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Active oxygen species can cause lung injury. Although a direct action on endothelial cells is proposed, the possibility exists that they might cause injury via mediators. We considered that active oxygen species would stimulate the generation of cyclooxygenase metabolites, which then alter pulmonary vasoreactivity and cause edema. We chemically produced hydrogen peroxide by adding glucose oxidase to a plasma- and cell-free, but beta-D-glucose-containing, solution, which perfused isolated rat lungs. Addition of glucose oxidase to the perfusate caused a marked decrease in pulmonary vasoreactivity, accompanied by an increase in the concentrations of prostacyclin, thromboxane A2, and prostaglandin F2 alpha. Pretreatment with catalase, a specific scavenger of hydrogen peroxide, preserved pulmonary vasoreactivity, inhibited the increase of the concentration of the measured prostaglandins, and prevented edema formation. Indomethacin effectively blocked lung prostaglandin production but neither prevented the decrease in vasoreactivity nor inhibited edema formation. From these data we conclude that hydrogen peroxide impaired pulmonary vasoreactivity and subsequently caused edema. Despite the fact that hydrogen peroxide stimulated lung prostaglandin production, cyclooxygenase-derived products neither caused the decrease in vasoreactivity nor the development of edema.

Reactive oxygen metabolites cause pulmonary vasoconstriction and activate arachidonic acid metabolism. We proposed that hydrogen peroxide, generated enzymatically in an insolated rat lung model, would cause vasoconstriction which was independent of circulating cells, but dependent on activation of the arachidonic acid cascade. Although hydrogen peroxide caused an increase in lung effluent thromboxane B2 concentration, indomethacin did not inhibit hydrogen peroxide induced vasoconstriction. In order to test the hypothesis that hydrogen peroxide activates the 5-lipoxygenase pathway, lung effluents were analyzed for 5-hydroxy-eicosatetranoic acid (5-HETE) using a sensitive, highly specific mass spectrometer technique. Glucose oxidase increased the 5-HEFE effluent concentrations and this was prevented by U60,257. We therefore conclude that hydrogen peroxide stimulates the 5-lipoxygenase pathway and that substances derived from this pathway are at least in part responsible for the hydrogen peroxide induced vasoconstriction in isolated rat lungs.

Both tumor necrosis factor (TNF) and platelet-activating factor (PAF) have been incriminated as mediators of endotoxic shock. Since TNF stimulates PAF synthesis in vitro, we tested the hypothesis that PAF mediates TNF-induced lung injury in vivo using specific PAF receptor antagonists. Intravenous infusion of purified human recombinant TNF resulted in peripheral neutrophilia, lymphocytopenia, and hemoconcentration, caused hemorrhagic injury to the cecum, and increased lung tissue levels of thromboxane B2 and 6-ketoprostaglandin F1 alpha. In addition, plasma glutathione disulfide (GSSG), an in vivo index of oxidative stress, was significantly increased. TNF (0.01-1 mg/kg) caused a dose-dependent increase in lung permeability-surface area product (PS) measured in isolated perfused lungs removed from rats 90 min after injection of TNF. [Lung PS in controls and after 0.01, 0.1, and 1.0 mg/kg of TNF were 0.022 +/- 0.001, 0.027 +/- 0.002, 0.033 +/- 0.001, and 0.036 +/- 0.005, respectively (P less than 0.05 from control for TNF 0.1 and 1 mg/kg).] Pretreatment of the rats with the PAF receptor antagonists WEB 2086 (10 mg/kg) and SRI 63-441 (10 mg/kg), at doses that previously protected against endotoxin-induced lung injury, did not significantly affect TNF-induced (0.1 mg/kg) changes in hematocrit, plasma GSSG, or lung PS. Moreover, WEB 2086 (10 mg/kg) did not inhibit TNF-induced (1 mg/kg) lymphocytopenia or the increases in lung tissue eicosanoid products. We conclude that TNF causes oxidative stress, eicosanoid activation, and acute lung injury in rats by a mechanism largely independent of PAF receptor activation.

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.

Recent studies have shown that vasodilator prostaglandins are continually produced by the isolated rat lung. We postulated that these vasodilators may contribute to maintenance of normal low pulmonary arterial pressure. Pulmonary pressure and cardiac output were measured in conscious dogs prior to and 30 to 60 min following administration of meclofenamate (2 mg/kg iv, followed by infusion at 2 mg . kg-1 . h-1) or the structurally dissimilar inhibitor RO-20-5720 (1 mg/kg iv, followed by infusion at 1 mg . kg-1 . h-1). The animals were also made hypoxic with inhalation of 10% O2 before and after inhibition. Time-control experiments were conducted in which only the saline vehicle was administered. Meclofenamate or RO-20-5720 caused an increase in mean pulmonary arterial pressure and total pulmonary resistance. Cardiac output and systemic pressure were unaffected. The mild hypoxic pulmonary pressor response observed was not affected by meclofenamate. Animals breathing 30% O2 to offset Denver's altitude also demonstrated increased pulmonary pressure and resistance when given meclofenamate. It is concluded that endogenous vasodilator prostaglandins may contribute to normal, low vascular tone in the pulmonary circulation.

Exposure to chronic hypoxia results in a lower resting heart rate and a blunted cardiovascular responsiveness to beta-adrenergic receptor stimulation. Possible effects of acclimatization to high altitude on the binding of [125I]iodohydroxybenzylpindolol to beta-adrenergic receptors on membranes of right and left ventricles of rat heart were determined. Chronic high-altitude exposure led to a decrease in the density of beta-adrenergic receptors in nonhypertrophied left ventricles as well as in hypertrophied right ventricles. The affinity of the receptor for the radioligand was not changed by the exposure to high altitude, suggesting that the properties of the receptor were not affected. Basal and isoproterenol-stimulated adenylate cyclase activities were decreased in membranes prepared from hearts and pulmonary arteries of rats acclimatized to high altitude. The loss of cardiac beta-adrenergic receptors in rats adapted to high altitude was prevented by the chronic coadministration of a low dose of DL-propranolol. The results suggest that changes in beta-adrenergic receptor density may partially explain the hemodynamic adaptation that occurs with chronic hypoxia. These decreases may be due to a loss of functional beta-adrenergic receptors caused by chronically elevated concentrations of circulating neurally released catecholamines.

Alveolar hypoxia causes pulmonary vasoconstriction; we investigated whether hypoxia could also impair pulmonary vasodilation. We found in the isolated perfused rat lung a delay in vasodilation following agonist-induced vasoconstriction. The delay was not due to erythrocyte or plasma factors, or to alterations in base-line lung perfusion pressure. Pretreating lungs with arachidonic acid abolished hypoxic vasoconstriction, but did not influence the hypoxia-induced impairment of vasodilation after angiotensin II, bradykinin, or serotonin pressor responses. Progressive slowing of vasodilation followed angiotensin II-induced constriction as the lung oxygen tension fell progressively below 60 Torr. KCl, which is not metabolized by the lung, caused vasoconstriction; the subsequent vasodilation time was delayed during hypoxia. However, catecholamine depletion in the lungs abolished this hypoxic vasodilation delay after KCl-induced vasoconstriction. In lungs from high altitude rats, the hypoxia-induced vasodilation impairment after an angiotensin II pressor response was markedly less than it was in lungs from low altitude rats. We conclude from these studies that (a) hypoxia impairs vasodilation of rat lung vessels following constriction induced by angiotensin II, serotonin, bradykinin, or KCl, (b) hypoxia slows vasodilation following KCl-induced vasoconstriction probably by altering lung handling of norepinephrine, (c) the effect of hypoxia on vasodilation is not dependent on its constricting effect on lung vessels, (d) high altitude acclimation moderates the effect of acute hypoxia on vasodilation, and (e) the hypoxic impairment of vasodilation is possibly the result of an altered rate of dissociation of agonists from their membrane receptors on the vascular smooth muscle.

The lung histamine concentration of rats exposed to chronic hypobaric hypoxia was higher than in normoxic sea-level control rats. This hypoxia-induced lung histamine increase was absent in adrenalectomized chronically hypoxic rats. Adrenalectomy, per se, did not influence lung histamine content. The results suggest that the lung histamine content of chronically hypoxic rats is under hormonal control. This control mechanism is not evident under normoxic conditions.

The effect of hydrodynamic shear stress on the adhesive interaction between neutrophils and endothelial cells in vitro was investigated using an apparatus similar to a cone-in-plate viscometer. Isolated bovine neutrophils were labeled with 111In and incubated with monolayers of cultured bovine pulmonary arterial endothelial cells in the presence of different degrees of shear stress. Physiologically relevant shear (less than 2 dyn/cm2) was associated with marked decrease in neutrophil adherence. Stimulation with 10% bovine zymosan-activated plasma increased adherence under static conditions but failed to increase adherence conducted during the application of shear stress. Inhibition of endothelial cell prostacyclin production by meclofenamate or aspirin failed to alter the response to shear. Incubation of neutrophils under static conditions for 10, but not 5, min however, markedly enhanced subsequent resistance to shear, suggesting that a time-dependent reaction between neutrophil and endothelial cell was required to induce an increase in the strength of adherence. Analysis of neutrophil migration underneath the monolayer indicated that such migration in no way accounted for resistance to shear, particularly since shear resistance was enhanced on serum-coated plastic as well as endothelial cells. We conclude that hemodynamic factors may play an important role in modulating neutrophil adherence to endothelium in both normal and inflammatory states.

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.

Sympathetic nerve stimulation can cause pulmonary vasoconstriction related to norepinephrine (NE) release. Because of recent reports that NE caused prostacyclin (PGI2) release from systemic arteries, we wondered whether NE caused pulmonary vascular PGI2 release and whether a feedback mechanism existed whereby PGI2 modulated NE-induced vasoconstriction. NE-induced PGI2 synthesis in rat main pulmonary artery rings was larger than that induced by KCl, passive stretch, or a thromboxane analogue, was alpha-adrenergic receptor dependent, and was enhanced by endothelium removal. The NE-induced PGI2 synthesis was not tightly coupled to the magnitude of the pulmonary artery ring contractile response, and inhibition of NE-induced PGI2 production by cyclooxygenase blockade in either the pulmonary artery ring preparation or in isolated rat lungs perfused with a physiological solution did not augment the magnitude of the contractile response. We concluded that NE is a potent stimulus for PGI2 synthesis in the rat main pulmonary artery ring and in the rat lung, yet PGI2 is not important as a modulator of NE-induced vasoconstriction in the rat lung.

Eicosanoids are metabolites of the essential fatty acid, arachidonic acid, which is an integral part of all cell membranes. Membrane damage (as in lung injury) and stretching of tissues are recognized stimuli for lung prostaglandin release, indicating a spectrum of possible roles of eicosanoids in the function of the normal and diseased lung. This article reviews the most pertinent issues regarding the roles of arachidonic acid metabolites in the lung.

Platelet-activating factor (PAF) administered to the pulmonary circulation in low dose (nanogram) has vasodilatory properties. Therefore, we investigated whether endogenous PAF plays a role in the control of tone in the pulmonary circulation. The PAF receptor antagonists, SRI 63-441 (2.6 X 10(-4) M) and L659,989 (1 X 10(-5) M), were the major investigative tools. In isolated perfused rat lungs, both agents caused a persistent increase in base-line perfusion pressure (Ppa), potentiated angiotensin II (ANG II) vasoconstriction, and potentiated hypoxic vasoconstriction (HPV). This potentiation of ANG II and HPV was found to be independent of circulating blood elements. Vasodilation in the presence of PAF blockade was also impaired. The combination of cyclooxygenase inhibition and PAF receptor blockade had an additive effect on ANG II vasoconstriction but did not cause more potentiation of HPV than achieved with PAF antagonism alone. In vivo, SRI 63-441 (10 mg/kg) caused only a transient increase in base-line Ppa without altering ANG II and hypoxic vasoconstriction. These findings support a vasodilatory role for endogenous PAF in the pulmonary circulation.

Platelet-activating factor caused rapid pulmonary vasoconstriction and edema in isolated lungs perfused with albumin-free salt solution devoid of formed blood elements. These effects may be due in part to the action of leukotrienes D4 and C4, which were identified by bioassay and high-pressure liquid chromatography in the lung effluent after stimulation by platelet-activating factor. These findings help illuminate some of the deleterious effects that platelet-activating factor elicits in anaphylactic reactions and possibly in other forms of lung injury.

Daily administration of propranolol to 9 chronically instrumented, trained dogs for 2 weeks caused significant (p less than 0.05) decreases in heart rate (70 +/- 8 to 57 +/- 6 beats/min), cardiac output (3.6 +/- 0.3 to 2.9 +/- 0.2 liters/min), pulmonary arterial pressure (15.7 +/- 0.5 to 10.0 +/- 0.5 mm Hg) and total pulmonary vascular resistance (4.6 +/- 0.6 to 3.3 +/- 0.4 units). Nadolol, a structurally dissimilar beta-adrenergic receptor antagonist, caused a similar decrease in total pulmonary resistance. Acute meclofenamate administration did not return to normal pulmonary arterial pressure and resistance in the dogs chronically treated with beta-adrenergic receptor blockers. We therefore conclude that chronic beta-adrenergic receptor blockade lowered pulmonary arterial pressure and resistance by a mechanism independent of cyclooxygenase. In addition, chronic beta-adrenergic receptor blockade did not affect the potential for hypoxic vasoconstriction.

The administration of platelet-activating factor (PAF) into the airway system of the lung is known to cause profound effects, yet little is known about the metabolism of this active lipid mediator. 3H-Labeled PAF administered into the airway of isolated rat lungs was rapidly and extensively metabolized. The tissue retained 96% of the administered radiolabel while the perfusate contained 4%. Characterization of the tissue retained lipid indicated metabolism into lyso-PAF (3.3%), phosphatidylcholine (82.3%), neutral lipid (1.7%) and intact PAF (10.2%). Analysis of tissue phosphatidylcholine by mass spectrometric techniques revealed metabolism of PAF to 1-0-hexadecyl-2-arachidonoyl-GPC, which represented 20-23% of the administered radiolabeled hexadecyl-PAF. These findings support the hypothesis that a relationship between PAF and arachidonate metabolism exists at the intact organ level. Autoradiographic analysis of the cellular distribution of the radiolabeled PAF metabolites in the lung tissue indicated labeling of two cell types, the alveolar type II cell and the nonciliated bronchiolar epithelial cell (Clara cell).

Persistent pulmonary hypertension of the newborn is a syndrome consisting of severe hypoxemia and pulmonary hypertension that appears within hours of birth. Since certain leukotrienes (C4, D4, and E4) are known to produce some of the features of persistent pulmonary hypertension of the newborn, including pulmonary vasoconstriction, bronchoconstriction, decreased lung compliance, and pulmonary edema, we studied five newborns with the syndrome to determine whether these leukotrienes were present in their airways. We found leukotriene C4 and leukotriene D4 in the lung lavage fluids of all five newborns who had the clinical diagnosis of persistent pulmonary hypertension and who required ventilatory assistance. In contrast, leukotrienes were not demonstrated in a control group of 14 infants requiring ventilatory assistance who did not have the clinical diagnosis of persistent pulmonary hypertension. We conclude that leukotrienes may have a role in persistent pulmonary hypertension of the newborn.

Measurements were made of the ratio of medial area to total arterial area in the muscular pulmonary arteries of a fossorial species, the hamster, and a non-fossorial control, the rat. It was found that the medial area was less in hamsters than in rats. In addition, chronic hypoxic exposure resulted in further medial thickening in rats, while the hamster was unaffected. The hamster has previously been shown to have a blunted hypoxic pulmonary pressor response, and the present data indicate that this relative unresponsiveness is associated with less pulmonary vascular smooth muscle. In addition, the insensitivity to hypoxia of the pulmonary vasculature and a lack of vascular luminal narrowing may tend to minimize increases in pulmonary vascular resistance during chronic hypoxia in the hamster, and thus be beneficial adaptations for a burrowing species.

Previous studies have documented direct vascular effects of opiate substances in the systemic circulation. Because opiate receptors have been identified in the lung, we wondered whether opiate substances might affect vasoreactivity in the lung circulation. We studied the pulmonary vascular effects of three opiate agonists: morphine, leucine-enkephalin, and dynorphin, as well as the opiate receptor antagonist naloxone, in isolated rat lungs perfused with a cell- and plasma-free salt solution. Because of previous reports of the smooth muscle effects of the methyl- and propylparaben preservatives in the naloxone preparation, we also studied the pulmonary vascular effects of these preservatives in the rat lung circulation. We found that morphine, a mu-receptor agonist, leucine-enkephalin, a delta-receptor agonist, and dynorphin, a kappa-receptor agonist, caused no immediate vascular effect when injected into the pulmonary artery. In addition, morphine did not affect the pulmonary vasoconstrictions induced by hypoxia, angiotensin II, or potassium chloride. The commercial preparation of naloxone, Narcan, caused a marked vasodilation during hypoxic pulmonary vasoconstriction. However, this effect was entirely attributable to the preservatives methyl- and propylparaben, as pure naloxone had no effect on either the baseline pulmonary vascular tone or the vasoconstrictive response to hypoxia. We conclude that opiate receptor agonists and antagonists do not affect vasoreactivity in the rat lung circulation and that the methyl- and propylparaben preservatives in Narcan are pulmonary vasodilators.

We tested the hypothesis that monocrotaline would activate arachidonic acid metabolism in rats. If activation occurred before the pulmonary hypertension developed, arachidonate metabolites could play a role in the hypertensive monocrotaline injury. We found that 1 wk after monocrotaline administration 6-ketoprostaglandin F1 alpha and leukotriene C4 were increased in lung lavages. At 3 wk when pulmonary hypertension was well developed, lung lavage contained increased 6-ketoprostaglandin F1 alpha and thromboxane B2. In addition, the number and activity of white blood cells in the lavages was increased, and abnormal alveolar macrophages were present. The lung extract contained slow-reacting substances including leukotriene D4. Indomethacin administration inhibited the formation of cyclooxygenase metabolites but did not prevent pulmonary hypertension. Diethylcarbamazine administration reduced the numbers and activity of inflammatory cells, increased pulmonary hypertension, prevented right ventricular hypertrophy, and inhibited the formation of slow-reacting substances. We concluded that arachidonate metabolism was activated before pulmonary hypertension developed, that the inflammatory cells in the alveolus accompanied the hypertensive process, and that diethylcarbamazine attenuated both the monocrotaline-induced inflammatory response and the pulmonary hypertension.

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)

Leukotriene C4 is produced during hypoxic pulmonary vasoconstriction and leukotriene inhibitors preferentially inhibit the hypoxic pressor response in rats. If lipoxygenase products are important in hypoxic vasoconstriction, then an animal deficient in arachidonic acid should have a blunted hypoxic pressor response. We investigated if vascular responsiveness was decreased in vascular rings and isolated perfused lungs from rats raised on an essential fatty acid deficient diet (EFAD) compared to rats raised on a normal diet. Rats raised on the EFAD diet had decreased esterified plasma arachidonic acid and increased 5-, 8-, 11-eicosatrienoic acid compared to rats raised on the normal diet (control). Compared to the time matched responses in control isolated perfused lungs the pressor responses to angiotensin II and alveolar hypoxia were blunted in lungs from the arachidonate deficient rats. This decreased pulmonary vascular responsiveness was not affected by the addition of indomethacin or arachidonic acid to the lung perfusate. Similarly, the pulmonary artery rings from arachidonate deficient rats demonstrated decreased reactivity to norepinephrine compared to rings from control rats. In contrast, the tension increases to norepinephrine were greater in aortic rings from the arachidonate deficient rats compared to control. Stimulated lung tissue from the arachidonate deficient animals produced less slow reacting substance and platelet activating factor like material but the same amount of 6-keto-PGF1 alpha and TXB2 compared to control lungs. Thus there is an association between altered vascular responsiveness and impairment of stimulated production of slow reacting substance and platelet activating factor like material in rats raised on an EFAD diet.

ARDS is a relatively common disorder whose high mortality has changed little over the past few years. Management remains supportive in essence, consisting of careful ventilatory management and attention to fluid balance and nutritional needs. Certain complications, especially sepsis, need to be prevented if possible. Currently, there is no pharmacologic intervention of proven benefit. Future improvements in the treatment of ARDS will depend on new research incorporating both laboratory and clinical approaches.

Histamine is an important amine within the lung. The purpose of the present study was to evaluate in rats the factors that affect the measurement of lung histamine concentration. We found that the lung content was not clearly altered by pentobarbital anesthesia or by freezing of the lung. Heparin administration and inhalational ether anesthesia appeared to result in elevated lung histamine contents by an unknown mechanism. Pulmonary edema rapidly lowered the histamine content. The histamine content is better related to DNA content than to the amount of protein in the lung.

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)

The adult respiratory distress syndrome is a condition of life-threatening organ failure triggered by blood-borne factors and challenges which arrive via the airways. Vascular damage is a necessary, but often not sufficient criterion for ARDS, which is observed in an acute and chronic form. There is a consensus that neutrophils and their products contribute to the pathogenesis of the syndrome, and that lung vascular tone regulation and endothelial and epithelial cell permeability are affected in ARDS. Whereas the precise roles of individual mediators for the development of ARDS are still ill-defined, a synergism between lipid mediators and other injurious principles is recognized. Chronic ARDS is a proliferative disorder which may require different treatment strategies than acute ARDS. Specific treatment modalities which inhibit the interaction between activated neutrophils and the lung endothelium, and surfactant replacement might have a future as early therapy approaches.

The effects of dietary polyunsaturated fats on chronic hypoxic pulmonary hypertension were assessed in rats fed fish oil, corn oil, or a lower fat, "high-carbohydrate" diet (regular) beginning 1 mo before the start of hypoxia (0.4 atm, n = 30 for each). Mean pulmonary arterial pressures were lower in the chronically hypoxic rats fed fish oil (19.7 +/- 1.8 mm Hg) than in the rats fed corn oil (25.3 +/- 1.6 mm Hg) or regular diets (27.5 +/- 1.5 mm Hg, P less than 0.05). The fish oil diet increased lung eicosapentaenoic acid 50-fold and depleted lung arachidonic acid 60% (P less than 0.0001 for each). Lung thromboxane B2 and 6-ketoprostaglandin F1 alpha levels were lower, and platelet aggregation, in response to collagen, was reduced in rats fed fish oil. Chronically hypoxic rats fed fish oil had lower mortality rates than the other hypoxic rats. They also had lower blood viscosity, as well as less right ventricular hypertrophy and less peripheral extension of vascular smooth muscle to intra-acinar pulmonary arteries (P less than 0.05 for each). The mechanism by which dietary fish oil decreases pulmonary hypertension and vascular remodeling during chronic hypoxia remains uncertain. The finding that a fish oil diet can reduce the hemodynamic and morphological sequelae of chronic hypoxia may have therapeutic significance.

Some human newborns have a syndrome characterized by irreversible pulmonary hypertension and severe hypoxemia and by medial hypertrophy and adventitial thickening of pulmonary arteries. We considered that newborn calves made severely hypoxic might reproduce features of the human disease. When 2-day-old calves were placed at 4,300 m simulated altitude, pulmonary arterial pressure was increased and could be reversed by 100% O2. However, after 2 wk at 4,300 m, pulmonary arterial pressures were suprasystemic and there was right-to-left shunting probably through the foramen ovale and a patent but restrictive ductus arteriosus. Suprasystemic pulmonary pressure and hypoxemia persisted with 100% O2 breathing. Morphometrical examination of the lung arteries showed a markedly thickened adventitia with cellular proliferation and collagen and elastin deposition. There was increased medial thickness and distal muscularization of the pulmonary arteries associated with decreased luminal diameter. The rapid development of severe pulmonary hypertension and poor responsiveness to O2 was associated with increased arterial wall thickness, particularly involving the adventitia. Thus the pulmonary arterial circulation in these calves, which were placed at high altitude for 2 wk, exhibited features resembling persistent pulmonary hypertension in newborn infants.

The effects of the cell-permeable dibutyryl derivative of cyclic AMP on the vascular reactivity of isolated perfused rat lungs were examined. In lungs perfused with homologous blood, pulmonary arterial infusion of db-cAMP (30 micrograms/min) inhibited hypoxia-induced vasoconstriction (IC50 = 6.3 X 10(-5) M) and vasoconstriction due to bolus injection of angiotensin II (IC50 = 8.2 X 10(-5) M). Cyclic AMP phosphodiesterase inhibition by aminophylline acted synergistically with db-cAMP in the reduction of hypoxia-induced vasoconstriction. Somatostatin, an inhibitor of adenylate cyclase, prevented the decay of hypoxic vasoconstriction typically observed in isolated lungs, suggesting that a rise in intracellular cAMP may occur during hypoxic vasoconstriction as a consequence of activation of the adenylate cyclase. In lungs perfused with cell and protein-free salt solution, db-cAMP inhibited both initial and prolonged vasoconstriction following bolus injection of 2 microgram leukotriene C4. Thus, db-cAMP inhibited pulmonary vascular reactivity nonspecifically.

We hypothesized that leukotrienes are involved in hypoxic pulmonary vasoconstriction, since they are pulmonary vasoconstrictors and cells capable of producing leukotrienes are located in the lung near resistance vessels. We investigated in isolated perfused rat lungs whether three structurally unrelated blockers [diethylcarbamazine citrate (DEC), U-60257, and FPL 55712] of leukotriene synthesis or action block hypoxic pulmonary vasoconstriction. DEC blocked ongoing and subsequent hypoxic pressor responses while minimally affecting the angiotensin II pressor response. The inhibition of the hypoxic pressor response by DEC was not affected by cyclooxygenase or H1-receptor blockers. Potassium-induced vasoconstriction, which is dependent on calcium entry, was largely blocked by verapamil but not by DEC, suggesting that DEC was not acting primarily as a calcium-entry blocker. U-60257 blocked the hypoxic pressor response without inhibiting the pressor response to angiotensin II or potassium chloride. FPL 55712, a leukotriene end-organ blocker, in a dose which inhibited vasoconstriction caused by exogenous leukotriene C4, inhibited the pressor response to hypoxia but not to angiotensin II. We conclude that leukotriene inhibitors preferentially inhibit hypoxic pulmonary vasoconstriction in isolated perfused adult rat lungs.

The recent availability of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the lung extract assayed for 6-keto-PGF1 alpha, 5-HETE and LTC4. By EIA lung tissue was found to contain large quantities of 6-keto-PGF1 alpha after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF1 alpha amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF1 alpha, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Pak) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher values obtained by RIA. LTC4 was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC4 levels were identical in unpurified lung extract and after Sep-Pak purification, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproducible results in lung tissue, although LTC4 and 5-HETE must be purified prior to analysis.

Leukotriene E4 (LTE4) appears to be a rather stable product of the lipoxygenase pathway. Its action in the pulmonary circulation is unknown. Therefore we investigated its effect on the circulation of isolated rat lungs perfused with a cell- and plasma-free solution. Synthetic LTE4 in doses from .15 micrograms to 5 micrograms/.25 ml .9% NaCl injected as a bolus in the pulmonary artery during normoxia caused a fast, transient perfusion pressure increase within seconds. This was followed by a slow rise in baseline perfusion pressure (normoxia) over 25 min. In addition, 5 micrograms LTE4 caused edematogenic lung damage. Injection of 1.5 micrograms LTE4 during hypoxic vasoconstriction caused fast, transient pressure rises, similar to normoxic conditions. 6-keto-PGF1 alpha and TXB2 were measured in the lung effluent before and after LTE4 injection. Neither 6-keto-PGF1 alpha nor TXB2 production changed after LTE4 injection. Meclofenamate (.5 micrograms/ml) increased the fast, transient and the slow, sustained pressure rise. We conclude that LTE4 caused direct pulmonary vasoconstriction unrelated to cyclooxygenase products.

The administration of monocrotaline to rats causes pulmonary vascular leak within 1 wk followed in 2-3 wk by perivascular proliferation and fatal pulmonary hypertension. Possibly blocking the proliferation might block the pulmonary hypertension, providing insight into its mechanism. Because heparin, given as an antiproliferative agent, reduced hypoxic pulmonary hypertension in mice, it might also block monocrotaline-induced pulmonary hypertension. Alternatively, anticoagulation could worsen the lung injury. We found that heparin (300 and 600 U/kg sc twice daily) inhibited clotting in rats given monocrotaline but did not change the vascular leak, the right ventricular pressure, the right ventricular hypertrophy, the increased medial thickness of the pulmonary arterioles, or the production of a slow-reacting substance of anaphylaxis-like material by the lungs. A nonanticoagulant heparin fragment (2 mg/kg sc twice daily), given to avoid anticoagulation also did not influence the monocrotaline injury. Thus neither anticoagulant nor nonanticoagulant heparin either attenuated or worsened the measured effects of monocrotaline.

Acetylcholine causes pulmonary vasodilation, but its mechanism of action is unclear. We hypothesized that acetylcholine-induced pulmonary vasodilation might be associated with prostacyclin formation. Therefore, we used isolated rat lungs perfused with a recirculating cell- and plasma-free physiological salt solution to study the effect of acetylcholine infusion on pulmonary perfusion pressure, vascular responsiveness and lung prostacyclin production. Acetylcholine (20 micrograms infused over 1 minute) caused immediate vasodilation during ongoing hypoxic vasoconstriction and prolonged depression of subsequent hypoxic and angiotensin II-induced vasoconstrictions. Both effects of acetylcholine were abolished by atropine pretreatment. The prolonged acetylcholine effect, but not the immediate response, was blocked by meclofenamate, an inhibitor of cyclooxygenase. The prolonged effect, but not the immediate response, of acetylcholine was associated with an increase in perfusate 6-keto-PGF1 alpha concentration. The acetylcholine stimulated increase in 6-keto-PGF1 alpha production was inhibited by meclofenamate and by atropine. Thus, blockade of prostacyclin production corresponded with blockade of the prolonged acetylcholine effect. In conclusion, acetylcholine caused in isolated rat lungs an immediate vasodilation and a prolonged, time-dependent depression of vascular responsiveness. Whereas both acetylcholine effects were under muscarinic receptor control, only the prolonged effect depended on the cyclooxygenase pathway and, presumably, prostacyclin synthesis.

Isolated lungs from high-altitude rats compared with those from low-altitude rats have blunted hypoxic pressor responses. Because calcium is essential for the hypoxic pressor response and has been shown to be altered in the pulmonary arterial smooth muscle during high-altitude exposure, we wondered whether altered calcium metabolism might account for the blunted response. We attempted to unmask differences between high- and low-altitude rat lungs by altering extracellular calcium. The addition of calcium to the blood perfusate augmented the hypoxic pressure response of high-altitude lungs and depressed that of low-altitude lungs. Calcium addition did not affect pressor responses to angiotensin II, but slowed vasodilation after both angiotensin injection and removal of the hypoxic stimulus. The maximal KCl-induced contraction was blunted in rings of main pulmonary artery from high-altitude rats, but augmented in their aortic rings. Submaximally constricted (40 mM KCl) pulmonary arteries from low- but not high-altitude rats further constricted with calcium addition. Aortic rings exhibited an inverse behavior. Thus, calcium addition augmented the blunted vasopressor response in high-altitude rats lungs, possibly by an effect on lung arteries. The aorta and main pulmonary artery differ in calcium handling.

Chronic exposure of rats to hypoxia resulted in a lower resting heart rate and a supranormal increase in heart rate in response to parasympathetic blockade by atropine. The density of muscarinic cholinergic receptors labeled by the antagonist [3H]quinuclidinyl benzilate was elevated significantly in the atria of animals kept hypoxic for 2-4 weeks. Chronic hypoxia did not change the affinity of the receptor for [3H]quinuclidinyl benzilate, the weight of the atria, or the amount of protein per atrial pair. Thus, the decrease in resting heart rate may be explained by the increase in the density of atrial muscarinic cholinergic receptors.

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.

Neonatal calves exposed to chronic hypobaric hypoxia develop severe pulmonary hypertension associated with altered vascular reactivity, cellular proliferation, and increased elastin and collagen production. We hypothesized that prostaglandin (PG) production would be decreased in the pulmonary arterial vessel wall of these calves. Further, because of the possibility that the hemodynamic stresses of hypoxic pulmonary hypertension might change along the longitudinal axis of the pulmonary circulation, we measured prostaglandin synthetic capability in tissues isolated from proximal pulmonary artery, distal pulmonary artery, and pulmonary vein. We found that PGI2 production was decreased in both proximal and distal pulmonary artery rings isolated from pulmonary hypertensive calves compared to controls. PGI2 production was greater in distal than in proximal lobar pulmonary artery. In contrast, pulmonary veins from hypertensive calves, which are protected from the hemodynamic stress of pulmonary arterial hypertension, did not demonstrate altered PGI2 production compared to controls. PGE2 production was also decreased in proximal hypertensive pulmonary arterial rings as compared to controls. To determine if this decrease in vessel wall production of prostaglandins was due to changes in cellular prostaglandin production, we studied prostaglandin production by the three major cell types comprising hypertensive and control arteries. Endothelial cells cultured from hypertensive main pulmonary artery produced less PGI2 than did those from control artery, and there appeared to be a shift from PGI2 production to PGE2 production in endothelial cells isolated from hypertensive artery. Explanted advential fibroblasts from hypertensive artery produced less PGE2 than did controls. Smooth muscle cell PGI2 production did not differ between cells isolated from hypertensive and control arteries in these brief 30-min incubations. We conclude that there is a relative deficit in PGI2 and PGE2 production in the pulmonary arteries of calves with hypoxia-induced pulmonary hypertension and speculate that this contributes to altered vascular tone and vessel remodeling.

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.

The distributions of mast cells and histamine along the canine pulmonary arterial tree were determined. Mast cell counts were made on toluidine blue stained histologic sections, and histamine concentration was measured in dissected arterial branches by an enzymatic-isotopic assay. Mast cell density and histamine concentration were greater in small muscular arteries compared to elastic arteries. In elastic arteries there was more histamine than would be expected by the number of mast cells found there, suggesting the presence of non-mast cell histamine in those vessels. In all arteries mast cells were located primarily in the adventitia. The finding of mast cells and histamine in greatest concentration in muscular pulmonary arteries suggests that these vessels are the major site of action of histamine and other mast cell contents within the pulmonary arterial tree.