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Leukotrienes (LT) are synthesized in the lung during asthmatic reactions and mediate certain inflammatory symptoms. Pulmonary metabolism and clearance of exogenously added peptidoleukotrienes were studied in nonasthmatics, asthmatics, and asthmatics challenged with allergen. [3H]LTC4 and [14C]dextran were instilled into the airways, and bronchoalveolar lavage fluid (BALF) was obtained 15 min later. After comparing the [3H]/[14C] ratio of the instilled solution with that in BALF, 77% of LT were found to have been removed from the airways of nonasthmatics, and 72% of unchallenged asthmatics. Allergen administration to asthmatics 1 min before LT instillation inhibited LT transfer out of the airways by 26%, compared with asthmatics not challenged with allergen. This decrease in the removal of LT from the airways during allergic reactions could potentiate the physiologic effects of LT produced in the airways. The predominant LT in BALF was LTE4, constituting 56% of the LT in asthmatics and 61% in nonasthmatics. The percentage of LTE4 in BALF increased to 87% in allergen-stimulated asthmatics (p < 0.05 compared with the two other groups), this again reflecting decreased transfer of LT out of the lung rather than an increase in metabolism. Urinary excretion of LT metabolites occurred rapidly, the majority being excreted excreted within 6 h after instillation. LTE4, the major urinary LT metabolite identified by high-performance liquid chromatography, was a similar percentage concentration in the three groups and, thus, can be accurately used as an index of LT synthesis.

It has been suggested that the von Willebrand factor antigen (vWF:Ag) may be a clinical marker for pulmonary endothelial cell injury. An ELISA was developed for the measurement of rat vWF:Ag. Rat lungs were isolated and perfused with a recirculating, blood-free, physiologic salt solution. Circulating levels of vWF:Ag and the eicosanoids thromboxane B2 (TXB2) and prostaglandin 6-keto F1-alpha (6-keto PGF1 alpha) were measured before and after different forms of insult. The addition of phospholipase C (PLC) or hydrogen peroxide (H2O2) to the perfusate caused lung damage as manifested by pulmonary artery pressure increase and pulmonary edema. This was paralleled by significant release of vWF:Ag, TXB2, and 6-keto PGF1 alpha. Increased hydrostatic pressure caused pulmonary edema without vWF:Ag and eicosanoid release. The addition of vasopressin to the perfusate caused vWF:Ag release but no lung injury and no release of eicosanoids. It is concluded that in the rat model, vWF:Ag release is a nonspecific marker for lung injury.

Recruitment of inflammatory cells to the lung capillaries has been proposed as an important step in the sequence of events that lead to acute lung injury. Frequently, in the clinical setting, bacteremia and sepsis syndrome precede the acute lung failure and endotoxin priming may represent a comparable paradigm, useful for experimental pursuit. Following addition of the chemotactic tripeptide FMLP (10(-9) to 10(-6) M) to the cell-free, salt solution perfusate of isolated rat lungs, only a small degree of vasoconstriction was observed. However, in lungs isolated from rats that received 2 mg/kg intraperitoneal Salmonella enteritidis endotoxin 2 h before lung perfusion, FMLP dose dependently caused a large, transient pulmonary pressor response, edema formation, and release of large amounts of thromboxane and leukotriene B4. Since in vitro priming with endotoxin, direct vascular injury by neutrophil elastase, nor direct stimulation with FMLP of pulmonary artery rings from endotoxin-pretreated rats, mimicked the effects of in vivo endotoxin priming, we conclude that the presence of inflammatory cells in the lung capillaries accounted for the large amount of eicosanoids produced by the lungs after FMLP stimulation. In fact, by retrograde lavage of the lung circulation with a collagenase solution, previously adherent cell clumps were mobilized and identified. These cell clumps, composed of red blood cells, neutrophils, and platelets, were not seen in the vascular lavage sediment obtained from unprimed control lungs. Indomethacin, a thromboxane antagonist, AA861, a 5-lipoxygenase inhibitor, and WEB 2086, a platelet-activating factor (PAF) antagonist, reduced the thromboxane synthesis and release after FMLP (10(-7) M) in in vivo endotoxin-primed lungs. None of the inhibitors employed exclusively inhibited only one particular eicosanoid mediator but rather affected the release of several mediators, suggesting a close link between the different synthetic arachidonic acid pathways. An inhibitor of phospholipase C (2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate), NCDC, but not an inhibitor of phospholipase D (Wortmannin) or of protein kinase C (staurosporine) inhibited the FMLP-stimulated pulmonary pressure rise and eicosanoid release in endotoxin-primed lungs in vivo. Our data suggest that eicosanoids (in particular thromboxane) released from cells trapped in the lung circulation, but not from constitutive lung cells, contribute to vasoconstriction and edema formation caused by the chemoattractant FMLP in endotoxin-primed lungs.

A group of 17 patients with allergic asthma was challenged with aerosolized antigen, and the excretion of the peptidoleukotriene metabolite leukotriene (LT) E4 in the urine was determined as an index of leukotriene synthesis. The allergen challenge caused a drop in FEV1 of 25 to 59% within the first 2 h in all patients. This was associated with an increase in urine LTE4 excretion during the subsequent 12 h. The amount of urinary LTE4 excreted increased from a 12-h baseline level of 46 +/- 6.8 ng to a postallergen challenge level of 92 +/- 13 ng. In a similar experimental protocol methacholine challenge alone did not significantly increase urinary LTE4 excretion. In comparing all individuals as a group, there was no significant positive correlation between the magnitude of the drop in FEV1 during the immediate asthmatic response (IAR) and the increased amount of LTE4 excreted in the urine (p = 0.08). These parameters were still not significantly correlated when airways reactivity (log PC20) was also considered (p = 0.057). However, when each individual was compared to self in the presence and absence of cromoglycate, a significant correlation was found to exist between the drop in FEV1 during the IAR, the excretion of urinary LTE4, and the airways reactivity. No correlation was found between the increase in urine LTE4 excreted during the 12 h following allergen challenge and the severity of late (3 to 12 h) responses to allergen, but there was a significant prolonged (12 to 36 h) elevated urine LTE4 excretion in those patients with the most severe late asthmatic response.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfidopeptide leukotrienes have been implicated in the pathogenesis of asthma because of their ability to induce bronchospasm, airways hyperreactivity, and increased mucus production. In the present study, the leukotrienes (LT) C4, D4, and E4 were measured in bronchoalveolar lavage fluid (BALF) before and 5 min after endobronchial allergen challenge in four subject groups: nonatopic nonasthmatic, nonatopic asthmatic, atopic nonasthmatic, and atopic asthmatic. As determined by high performance liquid chromatography (HPLC), after allergen challenge, the predominant sulfidopeptide leukotriene found in BALF from atopic asthmatics was LTC4. Smaller amounts of LTD4 and LTE4 were detectable. The baseline level of leukotrienes in the atopic asthmatics was 64 +/- 18 pg/ml, with measurable levels being found in nine of 11 samples. Atopic nonasthmatics had measurable levels in only one of seven baseline samples, whereas five of six nonatopic subjects had undetectable levels. Allergen challenge in atopic asthmatics resulted in significant increases in LTC4 over prechallenge levels (64 +/- 18 to 616 +/- 193 pg/ml) (p less than 0.01) and over levels in the three control groups after challenge (p = 0.0297). The atopic nonasthmatic group also had detectable leukotriene levels after allergen challenge (88 +/- 32 pg/ml), whereas leukotrienes remained undetectable in five of the six nonatopic samples. For comparison, histamine and the prostanoids prostaglandin D2 (PGD2) and thromboxane B2 (TxB2) were also measured in BALF. The levels of all three of these mediators increased in BALF from atopic asthmatics after allergen challenge. After allergen challenge, the best correlation was found between the levels in BALF for the prostanoids PGD2 and TxB2 (r = 0.88).(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic pulmonary infection/colonization caused by Pseudomonas aeruginosa accounts for much of the morbidity and mortality in cystic fibrosis (CF). The effect of chronic pulmonary P. aeruginosa infection on the pulmonary circulation has not been studied. Therefore, we investigated the effect of chronic P. aeruginosa infection on pulmonary hemodynamics in a rat model. Two groups of rats were inoculated with either agar beads containing 1.0 x 10(4) colony-forming units of P. aeruginosa (infected) or an equal volume of sterile beads alone (control). In vivo, pulmonary vasoreactivity measured as the percent change in total pulmonary resistance during hypoxia was decreased at 1 wk (22 +/- 7% versus 57 +/- 3%), 2 wk (29 +/- 5% versus 73 +/- 17%), 3 wk (41 +/- 8% versus 77 +/- 14%), and 6 to 9 wk (23 +/- 10 versus 53 +/- 7; p less than 0.05 all time points; mean +/- SEM) postinoculation in infected animals when compared with that in time-matched control animals. At 6 to 9 wk postinoculation, pulmonary artery pressure was significantly elevated in infected rats (25.8 +/- 1.6 versus 21.0 +/- 1.0 mm Hg; p less than 0.05) when compared with that in control animals. Histopathologic findings were characterized by bronchiectasis as well as by chronic bronchial, parenchymal, and perivascular inflammation at all time points in infected animals.(ABSTRACT TRUNCATED AT 250 WORDS)

It is known that reactive oxygen species cause lung injury in association with activation of arachidonate metabolism. Because metabolites of the cyclooxygenase pathway do not appear to mediate the injury, we considered that the 5-lipoxygenase pathway might be activated and that inhibition of the pathway could interfere with the development of the injury. Thus, we sought to induce an oxidant lung injury and to prevent such injury by inhibiting lipoxygenase pathway or by blocking leukotriene action. In isolated rat lungs, glucose oxidase added to a glucose-containing, cell-free perfusate was used to produce the injurious oxygen species. Lung edema occurred and increased with increasing oxygen tension in the inspired air. Light microscopy of the lung showed perivascular fluid cuffs, and electron microscopy showed endothelial cell damage. Measurements in the lung effluent showed that concentrations of 5-hydroxyeicosatetraenoic acid (5-HETE) and of cyclooxygenase metabolites increased after glucose oxidase administration; BW 755C, U60,257, and FPL 55712 inhibited the glucose-oxidase-induced lung edema. And U60,257 also inhibited the glucose-oxidase-induced increase in 5-HETE without concomitant inhibition of cyclooxygenase metabolites. Thus, glucose oxidase via generation of active oxygen species stimulated the lung 5-lipoxygenase pathway, and inhibitors of 5-lipoxygenase protected against the oxidant lung injury. Further, in these experiments, the injury occurred in the absence of circulating blood cells and was augmented by increasing the inspired oxygen concentration.

Leukotriene inhibitors preferentially inhibit the acute pressor response to hypoxia in isolated rat lungs. If leukotrienes are important in hypoxic pulmonary vasoconstriction, then inhibition of their synthesis could prevent the development of chronic hypoxic pulmonary hypertension. We found that diethylcarbamazine (DEC), a leukotriene synthesis blocker, reversibly inhibited acute hypoxic pulmonary vasoconstriction in the awake rat. Rats exposed to chronic hypobaric hypoxia showed pulmonary hypertension and the production of a slow-reacting substance compared with low altitude control rats. The higher of 2 doses of DEC blocked the pulmonary hypertension and the production of slow-reacting substance in all of the hypoxic rats treated. The lower dose of DEC attenuated the pulmonary hypertension in only some of the rats. Changes in weight gain, hematocrit, or generation of prostacyclin did not explain the prevention of the pulmonary hypertension by DEC. We conclude that diethylcarbamazine inhibits acute and chronic pulmonary hypertension in the intact rat.

It has been shown that lung distension can generate prostaglandins. To test the hypothesis that there may be a prostaglandin-mediated peripheral (alveolar duct) bronchodilator effect contributing to the increase in functional residual capacity (FRC) observed with positive end-expiratory pressure (PEEP), we applied PEEP to 8 beagle dogs at 2, 5, 5, 7.5, and 10 cm H2O, and measured FRC at each level and immediately after cessation of PEEP. This experiment was then repeated after the intravenous administration of indomethacin 10 mg/kg. The results indicate a significant reduction of FRC (p less than 0.05) at 5, 7.5, and 10 cm PEEP after the administration of indomethacin. Furthermore, FRC initially failed to return to baseline immediately after cessation of PEEP but did so after indomethacin. We conclude that endogenous prostaglandin production of PEEP but did so after indomethacin. We conclude that endogenous prostaglandin production may be partially responsible for the elevation of FRC with PEEP.

The patterns of lung eicosanoid production were investigated in five different species by stimulating isolated lungs with calcium ionophore A23187 (10(-5) M). The species studied were the rat, ferret, hamster, guinea pig, and rabbit. The eicosanoids measured included the cyclooxygenase metabolites 6-keto-PGF1 alpha, TxB2, and PGE2 and the lipoxygenase metabolites LTC4, LTD4, and LTB4. Since these metabolites are either retained in the lungs or released into the perfusate, eicosanoids were measured in lung tissue and in the perfusate. In all animal species, calcium ionophore stimulated the production of all the eicosanoid metabolites measured, although in different relative proportions and quantities. The sulfidopeptide LTC4 was predominantly retained in the lung tissue while other eicosanoids were distributed more evenly between tissue and perfusate, with tissue levels generally being higher. The ferret lung produced large quantities of eicosanoids, more than 90% being lipoxygenase products. In contrast, the guinea pig lung produced predominantly cyclooxygenase products, especially TxB2. The rat, hamster, and rabbit lungs showed a more even distribution of cyclooxygenase and lipoxygenase products. Species differences in the ability to produce lipid mediators in the lung may be important in determining the vasoconstrictive, bronchoconstrictive, or inflammatory response to physiologic stimuli.

Alveolar transfer of eicosanoids was studied in the rat after instillation of [3H] prostaglandins, leukotrienes, and arachidonate into the distal airways. The percentage of originally added label remaining in lung or lavage was determined 2 min after instillation, with the remainder of the label having been transferred out of the lung. Peptidoleukotrienes were found to be largely (greater than 85%) retained in the lung after 2 min, and even after 15 min, more than 50% of the added radiolabel remained in the lung. In clear contrast, greater than 90% of the radiolabeled prostaglandin (PG) D2 was removed from the lung within 2 min after instillation. PGE2, thromboxane B2, leukotriene (LT) B4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were also quickly removed from the lung, with less than 30% of the added label remaining in the lung after 2 min. Arachidonic acid and 6-keto-PGF1 alpha were removed more slowly from the lung, with 40 to 70% remaining in the lung 2 min after instillation. The arachidonic acid remaining in the lung was largely esterified into phospholipids, especially phosphatidylcholine. The 5-HETE radiolabel remaining in the lung was also associated in part with phosphatidylcholine. The radioactivity remaining in the lung after [3H] LTB4, 6-keto-PGF1 alpha, PGD2, and thromboxane B2 instillation reflected predominantly unmetabolized eicosanoids. The peptidoleukotrienes underwent a slow metabolism from LTC4 to LTD4 to LTE4. In addition, LTE4 was metabolized to a more polar product with chromatographic properties identical to those of N-acetyl LTE4. Rat lung slices and homogenates also metabolized peptidoleukotrienes via this pathway, although less N-acetyl LTE4 was produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent work with isolated blood vessels has emphasized the importance of intact endothelium when the relaxation of vascular smooth muscle is induced by acetylcholine (ACh). However, the physiologic significance of this endothelial-dependent ACh response in a complete organ circulation is unclear. We questioned whether diminished ACh vasodilation would result from damage of lung vascular endothelium and whether this response could be used as an indication of endothelial injury. We therefore induced pulmonary endothelial cell injury in one rat model by repeated injections of alpha-naphthyl thiourea (ANTU) and in a second rat model by exposing rats for 52 h to 100% oxygen at a barometric pressure of 760 torr (hyperoxia). Rats injected with Tween 80, the solvent for ANTU, or exposed to ambient Denver air served as the respective control animals. The isolated lungs of these rats were perfused with a recirculating cell- and plasma-free, physiological salt solution to study the effect of ACh or NaCl infusion on pulmonary perfusion pressure and vascular responsiveness. ANTU-treated rats demonstrated an intact vasodilatory response after ACh infusion when compared with the solvent control animals. The immediate pulmonary vasodilation after ACh infusion was slightly enhanced in the hyperoxic rat lung when compared with the rats exposed to ambient air, but there was no difference between these groups in the prolonged depression of vascular responsiveness to hypoxia or angiotensin II. Thus, in both models of lung endothelial cell injury, the pulmonary vascular responses to ACh were intact.(ABSTRACT TRUNCATED AT 250 WORDS)

During the last 4 decades hypoxic vasoconstriction has been acknowledged as an important pulmonary control mechanism that via matching ventilation and perfusion regulates one of the important physiologic parameters--PaO2. Whether the hypoxic vasoconstriction occurs directly in a localized vascular site with distinct biochemistry or whether it is mediated by a local hormone has not been resolved. Progress has been made because of the introduction of micropuncture techniques that allow direct pressure measurements in small segments of the lung vascular tree. Measurements of the force of contraction and membrane potential (118) and manipulation of the environment of pulmonary microvessels are the most recent progress in the elucidation of the mechanism of hypoxic vasoconstriction. Elements of energy, oxygen, and lipid metabolism appear to contribute to the biochemistry of this elusive response.

We investigated the effect of dibutyryl-cAMP pretreatment on endotoxin-induced hemodynamic changes and lung vascular injury in rats. In catheter-implanted, unanesthetized rats, intraperitoneal injection of Salmonella enteritidis endotoxin (2 mg/kg) decreased cardiac output and systemic blood pressure while increasing total pulmonary vascular resistance. Db-cAMP (1 mg given intraperitoneally every 30 min), although not significantly affecting cardiac output and systemic blood pressure, blocked the increase in total pulmonary resistance caused by endotoxin. Ninety minutes after intraperitoneal endotoxin injection, perfused lungs from endotoxin-treated rats exhibited increased pulmonary vascular permeability, as assessed by increased extravascular accumulation of 125I-albumin and water. Db-cAMP treatment in vivo markedly attenuated the increases in lung albumin leak index and wet-to-dry weight ratio caused by endotoxin without affecting lung microvascular pressures. This protective action of db-cAMP is not due to its effect on prostaglandin or leukotriene synthesis since endotoxin-stimulated increases in lung tissue 6-keto-prostaglandin F1 alpha, thromboxane B2 and leukotriene C4 were not inhibited. We conclude that db-cAMP blocks endotoxin-induced lung injury in the rat by a mechanism independent of eicosanoid products and speculate that agents that increase intracellular cAMP may be therapeutically useful in acute lung vascular injury.

We have proposed that the necessity of transporting large quantities of a relatively insoluble molecule, 02, from alveolar air to blood may have led to the evolution of an extremely thin but extensive membrane. The very structure of the membrane has required specialized machinery to maintain low vascular pressures within it, to divert blood flow to the better oxygenated areas, and to defend the membrane from external or internal attack. The purpose of this paper is to consider whether membrane lipids (platelet-activating factor and the metabolities of arachidonic acid) constitute some of this specialized machinery, and, if so, how this machinery might work.

The quantitative analysis of 5-hydroxyeicosatetraenoic acid (5-HETE) is one means by which to assess the activation of the 5-lipoxygenase pathway of arachidonic acid in biologic fluids including lung perfusates. Using combined gas chromatography-mass spectrometry (GC-MS) and an oxygen-18-labeled 5-HETE internal standard, a method of analysis has been developed that involves conversion of 5-HETE into its pentafluorobenzyl ester (PFB) which is purified and separated from 12- and 15-HETE PFB esters by thin-layer chromatography. Following isolation and trimethylsilyl ether formation, analysis on a short 5-m capillary GC column circumvents problems of thermal degradation. Negative ion chemical ionization results in abundant production of carboxylate anions (RCOO-) with little subsequent fragmentation; as little as 40 pg of 5-HETE (s/n approximately 10:1) could be detected in 5 ng of 1,1-[18O]2-5-HETE. Studies of the isolated rat lung perfused with a 4% albumin buffer revealed a significant increase in 5-HETE (750 pg/ml perfusate) when injury was induced by addition of glucose oxidase to the perfusion buffer (0.6 U/ml). This stimulated production of 5-HETE could be reversed by prior perfusion of the lung with the drug piriprost, an inhibitor of the 5-lipoxygenase cascade.