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Introduction: We recently demonstrated that the non-selective endothelin-1 (ET-1) receptor blocker tezosentan antagonizes ovine acute lung injury (ALI) following infusion of endotoxin or ET-1 by reducing the enhanced lung microvascular pressure, although we could not exclude the possibility of a simultaneous decline in microvascular permeability. In the present study, our aim was to find out if tezosentan reverses the rise in microvascular filtration coefficient (Kfc) in rat lungs that have been isolated and perfused 12 h after cecum ligation and puncture (CLP) or infusion of ET-1., Methods: Wistar rats (n = 42) were subjected to CLP. Postoperatively, rats were randomized to a CLP group (n = 7) and a CLP + tezosentan group (n = 7); the latter received tezosentan 30 mg/kg. A sham-operated group (n = 5) underwent laparotomy without CLP. Twelve hours postoperatively, the lungs were isolated and perfused with blood from similarly treated rats that also were used to assess plasma concentration of ET-1 and protein kinase Calpha (PKCalpha) in lung tissue. Additionally, isolated blood perfused lungs from healthy rats were randomized to a control group (n = 8), an ET-1 group (n = 7) subjected to pulmonary arterial injection of ET-1 10 nM, and an ET-1 + tezosentan group (n = 7) that received tezosentan 30 mg/kg. All lung preparations received papaverine 0.1 microg/kg added to the perfusate for vasoplegia. Pulmonary hemodynamic variables, Kfc and lung compliance (CL) were assessed., Results: After CLP, the plasma concentration of ET-1 increased. Papaverine abolished the vasoconstrictor response to ET-1 and the pulmonary vascular pressures remained close to baseline throughout the experiments. Both CLP and injection of ET-1 caused significant changes in Kfc and CL that were prevented in tezosentan-treated rats. Compared to sham-operated animals, CLP increased the content of PKCalpha by 50% and 70% in the cytosolic and the membrane fractions of lung tissue homogenates, respectively. Tezosentan prevented the upregulation of PKCalpha in the membrane fraction., Conclusion: In rat lungs isolated and perfused after CLP, tezosentan precludes both the increase in Kfc and the upregulation of PKCalpha in the membrane fraction of lung tissue.

The effects of methylprednisolone (MP) on the acute airway and pulmonary vascular responses induced by reactive oxygen species (ROS) were investigated in isolated, plasma-perfused rat lungs. ROS were generated by adding xanthine oxidase and hypoxanthine to the perfusate. MP was administered in 3 different ways: 1. Added to the perfusate (1 mg*ml-1) 5 min prior to xanthine oxidase and hypoxanthine, 2. Given as intraperitoneal injections (40 mg*kg-1) to lung donor rats 12 and 2 hours prior to the experiments, or 3. Combining 1 and 2. The lungs were perfused at constant volume inflow (15 ml*min-1). Pulmonary arterial pressure and transpulmonary pressure were followed for 30 min after addition of xanthine oxidase and hypoxanthine. ROS induced a powerful, acute broncho- and vasoconstriction, which was inhibited by addition of MP to the perfusate. Pretreatment with MP also inhibited the vascular and airway responses. Adding MP to the perfusate of pretreated lungs further reduced the ROS-induced smooth muscle constriction. In conclusion, MP inhibits vasoconstriction and bronchoconstriction induced by ROS in isolated rat lungs.

Extracorporeal membrane oxygenation (ECMO) may serve as extracorporeal lung assist (ECLA) in patients with acute respiratory failure (ARF) or as extracorporeal heart assist (ECHA) in patients with low output syndrome (LOS) after open heart surgery. From 1988 to 1992 seven patients underwent ECMO in our hospital; four suffered from ARF and three from LOS. Various bypass techniques were employed. Two ARF patients, aged 58 and 18 years, had veno-venous bypass; in the latter, ECMO was reinstituted as a veno-arterial bypass one week after weaning. In a three-year-old boy, the ECMO outflow tubing was primarily connected to the pulmonary artery, and shortly afterwards relocated to the common carotid artery. In a 31-year-old man with ARF, and three LOS patients, a 56-year-old woman, and two men aged 68 and 70 years, ECMO was veno-arterial with direct access to the ascending aorta. A heparin-coated system was used, and all but one patient, who was treated with warfarin, received a daily low dose of heparin, which was withdrawn after from one to nine days. Six patients were weaned off ECMO after 4.5 to 21 days. Three ARF patients recovered completely; the child died. In one LOS patient, ECMO was withdrawn due to a poor general condition. Two others were weaned off ECMO and the intra-aortic balloon pump, and the inotropic support was significantly reduced, but both died of multiple system organ failure. Although no firm conclusions can be drawn from these few case reports, the heparin-coated system used as ECLA appears promising, whereas ECHA seems to imply a poor prognosis in patients who are not candidates for cardiac transplantation.

The release and vascular effects of calcitonin gene-related peptide (CGRP) and endothelin-1 (ET-1) during acute alveolar hypoxia (O2 2%) were examined in isolated blood-perfused rat lungs. In 10 lungs, repeatedly ventilated with hypoxic gas for 5 min, samples from effluent blood were taken during hypoxia and analysed for plasma levels of CGRP-like immunoreactivity (-LI) and ET-1-LI. The plasma levels of ET-1-LI were significantly (p < 0.05) increased in hypoxic lungs (5.5 +/- 0.5 pmol l-1) compared with normoxic controls (3.7 +/- 0.56 pmol l-1). Plasma levels of CGRP-LI were significantly (p < 0.01) lower in hypoxic lungs (43.9 +/- 2.9 pmol l-1) than in normoxic controls (55.5 +/- 4.0 pmol l-1). No significant correlation was seen between perfusate peptide levels and pulmonary artery pressure (Ppa) during ventilation with normoxic or hypoxic gas. Infusion of the CGRP receptor blocker, CGRP, did not influence either the baseline Ppa or the development of the hypoxic pulmonary vasoconstriction response (HPV). In lungs undergoing HPV, 2 nmol l-1 ET-1 added to the perfusate, significantly reduced the hypoxic pressor response by 14 +/- 3% (p < 0.05), while addition of 200 nmol l-1 ET-1 caused no significant changes in HPV. CGRP 2 nmol l-1 caused no significant attenuation of HPV (8.9%), while 200 nmol l-1 CGRP significantly reduced HPV by 16 +/- 5% (p < 0.05). To conclude: acute alveolar hypoxia changes release of CGRP and ET-1 to the perfusate in isolated rat lungs. The results further suggest that CGRP and ET-1 are not involved in the development and regulation of the hypoxic pulmonary vasoconstriction response.

Unlike other vascular beds, lung vessels constrict when exposed to hypoxia. However, a marked difference has been noticed as regards the elicitability of hypoxic pulmonary vasoconstriction (HPV) in vivo as compared to in vitro models, like a preparation of isolated rat lungs; in the latter, HPV cannot be evoked from the onset of perfusion, but might be triggered gradually by repeated hypoxic challenges. The formation of adenosine, a potent dilator of most vascular beds, is enhanced during conditions of hypoxia or ischemia. Our hypothesis therefore was that pulmonary vasoconstriction was initially antagonized by tissue-adenosine accumulating during the circulatory arrest necessary for lung isolation, and then, gradually invigorated along with the elimination of adenosine during periods of perfusion with normally oxygenated blood. In a first series of isolated rat lungs, we studied release of adenosine in connection with the third and the sixth hypoxic challenges. Although the vascular responses were of significantly different size, there was no sign of increased adenosine formation during any of the two provocations, as assessed by release of its more stable metabolites hypoxanthine, xanthine and uric acid. In a series of tissue preparations taken at the height of a fully developed hypoxic pressor response and immediately frozen, we could not find significant changes in tissue level of adenosine, hypoxanthine and inosine as compared to controls that had never been exposed to hypoxic challenges. Further, we found no correlation between the size of pressor responses and concentrations of adenosine and its metabolites, in either blood or in lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic function of the lungs may be impaired in acute lung injuries. The present work examined the effect of toxic oxygen metabolites (TOM) on the pulmonary clearance of prostaglandin E2 (PGE2). Isolated rat lungs perfused with plasma were exposed to TOM, generated by xanthine oxidase (XO) and hypoxanthine (HX) in the perfusate. Inactivation of PGE2 was determined by superfusion bioassay technique. XO and HX (n = 6) reduced the inactivation of PGE2 from 78 +/- 4% (mean +/- SE) to 61 +/- 3%. This reduction was inhibited by the free radical scavengers superoxide dismutase and catalase, as well as by allopurinol, an inhibitor of XO. Neither hydrostatic lung edema nor perfusion per se decreased the inactivation of PGE2. Lungs pretreated with indomethacin still showed impaired PGE2 inactivation after exposure to XO and HX, indicating that a possible release of PGE2-like substances did not influence our findings. This study indicates that TOM may impair pulmonary metabolic function as shown by reduced inactivation of PGE2.

Toxic oxygen metabolites (TOM) have been suggested to be mediators of permeability edema associated with the adult respiratory distress syndrome (ARDS). Because corticosteroids have possible beneficial effects in ARDS, we have examined the effect of methylprednisolone (MP) on TOM-induced lung edema in isolated, plasma-perfused rat lungs. TOM were generated by adding xanthine oxidase (XO) and hypoxanthine (HX) to the perfusate. Microvascular permeability was assessed by fluid filtration rate (FFR). FFR was determined before and 30 min after administration of XO and HX by measuring the weight increase of the lungs for the last 3 min during a standard 5 min elevation of the outlet pressure. MP was administered in two different ways: 1) Added to the perfusate 5 or 60 min before XO and HX (0.1 and 1 mg ml-1), and 2) given as pretreatment to the rats 12 and 2 hr before preparation of the lungs (40 mg kg -1). XO and HX significantly increased FFR compared to lungs perfused with untreated plasma. Pretreatment with MP significantly attenuated the increase in FFR caused by XO and HX. Addition of MP to the perfusate also inhibited the effect of TOM. This latter protection occurred irrespective of when MP was added before XO and HX. However, when the highest dose of MP was added 5 min before XO and HX, there was a loss of the protective effect. In summary, this study provides evidence that MP may directly prevent microvascular injury induced by TOM in isolated perfused rat lungs. The effect was dependent on the dose of MP applied, but not on when MP was administered prior to exposure to TOM.

Effects of toxic oxygen metabolites (TOM) on the pulmonary vascular bed and airways were studied in isolated, plasma-perfused rat lungs. TOM were generated by xanthine oxidase (XO) (0.1 or 0.25 unit.ml-1) and hypoxanthine (HX) (1 mol.l-1). In vitro measurements by chemiluminescence indicated that the major oxygen metabolite generated by XO and HX was H2O2. Measurements of PO2 in the perfusate as an indicator of O2-consumption suggested that production of TOM by XO and HX was finished within 30 min. XO and HX induced an early dose-dependent bronchoconstriction and a late increase in transpulmonary pressure (Ptp). Pulmonary arterial pressure (Ppa) increased gradually and levelled off within 30 min with low-dose XO, but not with high-dose XO. As judged by weight increase of the lungs, interstitial edema occurred regularly. Allopurinol, an inhibitor of XO, blocked the lung responses caused by XO and HX. Catalase attenuated all lung responses induced by XO and HX, while superoxide dismutase had no effect. The hydroxyl radical scavenger dimethylsulfoxide abolished the increase in Ptp and attenuated the increase in Ppa, but did not consistently protect the lungs from edema development. This study shows that TOM induce vasoconstriction, bronchoconstriction and lung edema in plasma-perfused rat lungs, mainly due to generation of H2O2 and the hydroxyl radical.

Increased pulmonary vascular resistance (PVR) and microvascular hyperpermeability resulting in lung edema and arterial hypoxemia are mainstays in the development of adult respiratory distress syndrome (ARDS). The proposed pathophysiologic mechanisms include activation of complement and polymorphonuclear leukocytes secreting lysosomal enzymes, toxic oxygen metabolites (TOM) and eicosanoids. Platelets and coagulation factors are also involved, and in the most severe cases even monocytes are activated as reflected in release of thromboplastin. The latter may elicit disseminated intravascular coagulation (DIC). Under physiologic conditions lung blood flow is diverted from poorly to better oxygenated areas by way of hypoxic pulmonary vasoconstriction (HPV), thereby counteracting a decrease in arterial oxygenation. Many vasoactive substances have been proposed and again refuted as possible mediators of HPV. In this study we have focused on the following: histamine, catecholamines, arachidonates, calcium, phosphoinositides and TOM as well as endothelium-derived relaxing and constricting factors. Whether HPV is present in ARDS and whether it is advantageous or not seems to depend on the stage and extent of disease. We discuss possible interactions between HPV and ARDS mediators and between HPV and various vasoactive agents tested for therapeutic effects. Out of the abundance of mediators released, prostacyclin, prostaglandin E1, activated complement and platelet activating factor have been shown explicitly to inhibit HPV whereas others are suspected of doing so. In therapeutical use, prostacyclin has proved to reduce PVR and at the same time enhance cardiac output and oxygen delivery. In mild to moderate ARDS, improvement of arterial oxygenation has also been obtained employing almitrine bismesylate, a potentiator of HPV. Experimentally, adenosine effectively reduces increments in PVR and microvascular permeability with modest effects on systemic circulation. However, further investigations are warranted to decide whether adenosine or more specific blockers as, for instance, monoclonal antibodies against tumor necrosis factor should be integrated in ARDS therapy in the future.

The exact mechanism whereby hypoxic pulmonary vasoconstriction (HPV) is elicited is still unsettled. We have evaluated a possible role for toxic oxygen metabolites (TOM), employing a set-up of blood-perfused isolated rat lungs. HPV reflected as pulmonary arterial pressor responses, was evoked by alternately challenging the airways with a hypoxic- and a normoxic gas mixture, resulting in gradually increasing responses until a maximum was obtained. In a sequence of responses (mean +/- s.e. mean) increasing from 2.5 +/- 0.2 kPa to 3.2 +/- 0.1 kPa, administration to the perfusate of the inhibitor of xanthine oxidase (XO), allopurinol (AP) reduced the subsequent response to 2.5 +/- 0.2 kPa (P less than 0.001). By contrast, AP did not affect vasoconstriction induced by serotonin or bradykinin. In control experiments responses continued to increase after administration of hypoxanthine (substrate of XO). Neither pretreatment with daily injections of the antioxidant vitamin E for 3 days in advance, nor addition to the perfusate of the scavenger enzymes superoxide dismutase and catalase, or dimethylsulfoxide had any impact on HPV; the subsequent responses rose at the same rate and in the same way as before. Thus, the present study has shown that AP inhibition of XO depresses HPV. This could be due either to reduced production of TOM or to accumulation of purine metabolites. The absence of inhibitory effects of quenchers of TOM refutes a role for these metabolites in the elicitation of HPV. More likely, AP inhibits HPV by interfering with the purine metabolism.

The aim of the present work was to investigate whether prostaglandins (PGs) are synthetized and released from isolated blood-perfused rat and cat lungs secondary to vasoconstriction induced by alveolar hypoxia. The lungs were perfused with autologous blood with constant volume inflow via the pulmonary artery in a recirculating system. They were ventilated with constant volume positive pressure, and acute alveolar hypoxia was induced by ventilation with a gas containing 2% O2. A superfusion bioassay technique was used to measure PG-like activity in the perfusate from the lungs, the blood being re-oxygenated before reaching the assay tissues. The oxygenator prevented the perfusate hypoxia induced by ventilation hypoxia to affect the bioassay tissues. The assay tissues were rat stomach strip, rat colon and chick rectum. They were sensitive to calibrating doses of 0.5--1 ng/ml PGE2 and 1--2 ng/ml PGF2alpha. In another series of experiments PGs of the F-series were measured in lung tissue from normoxic and hypoxic lungs with radioimmunoassay technique. No increase in PG-like activity could be detected in the venous effluent by means of bioassay during hypoxia, nor was the lung tissue content of immunoactive PGF increased by hypoxia. The present findings indicate that alveolar hypoxia does not stimulate PG-synthesis in lungs, refuting that PGs are important mediators of the pulmonary vasoconstrictor response to alveolar hypoxia. It is concluded that PGs play no significant role in producing the pressor response to alveolar hypoxia.

Isolated rat lungs were perfused with homologous blood at constant volume inflow. The effect of ventilation hypoxia on pulmonary vascular resistance, preparation weight and reservoir volume (vascular capacitance) were studied. In some experiments also wet/dry weight ratio of the preparation was estimated (extravascular water content). There was no difference in this last parameter between hypoxic and normoxic lungs, thus alveolar hypoxia had no effect on tissue water content as previously described in intact rats. With forward perfusion small and transient changes in either direction were seen in preparation weight and reservoir volume, even though inflow pressure exceeded 5 kPa during alveolar hypoxia. With backward perfusion marked weight increases were seen, and if inflow pressure in this situation was above 3.5 kPa, the weight change was irreversible, thus indicating outward fluid filtration. It is concluded that the vessels responding to alveolar hypoxia are located on the arterial side of the pulmonary vasculature.

Reactive oxygen intermediates such as free radicals have been proposed to mediate lung injury. The present work examined whether or not enzymatically generated oxygen metabolites altered serotonin clearance. Isolated, plasma-perfused rat lungs were exposed to xanthine oxidase (XO) and hypoxanthine (HX). Pulmonary arterial pressure (Ppa) and lung weight were recorded. Fulminant edema was defined as a spontaneous weight increase exceeding 500 mg. Inactivation of serotonin was determined by superfusion bioassay. XO and HX reduced serotonin inactivation from 74 +/- 3% (mean +/- SEM) to 62 +/- 2%. This reduction was inhibited by the scavenger enzymes superoxide dismutase (SOD) and catalase and by allopurinol, an inhibitor of XO. Hydrostatic edema and perfusion per se did not decrease the pulmonary clearance of serotonin. XO and HX did not significantly alter Ppa. Fulminant edema developed in four of six lungs after exposure to XO and HX compared with none in the other groups. It was concluded that reactive oxygen intermediates inhibited serotonin inactivation in isolated rat lungs.

We have investigated the relative contribution of mechanical obstruction and hypoxia-induced vasoconstriction to the increased pulmonary vascular resistance (PVR) in atelectatic lungs. For this purpose we have utilized the previous observation that inhalation anesthetics inhibit the vasoconstrictor response to pulmonary hypoxia. The effects of halothane, enflurane and ether on PVR in atelectatic lungs have been explored. Two pairs of isolated rat lungs were perfused in series at constant flow. One of the preparations was made atelectatic by airway occlusion subsequent to ventilation with a high PO2 gas (95% O2). Ventilation of the other preparation continued with hypoxic gas (2% O2), resulting in a gradual increase in PVR in both preparations. When maximum PVR was reached, one of the above inhalation anesthetics was administered to the atelectatic lungs via the ventilated lung preparation. This caused a dose-dependent, reversible reduction of PVR. The same effect was observed when pulmonary arterial PO2 was increased (greater than 66.5 kPa). Histological examination revealed that two out of four preparations were completely atelectatic 1 h after airway occlusion, whereas atelectasis was nearly complete in the other two. In two groups, airways were occluded for 1 h. In the first group PVR increased to 163% (median) above baseline level, as found during ventilation with high PO2. High arterial PO2 reduced PVR in the atelectatic lungs to 50% (median) above baseline, whereas papaverine induced a further PVR reduction, to 7% (median) above baseline. In the other group, papaverine was given before airway occlusion, and PVR increased to 10% (median) above baseline. Comparison of the two groups shows that mechanical obstruction accounts for about 6% (10/163) of the overall rise in PVR during atelectasis.

Toxic oxygen metabolites (TOM) released from stimulated phagocytes and lung tissue have been shown to injure the pulmonary microcirculation. In the present study we evaluated microvascular injury caused by TOM in rat lungs perfused with plasma. The injury, as indicated by an increase in vascular permeability, was assessed by determining the fluid filtration rate (FFR) after paralysing the pulmonary vascular bed with papaverine (0.1 mg/ml). TOM were generated by adding xanthine oxidase (XO) (0.05-0.125 U/ml) and hypoxanthine (HX) (1 mmol/l) to the perfusate. FFR was measured before, 30 and 60 min after addition of XO and HX. The following interventions were done: 1. the H2O2-scavenger catalase, 2. substitution of the perfusate after 30 min, 3. BW 755 C, a combined lipoxygenase and cyclooxygenase inhibitor, and 4. indomethacin, a cyclooxygenase inhibitor. Addition of XO and HX caused FFR to increase from 14 +/- 4 mg/min (mean +/- s.e. mean) at the onset to 56 +/- 7 mg/min and 86 +/- 10 mg/min after 30 and 60 min, respectively. Replacing the perfusate with fresh plasma after 30 min caused a significant reduction in FFR at 60 min, from 86 +/- 11 mg/min to 58 +/- 10 mg/min. Catalase prevented the increase in FFR. Indomethacin and BW 755 C had no effect on the increase in FFR. We conclude that TOM induced a partly reversible increase in microvascular permeability of isolated rat lungs. From previous studies, the activity of XO was expected to cease after 30 min. Therefore it is suggested that secondary products of TOM propagate the lung injury. The increase in permeability was not mediated by arachidonic acid metabolites.