Effects of acid-base imbalance on pulmonary angiotensin-converting enzyme in vivo

The effects of acid-base balance disturbances on pulmonary endothelial angiotensin-converting enzyme (ACE) were studied in anesthetized mechanically ventilated rabbits. Enzyme function was estimated from [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) utilization under first-order reaction conditions during a single transpulmonary passage and expressed as 1) substrate metabolism (M), 2) Amax/Km (Amax being equal to the product of enzyme mass and the constant of product formation), and 3) (Amax/Km)/100 ml blood flow. When respiratory acidosis/alkalosis was produced by altering respiratory rate at constant airway pressure, substrate (BPAP) utilization varied proportionally to arterial pH and inversely proportionally to arterial PCO2 (PaCO2) (P less than 0.05). Percent BPAP metabolism (%M) ranged from 92 +/- 3 (respiratory alkalosis) to 85 +/- 3 (normal), 82 +/- 3 (respiratory acidosis), and 78 +/- 2% (severe respiratory acidosis). Amax/Km similarly decreased from 899 +/- 129 to 825 +/- 143, 601 +/- 74, and 450 +/- 34 ml/min, respectively, and (Amax/Km)/100 ml blood flow was reduced from 176 +/- 26 to 131 +/- 22, 111 +/- 12, and 97 +/- 5, respectively. However, when respiratory acidosis/alkalosis was produced by altering both respiratory rate and airway pressure, no changes were observed in either %M, Amax/Km or (Amax/Km)/100 ml blood flow. Similarly metabolic alkalosis or acidosis did not alter M, Amax/Km or (Amax/Km)/100 ml blood flow. These results indicate that pulmonary endothelial ACE function can be affected by acid-base disturbances, probably indirectly through changes in perfused microvascular surface area.     

Effects of alveolar pressure on lung angiotensin-converting enzyme function in vivo

Angiotensin-converting enzyme lines the luminal surface of pulmonary capillary endothelial cells. The metabolism of its synthetic substrate, 3H-benzoyl-L-phenylalanyl-L-alanyl-L-proline ([3H]BPAP) has been used as an indicator of pulmonary microvascular function. Because the flow-volume status of the pulmonary capillaries is dependent on intra-alveolar pressure, we have studied the effects of airway pressure on endothelial plasmalemmal angiotensin-converting enzyme function in rabbit lungs in vivo. Static inflation of the lungs to a pressure of 0 or 5 Torr did not change percent transpulmonary metabolism and Amax/Km ratio (defined as E X Kcat/Km and thus, under normal conditions, an indirect measure of perfused endothelial luminal surface area) compared with control measurements during conventional mechanical ventilation. When the inflation pressure was increased to 10 Torr, percent metabolism of [3H]BPAP remained unaltered but Amax/Km decreased to 60% of the control value. This decrease was in close relation to the decrease in pulmonary blood flow. Addition of 5 cmH2O positive end-expiratory pressure (PEEP) to the mechanical ventilation also decreased Amax/Km values and pulmonary blood flow but did not influence percent metabolism of [3H]BPAP. These results suggest that the detected alterations in apparent enzyme kinetics were more likely due to hemodynamic changes than to alterations in angiotensin-converting enzyme function. Thus high static alveolar pressures as well as PEEP probably reduced the fraction of perfused microvessels as reflected in changes in Amax/Km ratios. This information should prove useful in interpreting the response of pulmonary endothelial enzymes to injury.     

Effect of flow and surface area on angiotensin-converting enzyme activity in rabbit lungs

Pulmonary angtiotensin-converting enzyme (ACE) is located on the luminal surface of pulmonary microvasculature. Multiple indicator-dilution techniques have been used to measure pulmonary ACE activity in vivo and in isolated lungs. These studies suggest that ACE activity is depressed in several forms of acute lung injury. Depression of ACE activity may reflect impaired substrate delivery to enzyme sites because of flow-related reduction of perfused surface area. To assess the role of altered microvascular flow and surface area in the measurement of ACE activity, we utilized similar techniques to estimate the apparent Km and Vmax of pulmonary ACE in isolated, Krebs-perfused rabbit lungs. Km is an estimate of the affinity of a synthetic ACE substrate, [3H]benzoyl-phenyl-alanyl-alanyl-proline ([3H]BPAP), for ACE and should not be influenced by the rate of substrate delivery to luminal enzyme sites. Conversely, Vmax is an index of the number of ACE sites and should be influenced by perfusion changes that alter the number of perfused sites (recruitment or derecruitment). When isolated lungs were subjected to physiological maneuvers designed to increase or decrease perfused surface area, apparent Vmax increased or decreased respectively. Apparent Km was not altered by these maneuvers. Km and Vmax were independent of changes in perfusion rate when surface area was held constant. Thus these parameters should be useful in evaluating perfusion changes in normal and injured lungs.     

Early pulmonary endothelial enzyme dysfunction after phorbol ester in conscious rabbits

We investigated changes in angiotensin converting-enzyme (ACE) activity before and at 5, 15, 60, and 240 min after 20 micrograms phorbol myristate acetate/kg body wt iv in conscious rabbits. ACE activity was estimated in vivo from the single-pass transpulmonary metabolism of the synthetic substrate [3H]benzoyl-Phe-Ala-Pro [( 3H]BPAP) under first-order reaction conditions. Within 5 min after PMA administration, all animals developed profound granulocytopenia (15% of control) and moderate thrombocytopenia (57% of control), both lasting for the duration of the experiment. Concomitantly, there was a significant decrease in the transpulmonary metabolism of [3H]BPAP and the calculated apparent first-order reaction constant Amax/Km of ACE for [3H]BPAP. No histological evidence of lung injury was observed at these times. Since a concomitant fall in the permeability surface area product for urea was also observed, we considered that the apparent decline in ACE activity might have resulted from a reduction in perfused endothelial surface area. To resolve this, we studied the effect of PMA on the Km (a measure of enzyme affinity for its substrate) and Amax (a derivative of Vmax that is dependent upon total enzyme present and thus capillary surface area) of ACE and 5'-nucleotidase for [3H]BPAP and [14C]AMP, respectively. A significant increase in Km for both enzymes was observed at 1 h after PMA, whereas Amax was unaffected, suggesting that low-dose PMA may indeed produce endothelial cell enzyme dysfunction independent of its effect on capillary surface area. These results provide evidence of pulmonary capillary functional injury before or in the absence of structural endothelial damage.     

Pulmonary angiotensin-converting enzyme substrate hydrolysis during exercise.

We examined exercise-induced changes in indicator-dilution estimates of the angiotensin-converting enzyme first-order kinetic parameter, the ratio of a normalized maximal enzymatic conversion rate to the Michaelis constant (Amax/Km), which, under stable enzymatic conditions, will vary with the pulmonary vascular surface area accessible to vascular substrate, the extravascular lung water (an index of the proportion of lung tissue perfused), and the central blood volume (from pulmonary trunk to aorta). Experiments were performed in 10 mongrel dogs at rest and through two increasing levels of treadmill exercise, with the use of two vascular space tracers (labeled erythrocytes and albumin), a water space tracer ([1,8-14C]-octanediol), and a vascular endothelium surface area marker, benzoyl-Phe-Gly-Pro ([3H]BPGP), which is a pharmacologically inactive angiotensin-converting enzyme substrate. The exercise-induced increase in cardiac output was accompanied by a linear increase in central blood volume, and dilutional extravascular lung water rapidly increased to an asymptotic proportion close to 100% of postmortem vascular lung water. There was an average 55% [3H]BPGP hydrolysis, which did not vary with flow, and the computed Amax/Km increased linearly with exercise. We conclude that exercise results in complete lung tissue recruitment and increases the pulmonary vascular surface area available for BPGP hydrolysis linearly with flow, so that pulmonary vascular recruitment continues after full tissue recruitment.     

Kinetics of pulmonary angiotensin-converting enzyme and 5'-nucleotidase in vivo

Angiotensin-converting enzyme and 5'-nucleotidase line the luminal surface of pulmonary microvascular endothelium and participate in the synthesis and/or degradation of potent vasoactive substances. We applied Michaelis-Menten kinetics in simultaneous estimations of apparent constants Km and Amax (product of Vmax and microvascular plasma volume) of these two enzymes for the substrates 3H-labeled benzoyl-Phe-Ala-Pro and 14C-labeled 5'-AMP, respectively, in vivo. Values of angiotensin-converting enzyme for benzoyl-Phe-Ala-Pro (Km = 10-11 microM; Amax = 12-13 mumol X min-1) were somewhat higher than published estimates in vitro and changed predictably in response to the known enzyme inhibitor captopril. Kinetic values of 5'-nucleotidase for 5'-AMP (Km = 3-4 microM; Amax = 3-4 mumol/min) were substantially lower than those reported in vitro but also responded predictably to the competitive inhibitor of 5'-nucleotidase, adenosine 5'-[alpha, beta-methylene]diphosphate. These data offer in vivo estimates of enzyme kinetics that are useful in revealing enzyme behavior in their normal physiological environment and provide means of evaluating the action of pharmacological, physiological, and pathological modulators of enzyme activity, in vivo.     

Mechanisms of extracellular reactive oxygen species injury to the pulmonary microvasculature.

We investigated the effect of xanthine (X) plus xanthine oxidase (XO) on pulmonary microvascular endothelial permeability in isolated rabbit lungs perfused with Krebs buffer containing bovine serum albumin (5 g/100 ml). Addition of five mU/ml XO and 500 microM X to the perfusate caused a twofold increase in the pulmonary capillary filtration coefficient (Kf,c) 30 min later without increasing the pulmonary capillary pressure. This increase was prevented by allopurinol or catalase but not by superoxide dismutase or dimethyl sulfoxide. Because these data implicated hydrogen peroxide (H2O2) as the injurious agent, we measured its concentration in the perfusate after the addition of X and XO for a 60-min interval. In the absence of lung tissue and albumin, H2O2 increased with time, reaching a concentration of approximately 250 microM by 60 min. If albumin (5 g/100 ml) was added to the perfusate, or in the presence of lung tissue, the corresponding values were 100 microM and less than 10 microM, respectively. To understand the mechanisms of H2O2 scavenging by lung tissue, we added a 250 microM bolus of H2O2 to the lung perfusate. We found that H2O2 was removed rapidly, with a half-life of 0.31 +/- 0.04 (SE) min. This variable was not increased significantly by inhibition of lung catalase activity with sodium azide or inhibition of the lung glutathione redox cycle with 1-chloro-2,4-dinitrobenzene. However, inhibition of both enzymatic systems increased the half-life of H2O2 removal to 0.71 +/- 0.09 (SE) min (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular ATP signaling in the rabbit lung: erythrocytes as determinants of vascular resistance

Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). In lungs perfused with PSS containing RBCs, L-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, L-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis.     

Thromboxane mediates pulmonary hypertension and lung inflammation during hyperacute lung rejection.

The role of thromboxane (Tx) in hyperacute rejection of pig lung by human blood was studied in an ex vivo model, wherein lungs from juvenile piglets were perfused with fresh heparinized human blood. In this model, hyperacute lung rejection was characterized by an abrupt rise in pulmonary vascular resistance (PVR; >1 cmH2O x ml(-1) x min) and prolific Tx elaboration (>15 ng/ml) within 5 min and loss of function within 10 min. Although papaverine significantly blunted the rise in PVR (<0.2 cmH2O x ml(-1) x min), Tx production was not inhibited (>20 ng/ml), and florid tracheal edema was usually evident within 20 min. In contrast, both inhibition of Tx synthesis (Tx < 3 ng/ml) with OKY-046 and blockade of the Tx receptor with SQ-30741 (Tx > 20 ng/ml) were not only associated with significantly lower peak PVRs (<0.2 cmH2O x ml(-1) x min) but also with attenuated increase in lung wet-to-dry ratio and airway edema. In concert, elaboration of histamine and tumor necrosis factor was blunted, and median survival increased >10-fold to 2 h (SQ-30741) and >4 h (OKY-046). Depletion of the pig lung macrophages with dichloromethyl bisphosphonate in liposomes, but not Pall filtration of the human blood or liposomes alone, significantly inhibited Tx elaboration (<0.2 vs. >8 ng/ml for Pall filtration or liposomes) and blunted PVR elevation (<0.3 cmH(2)O x ml(-1) x min) during initial perfusion. C3a and histamine elaboration were inhibited, and median survival was significantly prolonged (>4 h). These findings implicate Tx in the inflammation associated with hyperacute lung rejection and demonstrate that pulmonary intravascular macrophages are critical to its elaboration.     

Angiotensin-converting enzyme activity in ovine bronchial vasculature.

Angiotensin-converting enzyme (ACE) plays a major role in the metabolism of bradykinin, angiotensin, and neuropeptides, which are all implicated in inflammatory airway diseases. The activity of ACE, which is localized on the luminal surface of endothelial cells (EC), has been well documented in pulmonary EC; however, few data exist regarding the relative activity of ACE in the airway vasculature. Therefore, we measured ACE activity in cultured EC from the sheep bronchial artery and bronchial mucosa (microvascular) and compared it with pulmonary artery EC. The baseline level of total ACE activity (cellular plus secreted) was significantly greater in bronchial microvascular EC (1.24 +/- 0.24 mU/106 cells) compared with bronchial artery EC (0.59 +/- 0.15 mU/106 cells; P < 0.05) and comparable to pulmonary artery EC (1.12 +/- 0.14 mU/106 cells; P > 0.05). Measured ACE activity secreted into culture medium for each cell type was 64-74% of total activity and did not differ among the three EC types (P = 0.17). Hydrocortisone (10 microg/ml; 48-72 h) treatment resulted in a significant increase in ACE activity in bronchial EC. Likewise, TNF-alpha (0.1 ng/ml) treatment markedly increased ACE activity in all cell lysates (P < 0.05). We confirmed the importance of ACE activity in vivo since, at the highest dose of bradykinin studied (10-8 M), bronchial artery pressure at constant flow showed a greater decrease after captopril treatment (36% before vs. 60% after; P = 0.05). These results demonstrate high ACE expression of the bronchial microvasculature and suggest an important regulatory role for ACE in the metabolism of kinin peptides known to contribute to airway pathology.     

OKY-046 prevents increases in LTB4 and pulmonary edema in phorbol ester-induced lung injury in dogs.

Thromboxanes (Txs) were implicated as possible participants in the altered microvascular permeability of acute lung injury when the Tx synthase inhibitor, OKY-046, was reported to prevent pulmonary edema induced by phorbol myristate acetate (PMA). Recently, however, we found that OKY-046, at a dose just sufficient to block Tx synthesis in intact dogs, did not prevent PMA-induced pulmonary edema but rather merely reduced it modestly. The present study was designed to explore other mechanisms whereby OKY-046 might prevent PMA-induced pulmonary edema. The finding that 5-lipoxygenase (5-LO) metabolites of arachidonic acid were increased within the lung after PMA administration, coupled with the report that OKY-046 inhibited slow-reacting substance of anaphylaxis formation, permitted formulation of the hypothesis that OKY-046, at a dose in excess of that required to inhibit Tx synthesis, inhibits the formation of a product(s) of 5-LO and, thereby, prevents edema formation. In vehicle-pretreated pentobarbital-anesthetized male mongrel dogs (n = 4), PMA (20 micrograms/kg i.v.) increased pulmonary vascular resistance (PVR) from 4.4 +/- 0.3 to 26.3 +/- 8.8 mmHg.l-1 x min (P < 0.01) and extravascular lung water from 6.7 +/- 0.5 to 19.1 +/- 6.2 ml/kg body wt (P < 0.05). Concomitantly, both TxB2 and leukotriene B4 (LTB4) were significantly increased in the lung. Pretreatment with OKY-046 (100 mg/kg i.v., n = 8) prevented PMA-induced increases in TxB2, LTB4, and pulmonary edema formation but did not prevent the increase in PVR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine prevents phorbol ester injury in rabbit lungs: role of leukotrienes and TNF.

The objective of this study was to determine whether adenosine (ADO) prevents phorbol myristate acetate- (PMA) induced lung injury by modulating peptidoleukotrienes (LT) and/or tumor necrosis factor (TNF) production. PMA significantly increased pulmonary vascular resistance (PVR, 275 +/- 4 to 447 +/- 30 cmH2O.1-1.min) and microvascular filtration coefficient.(Kf, 0.024 +/- 0.002 to 0.040 +/- 0.006 g.min-1.cmH2O-1) in isolated blood-perfused rabbit lungs. ADO (5 mumol/min) blocked the increases in PVR (257 +/- 9 to 283 +/- 26) and Kf (0.028 +/- 0.005 to 0.018 +/- 0.002). After PMA (30 min), perfusate levels of LTC4 + LTD4 increased by 15.3 +/- 2.1 pg/ml; LTE4 increased by 15.1 +/- 4.1 pg/ml. ADO reduced the increase in LTC4 + LTD4 to 2.7 +/- 6.1 pg/ml, but total LT increased by 31.9 +/- 16.6 pg/ml, implying that ADO enhanced the conversion of LTC4 and LTD4 to LTE4. MK-886 (L663,536), an LT synthesis inhibitor, blocked the increase in total LT (6.1 +/- 13.9 pg/ml) but did not reduce the PMA-induced increase in Kf (0.022 +/- 0.003 to 0.035 +/- 0.005) or PVR (238 +/- 11 to 495 +/- 21). After PMA administration, perfusate TNF levels were not different from the 10-fold increase observed in control experiments and were not reduced by ADO or MK-886. TNF production was independent of perfusate blood components and presumably due to low levels of endotoxin in the perfusate (70-90 ng/ml). These results indicate that ADO does not protect against PMA-induced acute lung injury by altering circulating levels of LT or TNF.     

Pulmonary angiotensin-converting enzyme kinetics after acute lung injury in the rabbit

Depression of lung endothelial cell metabolic function may be an early and sensitive indicator of lung damage. When such functions are measured in vivo, substrates injected usually must be limited to "trace" doses due to the significant hemodynamic effects of high doses of substrate. Under first-order conditions (i.e., trace doses) the enzyme or transport system rate constant Vmax/Km may be calculated, but independent estimates of each variable (Vmax and Km) are not available. We therefore used multiple indicator-dilution methods and higher substrate concentrations to apply a mathematical model, based on saturable kinetics that yield independent estimates of the apparent kinetic parameters Vmax and Km for pulmonary angiotensin-converting enzyme (ACE). We used the ACE substrate, [3H]benzoyl-phenylalanyl-alanyl-proline ([3H]BPAP) and made these measurements and also estimates of serotonin [5-hydroxytryptamine (5-HT)] removal, before and after acute lung injury induced by intratracheal administration of phorbol myristate acetate (PMA). PMA significantly depressed the percent 5-HT removal (62 +/- 3 to 44 +/- 4%) and BPAP percent metabolism (74 +/- 2 to 66 +/- 2), when trace amounts of either compound were injected as a bolus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of vasoconstriction, temperature, and flow on the kinetics of serotonin uptake in rabbit lungs

In the process of estimating the kinetic parameters of the pulmonary endothelial serotonin (5-HT) uptake, it is critically important to distinguish the effects of hemodynamic changes from endothelial injury. Therefore, the effects of changes in flow rate (1.7-5.0 ml/s), hemodynamics (vasoconstriction by norepinephrine), and temperature (39 vs. 33 degrees C) were investigated in isolated rabbit lungs. Indicator-dilution data were expressed in terms of the Michaelis-Menten equation for the two 5-HT uptake pathways in the preparation. The maximum uptake velocity (Vmax1) and the 5-HT concentration at half-maximum velocity (Km1) of the first pathway as well as the first-order constant (Vmax2/Km2) of the linear part of the second pathway were determined. Neither vasoconstriction nor flow variations had any effect on Km1, whereas increasing the flow rate caused extensive recruitment, with a concomitant increase in Vmax1 and Vmax2/Km2. Furthermore, all the kinetic parameters were significantly decreased at the lower temperature. We conclude that Km1 is independent of organ hemodynamics (vasoconstriction and flow) but susceptible to changes in 5-HT uptake capacity caused by a change in temperature. Vmax1 and Vmax2/Km2 respond to alterations in 5-HT uptake capacity and perfused organ volume. These are prerequisites to apply kinetic modeling as a method for the investigation of pulmonary endothelial function and integrity.     

Promethazine or DPPD pretreatment attenuates oleic acid-induced injury in isolated canine lungs

Oleic acid causes pulmonary edema by increasing capillary endothelial permeability, although the mechanism of this action is uncertain. We tested the hypothesis that the damage is an oxidant injury initiated by oleic acid, using isolated blood-perfused canine lung lobes. The lobes were dilated with papaverine and perfused in zone III with a constant airway pressure of 3 cmH2O. Changes in isogravimetric capillary pressure (Pc,i) and capillary filtration coefficient (Kf,C) were used as indices of alterations in microvascular permeability in lungs treated with silicone fluid (n = 3), oleic acid (n = 11), oleic acid after pretreatment with the antioxidants promethazine HCl (n = 11) or N,N'-diphenyl-p-phenylenediamine (DPPD; n = 4), or oleic acid following pretreatment with methylprednisolone (n = 4). Kf,C averaged 0.21 +/- 0.02 ml X min-1 X cmH2O-1 X 100 g-1 in control and increased to 0.55 +/- 0.05 and 0.47 +/- 0.05 when measured 20 and 180 min after the administration of oleic acid. When oleic acid was infused into lungs pretreated with promethazine, Kf,C increased to only 0.38 +/- 0.05 ml X min-1 X cmH2O-1 X 100 g-1 after 20 min and had returned to control levels by 180 min. Pretreatment with DPPD, but not methylprednisolone, similarly attenuated the increase in Kf,C following oleic acid. Silicone fluid had no effect on Kf,C. That oleic acid increases vascular permeability was also evidenced by a fall (P less than 0.05) in Pc,i from control when measured at 180 min in every group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced microvascular permeability of PMA-induced acute lung injury is not mediated by cyclooxygenase products.

Products of cyclooxygenase activity have been proposed to mediate the pulmonary hypertension and increased microvascular permeability associated with phorbol myristate acetate- (PMA) induced acute lung injury. Previously, we reported that thromboxane (Tx) does not mediate PMA-induced pulmonary hypertension in intact anesthetized dogs. In the present study, PMA was administered to isolated canine lungs perfused with autologous blood at constant flow to investigate a possible role for Tx in the PMA-induced increase in microvascular permeability. Changes in permeability were assessed by determining changes in the capillary filtration coefficient (Kfc). In lobes pretreated with papaverine to prevent PMA-induced increases in pulmonary vascular resistance, Kfc increased from a baseline value of 0.2 +/- 0.03 to 1.5 +/- 0.29 ml.min-1.cmH2O-1.100 g wet lobe wt-1 (P < 0.01) 30 min after PMA (5.8 x 10(-8) M, n = 10). Concomitantly, TxB2, the stable metabolite of TxA2, increased from 138 +/- 44 to 1,498 +/- 505 pg/ml (P < 0.05) in the blood. Both the selective Tx synthase inhibitor, OKY-046 (7 x 10(-4) M, n = 6), and the cyclooxygenase inhibitor, indomethacin (10(-4) M, n = 7), prevented the PMA-induced increase in TxB2, but neither compound attenuated the PMA-induced increase in Kfc. ONO-3708 (10(-6) M), a selective prostaglandin (PG) H2/TxA2 receptor antagonist, prevented the vasoconstriction resulting from administration of U-46619, a stable PGH2/TxA2 receptor agonist, but it did not prevent the PMA-induced increases in Kfc (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Propranolol and serotonin removal in lung injury

Indicator dilution technique was used to study effects of reduced vascular volume or acute injury on removal of low doses of [3H]propranolol and [14C]serotonin (5-hydroxytryptamine, 5-HT) by perfused rabbit lung. Glass-bead (500 micron) embolization doubled pulmonary arterial pressure (Ppa) at flow rates of 20, 50, and 100 ml/min, decreased volume of distribution by approximately 50%, and increased pulmonary vascular resistance by at least 60%. Before embolization, (flow rate 20 ml/min) removal of [3H]propranolol and [14C] 5-HT was 89 +/- 2 and 75 +/- 5%, respectively, and was unaltered by changes in flow rate. However, after embolization, [3H]propranolol and [14C]5-HT removal decreased in a flow-dependent manner, reaching 28 +/- 4 and 1 +/- 3% (P less than 0.05), respectively, at a flow rate of 100 ml/min. When phorbol myristate acetate (PMA, 200 nM) was perfused (50 ml/min) through the lungs for 15 min, Ppa increased from 13 +/- 1 to 25 +/- 2 cmH2O (P less than 0.05), whereas [3H]propranolol removal decreased from 92 +/- 1 to 75 +/- 5% (P less than 0.05) and [14C]5-HT removal decreased from 73 +/- 3 to 46 +/- 8% (P less than 0.05). The PMA also caused vasoconstriction, which could be partially blocked by adding papaverine (500 microM) to the perfusion medium. Under the latter conditions, Ppa increased to 19 +/- 1 cmH2O and [3H]propranolol removal was unaffected. However, the combination of PMA and papaverine reduced [14C]5-HT removal from 64 +/- 4 to 19 +/- 3%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary microvascular response to LTB4: effects of perfusate composition

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.     

Acute and long-term TNF-alpha administration increases pulmonary vascular reactivity in isolated rat lungs.

Tumor necrosis factor-alpha (TNF-alpha) causes pulmonary hypertension and arterial hypoxemia, but the mechanisms are unknown. We conducted two experiments to test the hypothesis that TNF-alpha alters pulmonary vascular reactivity, which in turn could cause either pulmonary hypertension or arterial hypoxemia. In experiment 1, rats were given acute or long-term injections of TNF-alpha (recombinant human) in vivo. Rats treated acutely received either saline or TNF-alpha (40 micrograms/kg iv in saline) 3 min (TNF-3 min; n = 8), 20 min (TNF-20 min; n = 8), or 24 h (TNF-24 h; n = 5) before the lungs were isolated. Rats treated chronically received injections of either saline or TNF-alpha (250 micrograms/kg ip in saline) two times per day for 7 days (TNF-7 days; n = 9). Lungs were isolated and perfused with Earle's salt solution (+2 g/l NaHCO3 + 4 g/100 ml Ficoll), and vascular reactivity was tested with acute hypoxia (3 min; 3% O2) and angiotensin II (ANG II; 0.025-0.40 micrograms). Pulmonary pressor responses to hypoxia were greater (P less than 0.05) in TNF-20 min and TNF-7 day groups. ANG II responses were increased (P less than 0.05) in TNF-7 day rats. In experiment 2, lungs were isolated and perfused and received direct pulmonary arterial injections of TNF-alpha (0.2, 2.0, and 20 micrograms) or saline, after stable responses to hypoxia and ANG II (0.10 microgram) were attained. Reactivity was not different between control and TNF-alpha rats before the injections, but TNF-alpha increased (P less than 0.05) responses to hypoxia and ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary vascular effects of sildenafil on the development of chronic pulmonary hypertension in the ovine fetus

We investigated the pulmonary vascular effects of prophylactic use of sildenafil, a specific phosphodiesterase-5 inhibitor, in late-gestation fetal lambs with chronic pulmonary hypertension. Fetal lambs were operated on at 129 +/- 1 days gestation (term = 147 days). Ductus arteriosus (DA) was compressed for 8 days to cause chronic pulmonary hypertension. Fetuses were treated with sildenafil (24 mg/day) or saline. Pulmonary vascular responses to increase in shear stress and in fetal PaO2 were studied at, respectively, day 4 and 6. Percent wall thickness of small pulmonary arteries (%WT) and the right ventricle-to-left ventricle plus septum ratio (RVH) were measured after completion of the study. In the control group, DA compression increased PA pressure (48 +/- 5 to 72 +/- 8 mmHg, P < 0.01) and pulmonary vascular resistance (PVR) (0.62 +/- 0.08 to 1.15 +/- 0.11 mmHg x ml(-1) x min(-1), P < 0.05). Similar increase in PAP was observed in the sildenafil group, but PVR did not change significantly (0.54 +/- 0.06 to 0.64 +/- 0.09 mmHg x ml(-1) x min(-1)). Acute DA compression, after brief decompression, elevated PVR 25% in controls and decreased PVR 35% in the sildenafil group. Increased fetal PaO2 did not change PVR in controls but decreased PVR 60% in the sildenafil group. %WT and RVH were not different between groups. Prophylactic sildenafil treatment prevents the rise in pulmonary vascular tone and altered vasoreactivity caused by DA compression in fetal lambs. These results support the hypothesis that elevated PDE5 activity is involved in the consequences of chronic pulmonary hypertension in the perinatal lung.