neutrophils in BAL fluid. FK-224 (10 mg/kg i.v.), a tachykinin antagonist at NK1 and NK2 receptors, significantly inhibited these changes. The thromboxane (Tx) B2 concentration was increased in BAL fluid after smoke exposure and was significantly inhibited by FK-224. OKY-046 (10 mg/kg i.v.), a Tx synthase inhibitor, significantly inhibited airway hyperresponsiveness but had no effect on neutrophil influx or plasma extravasation. The results suggest that neurogenic inflammation and the subsequent generation of Tx in the airway are important in the development of the airway hyperresponsiveness induced by cigarette smoke.

In the Mouse Antithrombotic Assay aspirin (30-300 mg/kg) protected mice from death by 15 and 42%, respectively. Four Ca++ channel blockers (nitrendipine, nicardipine, nifedipine and verapamil) were effective in reducing the mortality. At the dose of 100 mg/kg nitrendipine and nicardipine gave 80 and 85% protection respectively. Whereas aspirin almost suppressed completely thromboxane (Tx)B2 and platelet factor-4 release after collagen-epinephrine infusion, neither nitrendipine nor nicardipine modified TxB2 release and only reduced slightly platelet factor-4 release. The treatment with aspirin, nitrendipine or nicardipine did not counteract the fall in circulating platelets counted 1 min after the aggregation challenge, but at 3 min platelet count was significantly higher in aspirin-treated mice than in animals given either Ca++ channel blocker. Mouse platelet aggregation induced in vitro by the combination of collagen and epinephrine was inhibited in samples obtained from mice pretreated with aspirin but was unaffected by the treatment with nitrendipine or nicardipine. The i.v. injection of a 12.5% suspension of hardened red blood cells resulted in death of about 80% of mice within 1 to 2 min. Neither circulating platelet count nor plasma TxB2 level were modified significantly by red cell injection. Aspirin was ineffective whereas both Ca++ channel blockers lowered mortality to 50%. These data suggest that Ca++ channel blockers reduce the mortality in the Mouse Antithrombotic Assay by influencing factors other than platelet aggregation and/or Tx production. These factors might be important in mediating mortality occurring after infusion of hardened red cells.

Nitroglycerin and isosorbide dinitrate (ISDN), the most commonly used nitrate vasodilators, have been shown to possess antiplatelet properties. It has also been shown, interestingly, that their inhibition of aggregation (mostly upon ADP and adrenaline) occurs in vivo at concentrations 1-2 log orders lower than in vitro, and in the full therapeutic range. Many different hypotheses may explain such an in vivo/in vitro difference: 1) that nitrates induce prostacyclin synthesis; 2) that they synergize prostacyclin; 3) that they give rise to more active in vivo metabolites; 4) that some other requirement for their action, such as the availability of reducing thiols, may be a limiting factor in the in vitro setting. The discrimination among such hypotheses should contribute new insights into nitrate action at the platelet level. On the basis of experiments on cultured endothelial cells and vascular fragments, we had previously concluded against prostacyclin induction by nitrates. On the other hand, ISDN may decrease the IC50 for prostacyclin in aggregation by suprathreshold doses of various aggregating agents, and therefore, be synergistic with this endogenous antiplatelet substance. Compared to ISDN, the two longer-lived metabolites isosorbide-2- and isosorbide-5-mononitrates (IS-2-MN, IS-5-MN) appear remarkably different in terms of antiplatelet potency in vitro (minimum effective concentrations 10(-7)-10(-6)M for IS-2-MN, 10(-4)M for IS-5-MN, for ADP-and adrenaline-induced aggregation and thromboxane (TX) B2 production).(ABSTRACT TRUNCATED AT 250 WORDS)

This study tests whether activated complement leads to a selective entrapment of polymorphonuclear leukocytes (PMN's) in the lungs. Awake sheep were infused for 5 min with zymosan-activated plasma (ZAP, 2.5 mg/ml) at a rate of 5 ml/min into the superior vena cava (IV, n = 4) or intra-arterially into the aortic arch or femoral artery (IA, n = 8). At the end of IV infusion, leukocyte counts fell from 8,862 to 1,631/mm3 (P less than 0.01). PMN counts across the lungs decreased by 74%. There were increases in plasma thromboxane (Tx) B2 from 114 to 2,733 pg/ml (P less than 0.01), mean pulmonary arterial pressure from 12 to 42 mmHg (P less than 0.01), and physiological shunt from 13 to 25% (P less than 0.05). Within 1 h lymph TxB2 levels had risen from 301 to 4,916 pg/ml (P less than 0.01), lung lymph flow (QL) rose from 3.7 to 11.1 ml/30 min (P less than 0.05), lymph-to-plasma protein ratio (L/P) remained unchanged at 0.63, and lymph protein clearance increased from 2.3 to 7.5 ml/30 min (P less than 0.05). Leukosequestration, quantitated by capillary PMN counting and by assaying the granulocyte marker myeloperoxidase, occurred relative to sham animals (P less than 0.05) in the lung and spleen but not in other organs. Intra-arterial ZAP infusion led to changes that were similar in magnitude and timing to the IV group.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelets of patients with diabetes and no evidence of macroangiopathy produce normal amounts of thromboxane (Tx) B2 in vivo, whereas they usually show increased production in vitro. Since in vitro studies have been usually performed in citrated PRP, we tested the hypothesis that the discrepancy between in vivo and in vitro studies is due to the low concentration of plasma ionized calcium ([Ca2+]o) that is present in citrated PRP. In fact, low [Ca2+]o artifactually potentiates the platelet TxB2 production in vitro. Forty patients with diabetes mellitus and 37 matched controls were studied. Blood was anticoagulated with citrate, the thrombin inhibitor D-phenylalanyl-l-prolyl-l-chloromethylketone (PPACK) or both anticoagulants. Platelet aggregation, release of 14C-serotonin and TxB2 production were induced in platelet rich plasma (PRP) by several agonists. The following results were obtained: i) Citrated PRP: Arachidonic acid induced aggregation (p < 0.01) and TxB2 production (p < 0.02) were significantly greater in patients than in controls. No statistically significant differences were found with other agonists. ii) PPACK PRP: No statistically significant difference was found between diabetic platelets and controls. iii) PPACK plus citrate PRP: The results were not different from those obtained with citrate alone. Therefore, our results show that diabetic platelets produce normal amounts of TxB2 in vitro when the [Ca2+]o is physiological.

Isosorbide monitrates (IS-2-MN and IS-5-MN), hepatic metabolites of isosorbide dinitrate, inhibit platelet function in vitro very differently, with IS-2-MN being much more potent than IS-5-MN. To assess their antiplatelet properties in vivo and to compare time and dosage requirements, we infused both IS-2-MN and IS-5-MN for 30 minutes, on 2 separate days, into nine patients with stable coronary artery disease, at rates of 4 mg/hr (n = 4) and 8 mg/hr (n = 5). Two additional patients received IS-5-MN at 16 mg/hr. Platelet aggregation and thromboxane (TX) B2 generation in response to various agonists, drug plasma concentrations, and blood pressure were monitored throughout the study. A significant decrease in platelet aggregation and TXB2 production by adenosine diphosphate and adrenaline occurred in seven of nine patients receiving IS-2-MN and in 7 of 11 patients receiving IS-5-MN. Response was dose related, with more patients responding at 8 mg/hr to IS-2-MN (five of five) than to IS-5-MN (three of five), and was maximum at the end of the infusion time, corresponding to peak plasma levels. Patients responding to drug infusions with an inhibition of platelet function were characterized by a greater vascular responsiveness compared to nonresponders, since the decrease in systolic blood pressure (mean +/- SEM) was significantly greater in the former (15.4 +/- 3.2) than in the latter (2.5 +/- 2.1, p less than 0.05). Therefore both mononitrates, when administered at infusion rates between 8 and 16 mg/hr, are accompanied by a consistent inhibition of adenosine diphosphate- and adrenaline-induced aggregation and TX generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Indobufen is an antiplatelet drug able to inhibit thromboxane production and cyclooxygenase-dependent platelet aggregation by a reversible inhibition of cyclooxygenase. Indobufen exists in two enantiomeric forms, of which only d-indobufen is active in vitro in inhibiting cyclooxygenase. In order to verify that also inhibition of platelet function is totally accounted for by d-indobufen, ten patients with proven coronary artery disease (8 male, 2 female, age, mean +/- S.D., 58.7 +/- 7.5 years) were given, in random sequence, both 100 mg d-indobufen and 200 mg dl-indobufen as single administrations in a double-blind crossover design study with a washout period between treatments of 72 h. In all patients thromboxane (TX) B2 generation after spontaneous clotting (at 0, 1, 2, 4, 6, 8, 12, 24 h), drug plasma levels (at the same times), platelet aggregation in response to ADP, adrenaline, arachidonic acid, collagen, PAF, and bleeding time (at 0, 2, 12 h) were evaluated after each treatment. Both treatments determined peak inhibition of TXB2 production at 2 h from administration, with no statistical difference between the two treatments (97 +/- 3% for both treatments). At 12 h inhibition was 87 +/- 6% for d-indobufen and 88 +/- 6% for dl-indobufen (p = NS). Inhibition of TXB2 production correlated significantly with plasma levels of the drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies have demonstrated that urinary thromboxane B2 (TXB2) excretion (UTXB2) and glomerular production of TXB2 are enhanced in experimental diabetes and that selective inhibitors of TX synthesis prevent or delay the development of albuminuria. The present study was conducted to examine the contribution of platelet TXB2 production to the enhancement of UTXB2 and glomerular TXB2 production and to the pathogenesis of albuminuria in the partially insulin-treated moderately hyperglycemic (blood glucose, 200 to 400 mg/dL) streptozotocin-diabetic rat (SDR). Treatment of control rats or of SDR with diabetes of 5 months' duration with antiplatelet serum for 4 consecutive days reduced circulating platelet counts and serum TXB2 generation, an index of platelet cyclooxygenase activity, by 80% or greater, but reduced UTXB2 excretion by only 30%. UTXB2 and glomerular production of TXB2 of thrombocytopenic SDR remained markedly elevated compared with corresponding values from age-matched thrombocytopenic or platelet-replete, nondiabetic controls. Similarly, treatment of rats for 180 days with a dose of aspirin (ASA), which selectively inhibited platelet versus renal cyclooxygenase activity, reduced UTXB2 of both SDR and controls by 25% to 35%. The absolute reductions in UTXB2 induced by either ASA or thrombocytopenia in SDR were significantly greater than the absolute decrements in corresponding controls, suggesting that increased platelet TXB2 production in SDR may contribute to the enhanced UTXB2. However, as in the thrombocytopenic SDR, UTXB2 and glomerular production of TXB2 of SDR treated with ASA remained clearly above corresponding control values. Moreover, chronic ASA treatment failed to prevent the development of albuminuria in SDR.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of a peptide leukotriene receptor antagonist ONO-1078 on the production of thromboxane (Tx) B2 induced by leukotriene (LT) D4 and antigen challenge was examined in guinea pig lungs. LTD4 (1-1,000 nM) induced a concentration-dependent production of TxB2 in non-sensitized guinea pig lungs and ovalbumin challenge (0.01-100 micrograms/ml) produced TxB2 and peptide leukotrienes in a concentration-dependent manner in ovalbumin-sensitized guinea pig lungs. ONO-1078 inhibited LTD4 (100 nM)-induced TxB2 production with the IC50 value of 0.24 microM. Furthermore, ONO-1078 inhibited antigen (10 micrograms/ml)-induced TxB2 production with the IC50 value of 0.14 microM without effect on the production of peptide leukotrienes. These results suggest that ONO-1078 may prevent the antigen-induced production of TxB2 through the blockade of the activation of receptors by endogenously generated peptide leukotrienes.

Several studies have indicated that approximately 25 % of patients treated with aspirin exhibit high on-treatment platelet reactivity (HTPR), which is potentially associated with cardiovascular events (CVEs). However, this association is still controversial, since the mechanisms by which HTPR contributes to CVEs remain unclear and a no standardised definition of HTPR has been established. To determine whether HTPR is associated with CVE recurrence and what type of assay would best predict CVE recurrence, we conducted a multicentre prospective cohort study of 592 stable cardiovascular outpatients treated with aspirin monotherapy for secondary prevention. Their HTPR was determined by arachidonic acid- or collagen-induced aggregation assays using two different agonist concentrations. Residual cyclooxygenase (COX)-1 activity was assessed by measuring serum thromboxane (TX)B2 or urinary 11-dehydro TXB2. Shear-induced platelet thrombus formation was also examined. We followed all patients for two years to evaluate how these seven indexes were related to the recurrence of CVEs (cerebral infarction, transient ischaemic attack, myocardial infarction, unstable angina, revascularisation, other arterial thrombosis, or cardiovascular death). Of 583 patients eligible for the analysis, CVEs occurred in 69 (11.8 %). A Cox regression model identified several classical risk factors associated with CVEs. However, neither HTPR nor high residual COX-1 activity was significantly associated with CVEs, even by applying cut-off values suggested in previous reports or a receiver-operating characteristic analysis. In conclusion, recurrence of CVEs occurred independently of HTPR and residual COX-1 activity. Thus, our findings do not support the use of platelet or COX-1 functional testing for predicting clinical outcomes in stable cardiovascular patients.

1. BN 52021, an antagonist of platelet activating factor (PAF), was inactive against bronchoconstriction in guinea-pigs sensitized with low amounts of ovalbumin (OA) injected twice, at a 14 day interval and challenged i.v. 7 days later. 2. Serum IgG titers increased for 7 weeks after the booster injection at day 14 and returned to low levels at day 96. 3. Administered by the intratracheal (i.t.) route at 1 mg, BN 52021 failed to inhibit bronchoconstriction induced by the i.t. administration of OA to guinea-pigs tested 7, 28, 56 and 84 days after the booster injection, even when the titers of circulating IgG had declined with time. BN 52021 was also inactive against bronchoconstriction in guinea-pigs boosted at day 98 and tested 7 days later and against contractions and thromboxane (Tx) B2 and histamine release induced by OA-challenged parenchymal lung strips from the boosted guinea-pigs. 4. Sensitized unboosted guinea-pigs displayed reduced IgG serum titers. Used 21 or 70 days after the sensitizing injection, they did develop bronchoconstriction upon the i.t. instillation of OA, which was blocked by BN 52021. The latter also inhibited OA-induced contractions of lung parenchymal strips from these unboosted guinea-pigs. 5. When boosted and non-boosted guinea-pigs received OA i.t. and bronchoalveolar lavage fluid was collected 10 min later, the number of eosinophils increased markedly in boosted, but not in non-boosted guinea-pigs. 6. The booster injection of antigen thus modifies the response of the lung and PAF appears to be relevant for antigen-induced bronchoconstriction in unboosted animals, but loses its major role following the booster injection.

A radioimmunoassay method, using highly specific and sensitive rabbit antiserum, for the measurement of thromboxane (Tx)B2 in human plasma and urine, is described. Assay sensitivity was 5 pg; 50% displacement was achieved by 41 pg of cold TxB. Intra-assay variability was 10% and interassay variability 8,5%. Preliminary extraction and chromatography were necessary as direct radioimmunoassay of plasma although possible, was not reliable. The measurement of TxB2 reflects closely the synthesis of TxA2. The peripheral venous plasma TxB2 concentration in 13 young healthy volunteers (mean age 28 +/- 2 years) was 35 +/- 8 pg/ml; it was significantly higher in 14 older subjects (mean age 45 +/- 5 years) 101 +/- 14 pg/ml (p less than 0.001). Urinary TxB2 excretion was 141 +/- 15 pg/min in the younger group and 220 +/- 39 pg/min in the older group of normotensive subjects. There was a significant positive correlation between urinary TxB2 excretion and age (p less than 0.001). The increase with age of plasma and urinary TxB2 in man may have a pathophysiological importance.

We have previously demonstrated that the biologically important oxidant hydrogen peroxide (H2O2) triggers release and metabolism of arachidonic acid (AA) in the alveolar macrophage (AM). In this study, we evaluated the ability of glucocorticoids to inhibit rat AM AA metabolism stimulated by H2O2, as compared to the particulate zymosan. Methylprednisolone and other glucocorticoids failed to significantly inhibit release of AA stimulated by H2O2, while markedly reducing AA release in response to zymosan. Similarly, methylprednisolone only weakly inhibited synthesis of thromboxane (Tx)B2 stimulated by H2O2, while inhibiting zymosan-induced eicosanoid synthesis to a marked degree. On the other hand, the phospholipase inhibitor mepacrine strongly inhibited AA release and TxB2 formation stimulated by both H2O2 and zymosan, indicating that H2O2 induced AA metabolism is indeed susceptible to pharmacologic inhibition. The failure of glucocorticoids to inhibit AA metabolism stimulated by H2O2 in the AM may in part explain their inability to ameliorate oxidant-mediated lung inflammation and injury.

The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have used the U937 human monocytic cell line to explore the signal transduction mechanisms utilised by thrombin in these cells. In U937 cells differentiated into a macrophage-like phenotype, thrombin stimulated the formation of inositol trisphosphate (IP3) and the mobilisation of intracellular Ca2+ [( Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B2 synthesis in the differentiated cells. In contrast, the chemotactic peptide N-formyl-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxB2 synthesis under conditions where it evoked increases in IP3 formation and [Ca2+]i mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thrombin also failed to cause inhibitory guanine nucleotide binding protein (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. These results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB2 synthesis or cause Gi-mediated inhibition of adenylate cyclase in U937 cells contrasts with its effects in human platelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin receptor or receptor-effector coupling mechanism(s) present in other thrombin-responsive cells.

Prostaglandin synthetic profiles were studied in monolayers of highly enriched rabbit renal proximal tubular cells cultured in serum-free, hormone-supplemented, defined media. The cultures were initiated from glomeruli-free cortical suspensions. Cells in culture demonstrated morphologic and functional characteristics highly suggestive of proximal tubular cells. The basal and stimulated synthesis of immunoassayable prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 in response to various agonists, as well as the effect of two cyclooxygenase inhibitors, was assessed. Under both basal and stimulated conditions, PGE2 was the major product synthesized. PGF2 alpha and 6-keto-PGF1 alpha were synthesized to a lesser extent, and TxB2 was undetectable. The basal synthesis of PGE2 and PGF2 alpha in cultured cells was found to be higher than in isolated proximal tubular fragments by sevenfold and fivefold, respectively. Exogenous arachidonate, angiotensin II, and the divalent cation ionophore A23187 stimulated all three immunoassayable prostaglandins in a dose-dependent manner. Arginine vasopressin (10(-5) mol/L) had no stimulatory effect. In Ca++-free media or in the presence of 10(-5) mol/L Ca++ channel blocker, verapamil, the stimulatory effects of angiotensin II and A23187 were ameliorated. The stimulatory effect of angiotensin II was inhibited by saralasin (10(-5) mol/L), indicating that receptor binding could mediate PGE2 synthesis. Both indomethacin and sulindac sulfide (10(-5) mol/L) reversibly inhibited PGE2 synthesis.

The metabolism of arachidonic acid (AA) and the transfer of its metabolites was determined in in vitro perfused placental tissue from normal pregnancies and those complicated by maternal insulin-dependent diabetes mellitus (IDDM). 14C-labelled AA was recirculated in the fetal circulation for 60 min while 3H-AA was recirculated in the maternal circulation. Placental effluent was subjected to high performance liquid chromatography (HPLC) and analysis of dual-label scintillation counts. Placentae from IDDM pregnancies converted 3-6 times more radiolabelled AA to eicosanoids than did normal placentae. In addition, the transfer of eicosanoids into the opposing circulation was doubled in placentae from IDDM pregnancies compared to normal placentae. The predominant direction of eicosanoid transfer in both groups of placentae was in the fetal-to-maternal direction. The relative amounts of eicosanoids produced was also altered in placentae from IDDM pregnancies. Increased amounts of thromboxane (Tx) B2 and hydroxyeicosatetraenoic acids (HETEs) were present in both circulations of placentae from IDDM pregnancies. Levels of 6-keto prostaglandin F1a (6KPGF1a) were significantly reduced in both circulations in placentae from IDDM pregnancies. Thus, the ratio of TxA2 to PGI2 and the ratio of HETEs to PGI2 were both significantly increased in placentae from IDDM pregnancies. These results suggest an imbalance in eicosanoid production which may be relevant to abnormal placental structure and function in IDDM pregnancies.

Spontaneously hypertensive rats (SHR) were injected with streptozotocin (STZ-SHR) to induce diabetes. The effect of DP-1904, a thromboxane A2 synthetase inhibitor, on diabetic nephropathy was then studied by administering it for 5 months (1 or 10 mg/kg). DP-1904 did not affect renal 6-keto prostaglandin (PG)F1 alpha production in STZ-SHR, but markedly inhibited renal thromboxane (TX) B2 production, so that the 6-keto PGF1 alpha/TXB2 ratio was significantly increased (P less than 0.05). STZ-SHR showed significant uraemia and proteinuria, plus increases in urinary gamma-glutamyl-transpeptidase and urinary N-acetyl-beta-glucosaminidase. DP-1904 significantly decreased (P less than 0.01) the urinary changes. STZ-SHR also showed an increase in mesangial periodic acid-Schiff-positive substance and in relative renal weight, both of which were significantly inhibited by DP-1904 (P less than 0.05). Thus, DP-1904 inhibited both TXB2 production and the progression of renal damage in STZ-SHR.

The effects of platelet-activating factor (PAF) on prostanoid release during mesenteric ischemia-reperfusion-induced shock were investigated in anesthesized dogs 1) by measuring plasma levels of prostaglandin (PG)F2 alpha, 6-keto-PGF1 alpha and thromboxane (TX)B2 in the superior mesenteric vein during reperfusion following 2 hr occlusion of the superior mesenteric artery; 2) by monitoring the effects of BN 52021, a specific PAF receptor antagonist and indomethacin on hemodynamic parameters and prostanoid levels; and 3) by studying circulatory responses to PAF and PGF2 alpha injected into the superior mesenteric vein in the presence of BN 52021 or indomethacin. Restoration of the blood flow following 2 hr ischemia resulted in an immediate dramatic decrease in mean arterial blood pressure, with a concomitant increase in mean portal venous pressure, hematocrit values, and plasma prostanoid levels. Pretreatment of the animals either with BN 52021 (4 mg.kg-1) or indomethacin (2 mg.kg-1 plus 3 mg.kg-1hr-1) prevented the circulatory collapse and the increase in prostanoid levels during reperfusion. Administration of exogenous PAF (0.1 micrograms.kg-1) or PGF2 alpha (10 micrograms.kg-1) into the superior mesenteric vein evoked hypotension similar to that observed during reperfusion. Pretreatment of the animals with BN 52021 completely prevented the effects of PAF but failed to modify the responses to PGF2 alpha. Indomethacin at a dose that inhibited prostanoid formation was highly effective to attenuate the hypotensive response to exogenous PAF. These data suggest that prostanoid formation may be secondary to PAF release in circulatory collapse evoked by intestinal ischemia-reperfusion and give further support to the notion of the importance of PAF prostanoid interaction during ischemia-reperfusion-induced shock.