TxA2-mediated myocardial ischemia as a consequence of an acute lung inflammatory reaction in the rabbit.

Epidemiological studies link acute infection of the respiratory tract to a transient increased risk of acute myocardial infarction. The underlying mechanisms remain unknown. We hypothesized that vasoactive mediators produced by inflammatory cells in the lungs and drained in the coronary circulation may trigger acute myocardial ischemia. To test this hypothesis we used an experimental model in the rabbit. Injection of the bacterial-derived peptide N-formyl-Met-Leu-Phe (or N-formyl-Methionyl-Leucyl-Phenylalanine)(fMLP) in the jugular vein induced massive recruitment of both polymorphonuclear leukocytes (PMN) and platelets in the microcirculation of the lungs, accompanied by rapid and marked increase of leukotriene B4, cysteinyl leukotrienes and thromboxane (Tx) A2 in the aortic blood. In all animals, fMLP evoked ischemic electrocardiographic changes: within the first minute of infusion a profound depression of the ST segment and inversion of the T wave were observed. Mean aortic pressure and heart rate fell to 64.0 +/- 6.9 and 83.5 +/- 3.1% of the basal levels at 3 and 10 min, respectively. All these alterations were transient. Aspirin, prevented electrocardiographic ischemic changes, reverted bradycardia and hypotension but did not significantly modify either PMN or platelet recruitment nor leukotriene synthesis. Ridogrel, a Tx-synthase and receptor inhibitor, prevented ECG alterations and bradycardia, but did not prevent and even worsened hypotension; it blocked platelet, but not PMN, sequestration. Pretreatment of animals with intravenous high dose of aspirin prevented ridogrel-dependent hypotension and platelet inhibition, suggesting that PGI2 contributes to the effects of Tx-synthase and receptor inhibitor. In hypercholesterolemic rabbits, ECG alterations persisted longer than in normal controls. In summary, our results indicate that acute activation of PMN and platelets in the lungs provokes transient myocardial ischemia, in normal animals that is exacerbated in hypercholesterolemic rabbits. TxA2 appears to be the major mediator of this phenomenon. Moreover the data suggest that a balance between TxA2 and PGI2 plays a pivotal role in platelet activation and recruitment in our model.

Platelet contribution to leukotriene production in inflammation: in vivo evidence in the rabbit.

The contribution of platelets to arachidonic acid transcellular metabolism may represent an important pathway of leukotriene (LT) production. The aim of this study was to investigate the role of platelets on LT production in an acute inflammatory model in the rabbit. Preliminary experiments showed that rabbit whole blood (5 ml) stimulated in vitro with the calcium ionophore A23187 produced LTB4 (52.7+/-13.9 ng) and the mixed 5,12-DiHETE (7.25+/-0.75 ng). In A23187-stimulated thrombocytopenic blood, LTB4 was significantly reduced to 19.5+/-8.6 ng and 5,12-DiHETE was undetectable. Peptido-LTs were undetectable in both conditions. In experiments using washed cells, addition of thrombin-activated platelets to fMLP-activated PMN resulted in the appearance of 5,12-DiHETE and in more than twofold increase of LTB4 synthesis. When 3H-arachidonic acid-labelled platelets were mixed with unlabelled PMN and challenged with fMLP and thrombin, radioactive LTB4 and 5,12-DiHETE were produced, indicating that platelet-derived arachidonic acid was utilized by PMN 5-lipoxygenase. Intravenous infusion of fMLP (2.5 nmol/kg/min) in the rabbit induced marked granulocytopenia, thrombocytopenia and increased TxB2 plasma concentrations within 3 min. Electron microscopy of lungs showed morphologically activated and aggregated platelets occluding the capillary lumen. Activation and recruitment of circulating cells was accompanied by the production of LTB4 (peak levels at 1 min: 30.0+/-9.5 ng/ml) and LTE4 (peak levels at 10 minutes: 77.8+/-11.6 ng/ml). The areas under the blood concentration-time curve (AUC, ng min/ml) corresponded to 812+/-182 and 3692+/-658 for LTB4 and LTE4, respectively. In immunologically thrombocytopenic rabbits, the AUC for LTB4 (86.0+/-23.0) and LTE4 (1165+/-542) were both significantly different from controls while in rabbits treated with an anti-leukocyte antiserum, both LTB4 and LTE4 were similar to controls. This experimental model provides in vivo evidence that platelets, involved in an acute inflammatory event contribute to the transcellular production of LTs.

Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function.

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM, mean+/-s.e.mean), as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM, IC50 18.4+/-1.8 and 31+/-1.8 microM), and C5a (0.1 microM, IC50 41.6+/-3.5 and 42+/-8.3 microM), and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4), 6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.

Kinetics of endogenous leukotriene B4 and E4 production following injection of the chemotactic peptide FMLP in the rabbit.

The kinetic profiles of leukotriene B4 (LTB4) and E4 (LTE4) after intravenous administration (30 nmol/kg) of the inflammatory peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) were evaluated in male rabbits. LTB4 and LTE4 reached the maximal concentration of 84.2 +/- 60.0 and 162.2 +/- 51.4 nmol/L (mean +/- s.d.), at 2 and 5 min, respectively. The first elimination phase for LTB4 and LTE4, after FMLP administration, showed an apparent half-life of 24.6 +/- 6.7 and 36.9 +/- 13.0 min, respectively. The area under the blood concentration-time curve (AUC, nmol min/L) of LTB4 and LTE4 was 2178 +/- 1591 and 7627 +/- 3052, respectively. LTE4 and N-ac-LTE4 were the major components excreted in the urine, mostly in the first time interval (0-12 h) of urinary collection after FMLP treatment; 11-trans-LTE4 was recovered in the second interval (12-24 h). Two other more polar compounds, potential metabolites, were recovered in the first interval of urine collection. Knowledge of the kinetic characteristics of endogenously produced leukotrienes may be useful in understanding the role of these eicosanoids in inflammatory and thrombotic disease, as well as in evaluating the efficacy of drugs designed to modulate their production and effect.

[Hemostatic factors and family history of thrombosis in patients with a myocardial infarct: a case-control study. The participants in GISSI-2-Efrim. Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico]. / Fattori dell'emostasi e storia familiare di trombosi in pazienti con infarto del miocardio: studio caso-controllo. I patecipanti al GISSI-2-Efrim.

We studied a series of hemostasis factors in a group of patients selected from a cohort of 916 patients affected by MI from the GISSI-2 study population. Following a case-control design, 73 patients with a family history of thrombosis (the presence of at least two first degree relatives affected by MI and/or stroke before 65 years) were matched with MI patients with no family history of thrombosis. Blood collection could be performed 6 +/- 1 months after the acute phase following MI in 53 pairs of such patients. The presence of mixed disulphides (MDS) was significantly higher in patients with family history than in controls; MDS were detected in 7 cases and only in 1 control. No difference was found in contrast in the distribution of fibrinogen, factor VII, factor VIII, vWF, protein C, protein S, AT III, HC II, PAI-1, lipoprotein (a). Nevertheless, independently from the family history, in the whole population of MI patients studied, 21 cases of suspected deficiency of protein C were found. Sixteen out of 53 patients with family history of MI and/or stroke had a family history of MI only. In patients with family history of MI the t-PA antigen levels were significantly lower than in the control group (7.5 +/- 4.4 vs 11.1 +/- 3.5 ng/ml, t = -2.6, p < 0.02). In the whole population of MI patients and in patients with a family history of thrombosis t-PA antigen was positively correlated with PAI-1 antigen and vWF. The correlation with PAI-1 was lost in patients with family history of MI.(ABSTRACT TRUNCATED AT 250 WORDS)

The endoperoxides/TxA2 analogue, U46619, inhibits human polymorphonuclear leukocyte function.

The effects of the stable analogue of TxA2, U46619, on polymorphonuclear leukocyte (PMN) function were investigated. U46619, at micromolar concentrations, inhibited fMLP-stimulated aggregation, beta-glucuronidase release, and superoxide production. fMLP-induced LTB4 synthesis was also inhibited. U46619 did not modify intracellular Ca2+ increase induced by fMLP in Fura-2-loaded PMN, suggesting that early events of cell activation were not involved. In fact, U46619 also inhibited aggregation, beta-glucuronidase release, superoxide anion and LTB4 production induced by the calcium ionophore A23187. By comparison with the specific 5-lipoxygenase inhibitor, L-663,536, which prevented LTB4 synthesis without affecting degranulation, we excluded the impairment of PMN function by U46619 as a consequence of the reduction of this endogenous agonist. TLC separation of lipid extracts from [3H]-AA-loaded PMN, stimulated by A23187, showed significant reduction of the radioactivity associated with authentic free AA, suggesting that U46619 could interfere with mechanisms regulating AA release from membrane phospholipids. This suggestion is also supported by the observation that manoalide, a standard inhibitor of phospholipase A2, similarly to U46619, inhibits beta-glucuronidase release from stimulated PMN. Prostaglandin endoperoxides, produced by cells participating in inflammatory reactions, might therefore play a role in modulating PMN activities.

Polymorphonuclear leukocyte-platelet interaction: role of P-selectin in thromboxane B2 and leukotriene C4 cooperative synthesis.

In PMN/platelet suspensions stimulated by fMLP giant mixed aggregates are formed and TxB2 and LTC4 are synthesized as the result of the cooperation in the arachidonic acid (AA) metabolism during cell/cell contact. PMN-derived cathepsin G induced the expression of P-selectin on platelet surface. GE12, an antibody against P-selectin, significantly reduced mixed cell aggregates. GE12 did not affect platelet aggregation induced by PMN-derived supernatants, indicating that the inhibitory effect of GE12 on mixed cell aggregation depends on inhibition of PMN/platelet adhesion. GE12 significantly reduced TxB2 and LTC4 production in PMN/platelet mixed cell suspensions stimulated by fMLP. As previously reported, synthesis of 3H-TxB2 in 3H-AA-labeled PMN/unlabeled platelets indicates that platelets utilize 3H-AA from PMN. 3H-LTC4 production in unlabeled PMN/3H-AA-labeled platelets indicates that bidirectional routes are utilized in this system for LTC4 synthesis. GE12 significantly reduced 3H-TxB2 and 3H-LTC4 synthesis. These results show that cathepsin G released by activated PMN induces the expression of P-selectin on platelet membrane: this adhesive glycoprotein modulates cell-cell contact and transcellular metabolism of AA.

Simultaneous determination of leukotrienes B4 and E4 in whole blood and of leukotriene E4 in urine of rabbit by reversed-phase high-performance liquid chromatography.

Sulfidopeptide leukotrienes E4 (LTE4) and B4 (LTB4) were simultaneously extracted from rabbit whole blood with acetonitrile. LTC4 and LTD4 were converted to LTE4 by gamma-glutamyl transpeptidase and leucine-amino peptidase before extraction. LTE4 was extracted from urine with C18 Sep-Pak cartridges. The compounds were resolved and quantitated by reversed-phase high-performance liquid chromatography (HPLC) with a diode-array detector; in selected cases the collected fractions were assayed for LTB4 and LTE4 by specific enzyme immunoassay (EIA). The correlation factor of the measured increase in LTE4 concentrations and addition of incremental amounts of LTC4 to blood was r = 0.998; slope of 1.05 +/- 0.01 (mean +/- S.D.). Concentrations of LTE4 measured by HPLC correlated with those obtained with EIA (r = 0.996; slope = 0.98 +/- 0.03 and r = 0.991; slope = 0.97 +/- 0.04 in blood and urine, respectively). For blood LTB4 the correlation of HPLC versus EIA was r = 0.990; slope = 1.12 +/- 0.04. The method described is accurate and reproducible, allowing measurement of both LTB4 and LTE4 in whole blood after a single extraction procedure. Simultaneous measurement of these metabolites after specific stimulation or in pathological conditions is recommended for in vivo investigations of LTs production.

Pharmacokinetic profile of theophylline in isolated perfused liver of rabbits at different ages. Development of drug-metabolizing activity during ontogenesis.

The ontogeny of the biotransformation of exogenous and endogenous compounds has been mostly studied using liver cells and microsomal fractions. We have used liver perfusion for the first time to characterize the development of the total P-450 cytochrome-dependent system in the rabbit, with theophylline (TH) as tool substance. Livers of 0- to 60-day-old rabbits were perfused with TH (10 micrograms/ml) for 3 hr. Metabolizing enzymes (cytochrome P-450), ATP, glutathione, and glycogen were measured in liver tissue after perfusion. Lactate dehydrogenase, glutamic-oxalacetic transaminase, glucose, and urea were assayed in the medium throughout perfusion. The pharmacokinetic profile of TH was determined. The activity of total cytochrome P-450, as well as the intrinsic unbound clearance and TH metabolites production, increased following a similar sigmoidal pattern and reached a plateau around 30-45 days of the postnatal development of rabbit liver. The perfused tissue showed no signs of age-related hepatic damage or toxic effects of TH. Thus, the results in perfused liver predict its metabolic capacity during ontogenesis.

Transcellular metabolism of arachidonic acid: increased platelet thromboxane generation in the presence of activated polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMN) activated by n-formyl-methionyl-leucyl-phenylalanine (fMLP), in the presence of cytochalasin B, are able to induce activation of coincubated autologous platelets "via" cathepsin G released from the azurophilic granules. However, thromboxane (Tx) B2 production in this system cannot be completely explained by cathepsin G-stimulated platelet arachidonate metabolism. Indeed, the amount of TxB2 found in supernatants of platelet/PMN suspensions challenged with 1 mumol/L fMLP was twofold to fourfold higher than that measured when platelets were stimulated by supernatants from fMLP-activated PMN. In the present report, we analyzed the possibility that PMN-induced TxB2 production in this system is the result of transcellular metabolism of arachidonic acid (AA) between fMLP-activated PMN and cathepsin G-stimulated platelets. 3H-AA-labeled PMN were used to test if a transfer of AA or metabolite(s) occur from PMN to platelets. Our results showed that: (1) 3H-TxB2 and 3H-12-HHT are synthesized when 3H-AA-labeled PMN are activated mixed to unlabeled platelets; (2) total radioactivity released by fMLP-stimulated PMN is increased in the presence of platelets, whereas the membrane content of unesterified 3H-AA is reduced; (3) platelet cyclooxygenase inhibition completely prevents 3H-TxB2 synthesis; and (4) inhibition of cathepsin G-induced platelet activation with the antiprotease eglin C blocks the formation of 3H-TxB2. These data show that in the experimental system used, platelets use PMN-derived unmetabolized AA to synthesize TxB2.

Bioavailability of oral isbufylline in rabbits.

The bioavailability of isbufylline was assessed in male rabbits given 12 mg/kg i.v. (intravenous) or per os (oral) according to a randomized design. The concentrations of unbound (fu = 54.0) isbufylline were considered in plasma as a function of time, after i.v. and oral administration. After oral administration in saline solution, a mean absolute oral bioavailability of 59.6% was calculated. The drug is rapidly absorbed and the comparison of the kinetic profiles after i.v. and per os administration, revealed a rapid elimination: t1/2 27.3 and 28.8 min respectively, and a total body clearance of 67.06 ml/min/kg. Urinary recovery 0-48 h accounted for less than 1% of the dose.

Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography-mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies.

Pharmacokinetics of theophylline in the newborn and adult rabbit. In vivo and isolated perfused liver approaches.

The isolated perfused liver technique is the in vitro system most nearly comparable to the intact liver for experimental investigations on drug metabolism. The model currently used employs liver from different species, but only adults. For the first time, we have set up an experimental investigation involving perfusion of the liver of newborn animals. Using theophylline (TH) as tool drug, an in vivo/in vitro and adult/newborn disposition study was made in the rabbit. After a 10 mg/kg dose iv to adult rabbits and ip to rabbits at birth, the pharmacokinetic profile of TH was analyzed during liver perfusion at comparable TH concentrations in the medium. A few biochemical variables were recorded. No age-related differences were observed in the release of glutamic-oxalacetic transaminase and lactate dehydrogenase over the perfusion time. O2 consumption was higher in adults than in newborns, in accordance with the lower metabolic capacity of the neonatal liver, supported by the lower values of cytochrome P-450, cytochrome c, and glutathione. In vivo and in vitro values were close in adults and newborns for half-life (average 5.2 vs. 5.4 and 27 vs. 35 hr, respectively) and intrinsic clearance of TH (13 vs. 11 and 0.032 vs. 0.021 ml/min). The qualitative and quantitative TH metabolic patterns in the medium and in vivo also were close in adult animals. Only unchanged TH was detected in newborn perfusate. The isolated perfused liver technique appears to offer a reliable model for studying the in vitro ontogeny of drug metabolism, and for making in vitro and in vivo physiological and pharmacological comparisons.

Placental transfer and tissue distribution of vitamin E in pregnant rabbits.

alpha-Tocopherol (vitamin E) is widely prescribed in neonatal intensive care units, in large doses and by different schedules, for the prevention of retrolental fibroplasia, intraventricular haemorrhage, bronchopulmonary dysplasia, and haemolytic anaemia. Since the efficacy of the drug in premature newborns seems related to early administration, the physicochemical characteristics of the drug itself and available formulations limit the major therapeutic aim of promptly raising levels of vitamin E in premature babies during the early hours of life. It has thus been suggested that vitamin E be given to the mother before delivery to produce higher drug concentrations in the newborn. To see whether this would work, the tissue distribution and transplacental transfer of vitamin E were studied in six pregnant rabbits at steady-state after an i.v. bolus + infusion to give a mean venous blood concentration of about 325 mumol l-1 of alpha-tocopherol acetate, corresponding to about 30 mumol l-1 of alpha-tocopherol. Endogenous levels were measured in six control pregnant rabbits. In treated animals alpha-tocopherol was increased in liver, spleen, placenta, lung, mammary gland, blood, and bile but not in brain, heart, fat, muscle or adrenals probably because distribution into these tissues is very slow. Vitamin E levels in the placenta of treated mothers were 15 times those of control rabbits, but the vitamin was not detectable in amniotic fluid and only very low levels were found in fetal blood. These findings do not indicate any advantage of giving mothers alpha-tocopherol acetate before delivery.

Thiopental efficacy in phenobarbital-resistant neonatal seizures.

The successful treatment with thiopental (10 mg/kg, i.v.) of 9 severely asphyxiated newborns, under artificial ventilation, with neonatal seizures resistant to phenobarbital, is reported in this pilot study. The clinical and electroencephalogram control of seizures was prompt and resolute. No adverse effect on cardiovascular function (heart rate, blood pressure) was observed. The terminal half-life of thiopental averaged 9 h, the total plasma clearance 0.20 l/h/kg, and the steady-state volume of distribution 3.6 l/h/kg. The kinetic profile of the drug compared to phenobarbital and phenytoin in newborns suggests that its action is quicker and shorter lasting. Thus, from these findings, thiopental may offer a useful and handy approach for the safe and effective treatment of phenobarbital-resistant neonatal seizures.

Placental transfer and tissue distribution of thiopental in the pregnant rat.

The pharmacokinetic profile of thiopental was studied in pregnant rats after an iv bolus dose of 15 mg/kg. The unbound concentration-time profile of the drug in maternal plasma, placenta, fetal brain, fetal carcass, and amniotic fluid was described, developing an adequate pharmacokinetic model. Maternal plasma levels of thiopental fell rapidly after injection, distributing into tissues (half-life of distribution phase averaged 3 min). Thiopental crossed the placenta and entered the fetal body (brain included) and amniotic fluid. Peak levels were seen within 10 min of injection and declined in all tissues parallel to maternal plasma (rate constant range 0.012-0.017 min-1). The concentrations of drug in the fetal unit were smaller than in the central compartment and maternal plasma. However, the absolute transfer ratios (calculated using the pharmacokinetic parameters obtained from the model) and the relative exposure ratios (as the ratio of the area under the unbound concentration-time curve in tissue to that in maternal plasma) suggested that fetuses were exposed to a potentially efficacious level of the drug. The model formulated to describe the tissue distribution of thiopental may offer a useful approach for analysis of the kinetic profile of other compounds administered during pregnancy or at delivery in rats and other species.

Bioavailability of intramuscular vitamin E acetate in rabbits.

The bioavailability of alpha-tocopherol acetate and alpha-tocopherol (vitamin E) was assessed in male rabbits given 50 mg kg-1 doses according to a randomized design. After intramuscular injection of alpha-tocopherol acetate in colloidal aqueous solution, a mean absolute bioavailability of 65% was calculated for the acetate and 35% for the physiologically active compound, alpha-tocopherol. Comparison of the kinetic profiles after intravenous and intramuscular administration of the acetate and intravenous injection of alpha-tocopherol, revealed absorption of alpha-tocopherol acetate from the site of injection and hydrolysis of the acetate to be potential limiting steps in the bioavailability of alpha-tocopherol. Intramuscularly injected alpha-tocopherol acetate in olive oil (the only formulation available in a few European countries) proved completely bio-unavailable. It thus appears necessary to re-assess the utility of current vitamin E supplementation, since the only formulations available offer poor bioavailability.