In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells

A single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary microvascular permeability. Endotoxin-induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels. In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin. Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin. We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium. Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species. Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis. In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis. Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel. To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study.     

Verapamil attenuates lung vascular responses to endotoxin in sheep

Experiments were conducted on five chronically instrumented unanesthetized sheep to determine the effects of verapamil, a calcium channel inhibitor, on the pulmonary hemodynamic and microvascular permeability responses to endotoxemia. Paired control endotoxemia experiments (E) and endotoxemia with verapamil treatment (30-60 micrograms.kg-1.min-1) experiments (V + E) were conducted on each sheep in random order. In the V + E experiments sheep were pretreated with a continuous intravenous infusion of verapamil 1.5-2.0 h before endotoxin infusion (1.0 microgram/kg, given over 15 min). Verapamil significantly increased base-line pulmonary arterial pressure, left atrial pressure, lung lymph flow rate, and circulating blood leukocyte levels and significantly decreased base-line cardiac output. During the endotoxin response, verapamil significantly attenuated both phase I pulmonary arterial hypertension and phase II lung lymph flow rate compared with control endotoxin experiments. The results indicate that verapamil attenuates both the pulmonary hemodynamic and increased lung microvascular permeability response to endotoxin in sheep. In a series of in vitro experiments, verapamil was found to be a potent inhibitor of phorbol myristate acetate-induced superoxide production in isolated sheep granulocytes. These data suggest that the beneficial in vivo effects of verapamil during endotoxemia may in part be due to its inhibition of increased free cytosol calcium concentration and/or inhibition of toxic O2 metabolite production.     

Effects of dibutyryl cAMP on pulmonary air embolism-induced lung injury in awake sheep

To assess the role of intracellular adenosine 3',5'-cyclic monophosphate (cAMP), we tested the effects of dibutyryl cAMP (DBcAMP), an analogue of cAMP, on lung injury induced by pulmonary air embolism in awake sheep with chronic lung lymph fistula. We infused air (1.23 ml/min) in the pulmonary artery for 2 h in untreated control sheep. In DBcAMP-pretreated sheep DBcAMP was infused (1 mg/kg bolus and 0.02 mg.kg-1.min-1 constantly for 5 h); after 1 h from beginning of DBcAMP administration the air infusion was started. After the air infusion, pulmonary arterial pressure (Ppa) and lung lymph flow rate (Qlym) significantly increased in both groups. DBcAMP-pretreated sheep showed significantly lower responses in Qlym (2.7 X base line) compared with untreated control sheep (4.6 X base line); however, Ppa, left atrial pressure, and lung lymph-to-plasma protein concentration ratio were not significantly different between the two groups. Although plasma and lung lymph thromboxane B2 and 6-ketoprostaglandin F1 alpha concentrations increased significantly during the air infusion, DBcAMP-pretreated sheep showed significantly lower responses. Thus DBcAMP infusion attenuated pulmonary microvascular permeability induced by air embolism. We conclude that pulmonary vascular permeability is in part controlled by the intracellular cAMP level.     

Respiratory failure after endotoxin infusion in sheep: lung mechanics and lung fluid balance

Infusion of Escherichia coli endotoxin (0.12-1.5 micrograms/kg) into unanesthetized sheep causes transient pulmonary hypertension and several hours of increased lung vascular permeability, after which sheep recover. To produce enough lung injury to result in pulmonary edema with respiratory failure, we infused larger doses of E. coli endotoxin (2.0-5.0 micrograms/kg) into 11 chronically instrumented unanesthetized sheep and continuously measured pulmonary arterial, left atrial and aortic pressures, dynamic lung compliance, lung resistance, and lung lymph flow. We intermittently measured arterial blood gas tensions and pH, made interval chest radiographs, and calculated postmortem extravascular bloodless lung water-to-dry lung weight ratio (EVLW/DLW). Of 11 sheep 8 developed respiratory failure; 7 died spontaneously 6.3 +/- 1.1 h, and one was killed 10 h after endotoxin infusion. All sheep that had a premortem room air alveolar-arterial gradient in partial pressure of O2 (PAo2-Pao2) greater than 42 Torr (58 +/- 5 (SE) Torr) died. Of eight sheep that had radiographs made, six developed radiographically evident interstitial or interstitial and alveolar edema. Pulmonary artery pressure rose from base line 22 +/- 2 to 73 +/- 3 cmH2O and remained elevated above baseline levels until death. There was an initial fourfold decrease in dynamic compliance and sixfold increase in pulmonary resistance; both variables remained abnormal until death. EVLW/DLW increased with increasing survival time after endotoxin infusion, suggesting that pulmonary edema accumulated at the same rate in all fatally injured sheep, regardless of other variables. The best predictor of death was a high PAo2-Pao2. The marked increase in pulmonary resistance and decrease in dynamic compliance occurred too early after endotoxin infusion (15-30 min) to be due to pulmonary edema. The response to high-dose endotoxin in sheep closely resembles acute respiratory failure in humans following gram-negative septicemia. Respiratory failure and death in this model were not due to pulmonary edema alone.     

Human recombinant tumor necrosis factor alpha infusion mimics endotoxemia in awake sheep

The macrophage-derived cytokine tumor necrosis factor alpha (TNF alpha) has been proposed as the major mediator of endotoxin-induced injury. To examine whether a single infusion of human recombinant TNF alpha (rTNF alpha) reproduces the pulmonary effects of endotoxemia, we infused rTNF alpha (0.01 mg/kg) over 30 min into six chronically instrumented awake sheep and assessed the ensuing changes in hemodynamics, lung lymph flow and protein concentration, and number of peripheral blood and lung lymph leukocytes. In addition, levels of thromboxane B2, 6-ketoprostaglandin F1 alpha, prostaglandin E2, and leukotriene B4 were measured in lung lymph. Pulmonary arterial pressure (Ppa) peaked within 15 min of the start of rTNF alpha infusion [base-line Ppa = 22.0 +/- 1.5 (SE) cmH2O; after 15 min of rTNF alpha infusion, Ppa = 54.2 +/- 5.4] and then fell toward base line. The pulmonary hypertension was accompanied by hypoxemia and peripheral blood and lung lymph leukopenia, both of which persisted throughout the 4 h of study. These changes were followed by an increase in protein-rich lung lymph flow (base-line lymph protein clearance = 1.8 +/- 0.4 cmH2O; 3 h after rTNF alpha infusion, clearance = 5.6 +/- 1.2), consistent with an increase in pulmonary microvascular permeability. Cardiac output and left atrial pressure did not change significantly throughout the experiment. Light-microscopic examination of lung tissue at autopsy revealed congestion, neutrophil sequestration, and patchy interstitial edema. We conclude that rTNF alpha induces a response in awake sheep remarkable similar to that of endotoxemia. Because endotoxin is a known stimulant of TNF alpha production, TNF alpha may mediate endotoxin-induced lung injury.     

Intravascular macrophage depletion attenuates endotoxin lung injury in anesthetized sheep.

We recently showed that we can selectively and safely deplete most (average 85%) of the pulmonary intravascular macrophages in sheep by intravenously infusing liposomes containing dichloromethylene bisphosphonate. After a 1-h stable baseline, we made a 6-h comparison after a 30-min intravenous endotoxin infusion (1 microg/kg) between six anesthetized control lambs and six anesthetized lambs in which the intravascular macrophages had been depleted 24 h previously. Three of the control lambs had been macrophage depleted and allowed to recover their intravascular macrophage population for >/=2 wk. After depletion, both the early and late pulmonary arterial pressure rises were dramatically attenuated. Our main interest, however, was in the acute lung microvascular injury response. The early and late rises in lung lymph flow and the increase in lung lymph protein clearance (lymph flow x lymph-to-plasma protein concentration ratio) were >90% attenuated. We conclude the pulmonary intravascular macrophages are responsible for most of the endotoxin-induced pulmonary hypertension and increased lung microvascular leakiness in sheep, although the unavoidable injury of other intravascular macrophages by the depletion regime may also contribute something.     

Diethylcarbamazine on pulmonary vascular response to endotoxin in awake sheep

Diethylcarbamazine (DEC) is an inhibitor of lipoxygenase, with protective effects in several experimental models of anaphylaxis and lung dysfunction. The hypothesis of this study was that DEC would alter the pulmonary response to endotoxin infusion, especially the prolonged pulmonary hypertension, leukopenia, hypoxemia, and high flow of protein-rich lung lymph. We prepared sheep for chronic measurements of hemodynamics and collection of lung lymph. In paired studies we gave six sheep endotoxin (0.5 micrograms/kg iv) either with or without DEC. DEC was given (80-100 mg/kg iv) over 30 min followed by a continuous infusion at 1 mg X kg-1 X min-1. Endotoxin was given after the loading infusion of DEC, and variables were monitored for 4 h. The response to endotoxin was characterized by pulmonary hypertension, leukopenia, hypoxemia, and elevations of thromboxane B2 and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha). Lymph flow and protein content reflected hemodynamic and permeability changes in the pulmonary circulation. DEC did not significantly modify the response to endotoxin by any measured variable, including pulmonary arterial and left atrial pressures, cardiac output, lymph flow and protein content, alveolar-to-arterial PO2 difference, blood leukocyte count, and lymph thromboxane B2 and 6-keto-PGF1 alpha. We could not find evidence of release of leukotriene C4/D4 by radioimmunoassay in lung lymph after endotoxin infusion with or without DEC treatment. We conclude that lipoxygenase products of arachidonic acid may not be a major component of the pulmonary vascular response to endotoxin.     

Prostaglandin E2 attenuation of sheep lung responses to endotoxin

Prostaglandin (PG) E2 can inhibit inflammatory responses of neutrophils and lymphocytes, including eicosanoid release. Diffuse lung injury after endotoxemia in sheep is accompanied by sequestration of neutrophils and lymphocytes in the lungs, and eicosanoids mediate some of the pathophysiology of the response. To determine whether exogenous PGE2 could prevent the endotoxin response, we measured pulmonary hemodynamics, gas exchange, and lung lymph responses to infusion of Escherichia coli endotoxin (0.5 micrograms/kg iv over 30 min) in unanesthetized sheep in the presence and absence of PGE2 (0.5 micrograms.kg-1.min-1) infused intravenously for 4 h beginning 0.5 h before endotoxin infusion. We also measured lung lymph concentrations of thromboxane B2 (TxB2) and prostacyclin metabolite, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), by radioimmunoassay and leukotriene B4 (LTB4) by gas chromatography-mass spectrometry. PGE2 decreased endotoxin-induced pulmonary hypertension and hypoxemia and markedly attenuated the lymph flow and lymph protein clearance responses. PGE2 also attenuated endotoxin-induced increases in lung lymph TxB2 and 6-keto-PGF1 alpha and decreased lymph LTB4 flow after endotoxin without decreasing lymph LTB4 concentrations. We conclude that PGE2 infusion attenuates lung dysfunction caused by endotoxemia, possibly by preventing endogenous release of other eicosanoids.     

Effect of endotoxemia on hypoxic pulmonary vasoconstriction in unanesthetized sheep

This study examined the effect of acute endotoxemia on hypoxic pulmonary vasoconstriction (HPV) in awake sheep. Thirteen sheep were chronically instrumented with Silastic catheters in the pulmonary artery, left atrium, jugular vein, and carotid artery; with a Swan-Ganz catheter in the main pulmonary artery; with a chronic lung lymph fistula; and with a tracheostomy. Base-line HPV was determined by measuring the change in pulmonary vascular resistance (PVR) while sheep breathed 12% O2 for 7 min. Concentrations of immunoreactive 6-keto-PGF1 alpha and thromboxane B2 (TXB2) were measured in lung lymph during the hypoxic challenge. Escherichia coli endotoxin (0.2-0.5 micrograms/kg) was infused intravenously. Four hours after endotoxemia, HPV was measured. In five sheep, meclofenamate was infused at 4.5 h after endotoxemia and HPV measured again. During the base-line hypoxic challenge, PVR increased by 36 +/- 9% (mean +/- SE). There was no significant change in lung lymph 6-keto-PGF1 alpha or TXB2 levels with hypoxia. Twelve of the 13 sheep showed a decrease in HPV 4 h after endotoxemia; the mean change in PVR with hypoxia was -8 +/- 5%, which was significantly (P less than 0.05) reduced compared with base-line HPV. The infusion of meclofenamate at 4.5 h after endotoxin did not restore HPV.     

Effects of thromboxane synthase inhibition on air emboli lung injury in sheep

We tested the effects of OKY-046, a thromboxane synthase inhibitor, on lung injury induced by 2 h of pulmonary air infusion (1.23 ml/min) in the pulmonary artery of unanesthetized sheep with chronic lung lymph fistula so as to assess the role of thromboxane A2 (TxA2) in the lung injury. We measured pulmonary hemodynamic parameters and the lung fluid balance. The concentrations of thromboxane B2 (TxB2) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) in plasma and lung lymph were determined by radioimmunoassay. Air infusion caused sustained pulmonary hypertension and an increase in pulmonary vascular permeability. The levels of TxB2 and 6-keto-PGF1 alpha in both plasma and lung lymph were significantly elevated during the air infusion. TxB2 concentration in plasma obtained from the left atrium was higher than that from the pulmonary artery at 15 min of air infusion. When sheep were pretreated with OKY-046 (10 mg/kg iv) prior to the air infusion, increases in TxB2 were prevented. The pulmonary arterial pressure, however, increased similarly to that of untreated sheep (1.8 X base line). The increase in lung lymph flow was significantly suppressed during the air infusion. Our data suggest that the pulmonary hypertension observed during air embolism is not caused by TxA2.     

Sepsis in sheep reduces pulmonary microvascular sieving capacity

The changes in pulmonary microvascular permeability in sheep, after infusion of live Escherichia coli, were studied using estimations of the osmotic reflection coefficients (sigma) for total protein, albumin, immunoglobins (Ig) G and M and based on these estimations equivalent pore dimensions were calculated. A chronic lung lymph fistula was prepared in seven sheep. After a base-line period, left atrial pressure (Pla) was increased. E. coli (10(9) X kg body wt) were given after attaining filtration independent L/P values. The sigma's for the normal lung were calculated to 0.73 for total protein and to 0.65, 0.76, and 0.91 for albumin, IgG, and IgM, respectively. The equivalent pore radii were determined to 50 and 175 A with 35% of the filtration accounted for by the large pores. After bacterial infusion, the sigma's for total protein, albumin, IgG, and IgM decreased significantly from preseptic values to 0.58, 0.50, 0.64, and 0.83, respectively. After sepsis the small pores were 50 A and the large pores 200 A with 49% of total volume flow at maximum lymph flows occurring through the large pores. Assuming a constant small-pore population the large-pore number increased 32% after bacterial infusion. These results indicate that pulmonary microvascular permeability may have increased due to the sepsis.     

Effects of thromboxane synthase inhibition on tumor necrosis factor-induced lung injury in sheep.

To examine the role of thromboxane (Tx) A2 in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we tested the effects of OKY-046, a selective thromboxane synthase inhibitor, on pulmonary hemodynamics, lung lymph balance, circulating leukocytes, arterial blood gas analysis, and TxA2 (as TxB2) and prostacyclin (as 6-keto-prostaglandin F1 alpha) levels in plasma and lung lymph after TNF infusion in awake sheep. Infusion of human recombinant TNF (3.5 micrograms/kg) into a chronically instrumented awake sheep caused a transient increase in pulmonary arterial pressure (Ppa). The Ppa peaked within 15 min of the start of TNF infusion from 23.3 +/- 1.1 (SE) cmH2O of baseline to 42.3 +/- 2.3 cmH2O and then decreased toward baseline. The pulmonary hypertension was accompanied by transient hypoxemia, peripheral leukopenia, and the increases in TxB2 in plasma and lung lymph. These changes were followed by an increase in flow of protein-rich lung lymph, consistent with an increase in pulmonary microvascular permeability. OKY-046 significantly prevented the rises of Ppa and TxB2 concentrations in plasma and lung lymph during early phase after TNF infusion. OKY-046, however, did not attenuate the increase of lung lymph flow, transient hypoxemia, and leukopenia. From these data, and by comparison with our previous studies of OKY-046-pretreated sheep during endotoxemia, we conclude that TxA2 has an important role of the increase in the early pulmonary hypertension, but it is not related to the early hypoxemia, leukopenia, and lung lymph balances in TNF-induced lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal recovery after passage of the pulmonary circulation assessed by deconvolution

We determined the effect of H2O2 on both the physiological and biochemical lung changes seen in the adult sheep after endotoxin. Fourteen unanesthetized adult sheep with chronic lung lymph fistula were given Escherichia coli endotoxin (1 microgram/kg) over 30 min. Seven sheep were given catalase (32,500 U/kg body wt) as an intravenous bolus 30 min before endotoxin. Four sheep were given catalase alone. Oxidant lung changes were measured using arterial plasma conjugated dienes and lung tissue malondialdehyde (MDA) content, both reflecting the lipid peroxidation process. Animals were killed 5 h after endotoxin. We found that endotoxin alone caused an early increase in pulmonary arterial pressure lung lymph flow (QL), plasma thromboxane B2, 6-keto-prostaglandin F1 alpha, and plasma conjugated dienes. A decrease in cardiac output and arterial PO2 was also seen. A three- to four-fold increase in protein-rich QL was noted at 3-4 h as well as a continued increase in arterial conjugated dienes. Lung MDA and water content were also significantly increased from base line. Catalase pretreatment significantly attenuated both the physiological changes and the prostanoid and conjugated diene release. Lung MDA and water content also remained at base line. We conclude that H2O2 plays a major role in endotoxin-induced lung injury as well as the resulting lipid peroxidation process.     

Effect of progressive exercise on lung fluid balance in sheep

The purpose of this study is to determine the roles of cardiac output and microvascular pressure on changes in lung fluid balance during exercise in awake sheep. We studied seven sheep during progressive treadmill exercise to exhaustion (10% grade), six sheep during prolonged constant-rate exercise for 45-60 min, and five sheep during hypoxia (fraction of inspired O2 = 0.12) and hypoxic exercise. We made continuous measurements of pulmonary arterial, left atrial, and systemic arterial pressures, lung lymph flow, and cardiac output. Exercise more than doubled cardiac output and increased pulmonary arterial pressures from 19.2 +/- 1 to 34.8 +/- 3.5 (SE) cmH2O. Lung lymph flow increased rapidly fivefold during progressive exercise and returned immediately to base-line levels when exercise was stopped. Lymph-to-plasma protein concentration ratios decreased slightly but steadily. Lymph flows correlated closely with changes in cardiac output and with calculated microvascular pressures. The drop in lymph-to-plasma protein ratio during exercise suggests that microvascular pressure rises during exercise, perhaps due to increased pulmonary venous pressure. Lymph flow and protein content were unaffected by hypoxia, and hypoxia did not alter the lymph changes seen during normoxic exercise. Lung lymph flow did not immediately return to base line after prolonged exercise, suggesting hydration of the lung interstitium.     

Dose-dependent effects of a pyridoquinazoline thromboxane synthetase inhibitor on arachidonic acid metabolites and hemodynamics during E. coli endotoxemia in anesthetized sheep

We investigated the effects of a new pyridoquinazoline thromboxane synthetase inhibitor infused before administering Escherichia Coli endotoxin into 18 anesthetized sheep with lung lymph fistulas. In normal sheep increasing plasma Ro 23-3423 concentrations were associated with increased plasma levels of 6-keto-PGF1 alpha, a reduced systemic vascular resistance (SVR, r = -0.80) and systemic arterial pressure (SAP, r = -0.92), the mean SAP falling from 80 to 50 mm Hg at the 20 and 30 mg/kg doses. Endotoxin infused into normal sheep caused transient pulmonary vasoconstriction associated with increased TxB2 and 6-keto-PGF1 alpha levels while vasoconstriction and TxB2 increase were significantly inhibited by pretreatment with Ro 23-3423 in a dose-dependent manner. When compared to controls, plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 after endotoxin infusion were increased several-fold by administering Ro 23-3423 up to plasma levels of 10 micrograms/ml. Doses over 30 mg/kg with blood levels above 10 micrograms/ml reduced plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, suggesting cyclooxygenase blockade at this dose. The peak 6-keto-PGF1 alpha levels at 60 min after endotoxin infusion in sheep with Ro-23-3423 levels below 10 micrograms/ml were associated with the greatest systemic hypotension due to a reduced SVR (r = -0.86). After endotoxin infusion the leukotrienes B4, C4, D4 and E4 in lung lymph were assayed by radioimmunoassay and high pressure liquid chromatography and remained at baseline values.     

Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.     

Alpha-adrenergic agents have little effect during air embolism lung injury in awake sheep

Since it is not clear whether alpha-adrenergic receptors can modulate lung microvascular liquid and protein leakiness, we studied the effects of alpha-adrenergic receptor stimulation or blockade on lung filtration under base-line conditions and during the acute lung injury caused by a 4-h infusion of venous air emboli in six unanesthetized, chronically instrumented sheep with lung lymph fistulas. During the experiments we continuously infused the alpha-adrenergic receptor agonist phenylephrine hydrochloride (1.0 microgram X kg-1 X min-1 iv) or the alpha-adrenergic receptor antagonist phentolamine mesylate (1.0 mg X kg-1 X min-1 iv), and we measured pulmonary vascular pressures, cardiac output, lung lymph flow, and the lymph-to-plasma protein concentration ratio. During air embolism, alpha-receptor stimulation increased pulmonary vascular resistance and decreased lung lymph flow by 25%; alpha-receptor blockade had the opposite effects. During recovery, neither agent significantly affected pulmonary hemodynamics or lymph flow. Our results indicate that alpha-adrenergic receptors are active during air embolism and modulate pulmonary filtration by causing arteriolar constriction, which reduces the surface area or the perfusion pressure in the pulmonary microvascular bed. They may also affect venous smooth muscle tone. We found no evidence that alpha-adrenergic receptors modulate lung microvascular liquid or protein leakiness directly.     

Estimation of equivalent pore radii in pulmonary microvasculature after lung lymph fistula preparation

The effect of lung lymph fistula preparation on pulmonary microvascular permeability was investigated in sheep. Acutely prepared animals (n = 9) were compared with animals with a chronic lung lymph fistula (n = 5). The osmotic reflection coefficients (sigma) for total protein, albumin, immunoglobins (Ig) G and M, and the equivalent pore dimensions were calculated. Data were achieved at maximal possible lymph flows (QL) following elevation of left atrial pressure. In sheep with a chronic lung lymph fistula sigma's for total protein, albumin, IgG, and IgM at maximal lymph flows were 0.76 +/- 0.01, 0.65 +/- 0.09, 0.79 +/- 0.03, and 0.91 +/- 0.01, respectively. In the acutely prepared group the minimum lymph-to-plasma protein concentration for total protein was 0.39 +/- 0.06, corresponding to a sigma of 0.61 +/- 0.01. The sigma for albumin, IgG, and IgM were 0.48 +/- 0.04, 0.64 +/- 0.02, and 0.87 +/- 0.01, respectively. The equivalent pore radii in the chronic group were determined to be 54 and 190 A with 29% of the filtration accounted for by large pores. In the acute group the small pores were 56 A and the large pores 175 A with 53% of total volume flow at maximum lymph flows occurring through the large pores. Assuming a constant small-pore population the large pore number increased 4.5 times after surgery. For total protein, IgG, and IgM, sigma's in the acutely prepared group were significantly lower than in the control group. These results thus indicate that surgical preparation of a lung lymph fistula in sheep may cause acute increases in pulmonary microvascular permeability.     

Comparison of afferent and efferent lung lymph in the sheep

Efferent lymph collected from the caudal mediastinal lymph node (CMN) in the sheep lung lymph fistula model has been reported to represent free pulmonary interstitial fluid. Studies that utilize this model assume that nodal transit does not alter the composition of lymph. We collected afferent lymph from the tracheobronchial node (TBN) while simultaneously collecting CMN efferent lymph in acutely prepared sheep. We compared afferent and efferent lymph protein concentrations (CA and CE) and changes in flow rates (QLA and QLE) during base line and periods of elevated left atrial pressure (Pla). As a result of elevated Pla, QLA and QLE increased and the afferent lymph-to-plasma protein concentration ratio (CA/Cp) and the efferent lymph-to-plasma protein concentration ratio (CE/Cp) fell. The CA/Cp was significantly lower than the CE/Cp during base line (0.67 vs. 0.80) and periods of elevated Pla (0.41 vs. 0.61). Although we cannot exclude regional permeability differences, the difference between CA/Cp and CE/Cp is most likely due to the concentration of lymph within the CMN. Our data suggest nodal modification of CA is correlated with the afferent lymph-to-plasma colloid osmotic pressure ratio (pi A/pi p) and further suggest that nodal alteration of lymph during elevated Pla is due to the influence of decreased pi A/pi p at the blood-to-lymph barrier. We conclude that afferent lymph is a more accurate representation of lung free interstitial fluid because collection of pulmonary afferent lymph obviates the complications introduced by the CMN. Studies utilizing efferent lymph may have overestimated lung microvascular permeability in the acute sheep preparation.     

Endotoxemia produces an increase in arterial but not venous lipid peroxides in sheep

Our purpose was to determine the effect of an endotoxin-induced lung injury on circulating lipid peroxides. We measured both malondialdehyde (MDA) and conjugated dienes (as optical density at 233 nm) in aortic and venous plasma and lung lymph in 10 unanesthetized sheep given 1 microgram/kg of Escherichia coli endotoxin. Total lipids and prostanoids 6-ketoprostaglandin F1 alpha and thromboxane B2 were also measured. Six control sheep were also studied. Animals were monitored for a 12-h period and then killed, and lung tissue MDA was determined. A two-phase endotoxin response was noted with an initial pulmonary hypertension followed by a steady-state increase in protein-rich lung lymph flow (QL) between a 3- and 6-h period. Aortic plasma MDA was significantly increased from a base line of 4.8 +/- 1.4 to 8.9 +/- 1.6 and 7.5 +/- 1.3 nmol/ml at 1 and 4 h post-endotoxin. Aortic plasma conjugated dienes increased in all 10 sheep post-endotoxin. Venous levels of both MDA and conjugated dienes were not significantly increased. Lung QL increased two- to three-fold. Lung lymph MDA increased significantly at 1 h post-endotoxin. Lymph conjugated dienes decreased. Plasma and lymph lipid peroxide levels returned to base line by 12 h in most animals. However, tissue MDA remained significantly increased in all sheep from base line of 45 +/- 9 to 85 +/- 14 nmol/g tissue. We conclude that both MDA and conjugated dienes are transiently released into aortic plasma during endotoxin-induced oxidant lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)