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5 min (4°) and the pellets discarded. Acetone (1.5 volumes) is added to the supernatants which contain 12-HETE-1,20-dioic acid. Protein precipitation, acidification, and lipid extraction are carried out as described above. In this instance, 12-HETE-1,20-dioic acid is purified by HPLC on the txBondapak C1s column at 25° with a solvent system consisting of methanol/water/acetic acid (65 : 35 : 0.01, v/v/v), pH 6, at 0.42 ml/min. It is of critical importance to realize that 12-HETE-1,20-dioic acid undergoes spectral alterations if maintained and/or dried from the above acidic eluting solvent. Spectral integrity is best preserved by adding NH4OH to an approximate concentration of 0.4 mM after collection. Drying is then accomplished by rapid, direct flow of argon into the sample tube. The compound is then stored at - 7 0 ° in the dry state under argon. 8 Acknowledgments I would like to acknowledge the research and intellectual collaboration of the following colleagues: Lenore B. Sailer, Harris L. Ullman, Naziba Islam, M. Johan Broekman, M. Teresa Santos, and Juana Valles. I would also like to acknowledge the expertise provided by Ms. Evelyn M. Ludwig with regard to preparation of this manuscript. Research work mentioned in this chapter was supported by grants from the Veterans Administration, National Institutes of Health Grant HL-18828-14 SCOR, the Edward Gruenstein Fund, the Sallie Wichman Fund, and the S.M. Louis Fund.
[65] I s o l a t e d
P e r f u s e d R a t L u n g in A r a c h i d o n a t e S t u d i e s
By SHIn-WEN CHANG and NO~ERT F. VOELKEL
The isolated perfused rat lung preparation has been used for the past 20 years by both physiologists and pharmacologists interested in the circulatory, biochemical, and metabolic aspects of this complex organ. In particular, studies of arachidonate metabolism in isolated perfused rat lungs have yielded important information regarding the significant in vivo metabolic pathways of arachidonic acid metabolites, the action of various eicosanoids in the physiological regulation of the pulmonary circulation. In this section, we will detail the method of isolated lung perfusion currently used in our laboratory. We will also highlight some important results related to arachidonate metabolites obtained using this preparation. METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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General Methodology Lung Isolation and Perfusion
Pathogen-free, young adult Sprague-Dawley rats with body weight from 250 to 350 g are anesthetized with an ip injection of pentobarbital sodium (70 mg/kg). After removal of the overlying skin, a median incision is made in the center of the neck and the trachea exposed by blunt dissection. The trachea is partially transected and a 15-gauge blunt metal cannula is inserted and secured with a 0-silk suture. Following tracheal cannulation, ventilation is initiated with a small animal ventilator using a humidified gas mixture containing 21% 02, 5% CO2, and 74% nitrogen. The ventilator is set at a rate of 55 breaths per minute, using either a pressurelimited ventilator with a maximal inspiratory pressure of 8 cm water, or a volume-limited ventilator with a tidal volume of 2 to 4 ml/breath (approximately I ml/100 g body weight). We do not impose a positive endexpiratory pressure (PEEP) initially to allow partial lung collapse and to minimize the chance of nicking the lung during chest opening and lung removal. The sternum is split at the midline using a blunt-tipped scissors, taking care to avoid the ventilated lungs. The chest is then opened with a rib spreader and 100 units of heparin (in 0.5 ml saline) are immediately injected into the right ventricle to prevent intravascular clotting. Using a small, curved-tip hemostatic forceps, a suture is placed around the outflow tracts of both aorta and pulmonary artery and secured with a loose knot. At this time, it is prudent to be sure that the perfusion circuit is filled with the perfusate solution and that no air bubbles remain in the tubing. A small nick is made in the right ventricular-free wall and the pulmonary artery cannula (mounted at the end of the perfusion tubing) is introduced into the pulmonary artery through the preexisting ligature. The aorta and pulmonary artery are then tightly ligated. Another incision is made at the cardiac apex to allow insertion of a second cannula into the left ventricule to collect effluent perfusate coming from the lungs. The trachea, lungs, and the heart are removed en bloc from the chest cavity and suspended by the tracheal tubing in a constant-temperature, humidified chamber (Fig. 1). Perfusion is begun with a peristaltic Holter pump, starting at a slow rate, and gradually increasing to a final rate of 0.03 ml/g body weight/min. If blood-free perfusion is desired, the initial 50 ml of the perfusate containing residual plasma and blood cells are discarded prior to initiation of recirculation. When blood is used to perfuse the lungs, recirculation of the perfusate can be established from the beginning. Pulmonary artery perfusion pressure is measured (via a side arm of the inflow perfusion tubing) with a Statham P23AA transducer, the signal is amplified, and continu-
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• L • Weight
~
. . . . . . . . . . . . . . . . . . . . .
~
c
Ventilator
!
FIG. 1. Isolated peffused rat lung. The lungs are suspended on a weight transducer within
a humidified chamber, and ventilated with a small animal ventilator. The perfusate is recirculated using a Holter peristaltic pump. PEEP, Positive end-expiratory pressure; PPA, pulmonary arterial pressure.
ously recorded. Since perfusion rate is constant, changes in perfusion pressure reflect changes in pulmonary vascular resistance. At this time, the lungs are hyperinflated to remove any residual atelectasis and PEEP is initiated at 2 to 3 cm water. In a long perfusion protocol, periodic (every 30-40 min) hyperinflation of the lungs may be necessary to prevent atelectasis. Throughout the experiment, the lungs and the perfusate are maintained at 37° to 38° by recirculation of warmed water through waterjacketed chambers around the lungs, the humidification unit, and the perfusate reservoir.
Choice of Perfusate Depending on the experimental design, the lungs can be perfused with whole blood, plasma, or a blood- and plasma-free physiological salt solution (PSS). Initial perfusion pressure averages around 12-16 mmHg with blood perfusate and 5-7 mmHg with PSS, using the ventilation and perfusion parameters outlined above. Blood-perfused lungs closely resemble
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the in vivo state, maintain excellent vascular reactivity, and are often used in the studies of hypoxic pulmonary vasoconstriction. Ether-anesthetized, retired breeder rats are used as blood donors. Approximately 8-10 ml of whole blood may be removed from each donor rat via percutaneous cardiac aspiration. In general, 30 ml of whole blood are sufficient for each perfused lung experiment. However, in studies assessing the vascular action of lipid mediators or measuring lung metabolism of eicosanoids, binding of the mediator or metabolites to plasma proteins and metabolism by blood cells may complicate the interpretation of results. For these studies, perfusion with PSS containing either albumin or Ficoll is recommended. We use a modified Krebs-Henseleit PSS containing: 119 mM NaCI, 4.7 mM KCI, 1.17 mM MgSO4, 19.1 mM HaHCO3, 1.18 mM KH2PO4, 1.6 mM CaC12, and 5.5 mM glucose. Immediately prior to use, the solution is equilibrated with a gas mixture containing 95% 02 and 5% CO2 and Ficoll 70 (a synthetic copolymer of sucrose and epichlorohydrin with a molecular weight of 70,000) is dissolved to 40 g/liter. In experiments where bovine serum albumin is used in place of Ficoll, the PSS is modified to 22.6 mM NaHCO3 and 3.2 mM CaCI2. The use of PSS-Ficoll as perfusate is appropriate for short-term experiments in which mediator binding to albumin is a concern. Lungs effused with albumin-free perfusate (i.e., PSS-FicoI1) are more susceptible to the development of edema, especially with perfusion periods greater than 2-3 hr.
Stability of Preparation The major factors that affect the performance and stability of isolated perfused lung preparations are: (1) health status of the experimental animal, (2) technical expertise and care during lung isolation and removal, (3) perfusate composition, and (4) length ofperfusion time. Viral infections are not infrequent in rat colonies and superimposed bacterial pneumonitis can follow and complicate the viral respiratory tract infection. Lungs with macroscopic consolidation or scarring should be discarded as they develop edema easily and may have elevated baseline levels of eicosanoids. Extreme care must be taken not to traumatize the lung tissue during the isolation procedure. Some degree of flow interruption is unavoidable while the lung is removed from the chest cavity but, with practice, this ischemic period can be as short as 3-4 minutes; continued ventilation during this period of time should prevent any ischemia reperfusion-related problems. Some laboratories have reported rapid development of spontaneous edema in isolated lungs and the question is raised whether isolated lungs are unavoidably injured. In our experience, however, spontaneous edema
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is rare when healthy lung donor rats are used and stable and reproducible lung preparations are consistantly obtained. Moreover, we have recently found comparable values for lung permeability-surface area product between isolated rat lungs perfused with either blood or PSS-albumin and intact rats. i Blood-perfused lungs often maintain excellent vascular reactivity and remain edema free for up to 4 hr of perfusion. Even in lungs perfused with PSS-albumin, significant edema is not observed up to 4 hr of perfusion. 2 Although lungs perfused with PSS-Ficoll are somewhat more susceptible to edema than those perfused with PSS-albumin or blood, they should remain stable for up to 2 hr of perfusion. An important complication is the occasional development of ventricular spasm, which is observed most commonly between 5 and 20 rain after the initiation of lung perfusion and account for most cases of "spontaneous" edema that we have observed. Presumably, postmortem spasm in the heart muscle partially or completely interrupts the flow of effluent perfusate out of the left atrium, resulting in increased pulmonary venous and microvascular pressures. If uncorrected, the lungs rapidly become edematous. When ventricular spasm is suspected, the ventricle should be firmly squeezed with a tweezer and the left ventricular cannula repositioned. This should be followed by a rapid decrease in the pulmonary arterial pressure and a marked decrease in the size of the congested left atrial appendage. This complication can often be prevented by vigorously disrupting the mitral valve apparatus during the insertion of the left ventricular cannula and by a close monitoring of the lung preparation.
Physiological Parameters Mean pulmonary artery pressure is continuously measured and recorded on a strip chart recorder. Left atrial pressure is typically set at 0 cm water but can be varied by simply raising the platform supporting the perfusate reservoir. In some experiments, setting the left atrial pressure at 5 cm water may be desirable to fully recruit the rat lung vasculature. Since perfusion rate and left atrial pressure are fixed, changes in the pulmonary artery pressure reflect changes in total pulmonary vascular resistance. The total vascular resistance can be further partitioned to pre- and postcapillary sites by measuring the pulmonary microvascular pressure (Pmv). This is done by using the double occlusion technique initially described by Dawson and colleagues 3 and recently validated against the traditional t j. Czartolmna, N. F. Voelkel, and S. Chang, F A S E B J . 3, All40. A. B. Fisher, C. Dodia, and J. Linask, Exp. Lung Res. 1, 13 (1980). 3 C. A. Dawson, J. H. Linehan, and D. A. Rickaby, Ann. N. Y. Acad. Sci. 384, 90 (1982).
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gravimetric technique by Townsley et al. 4 The inflow and outflow tubings are simultaneously clamped for a period of 20 sec and the difference between this equilibration pressure and a stop-flow pressure is taken to be Pmv. This pressure averages around 2-2.5 mmHg in PSS-Ficoll perfused lungs and increases to 4.6 -+ 0.4 mm Hg after a bolus injection of LTC4 (2/~g) into the pulmonary artery.5 In experiments investigating mediator effects on lung edema, the isolated lungs can be suspended from a force-displacement transducer for continuous measurement of lung weight. Alternatively, the perfusate reservoir can be placed on a scale and changes in lung weight can be inferred by the loss of perfusate in a recirculating system. At the end of the experimental protocol, the lungs are dissected from the mediastinal tissues, weighed, and the wet lung-to-body weight ratio can be taken as a rough estimate of fluid accumulation. If lung tissues are not used for biochemical analysis, the lungs can be dried to constant weight and the lung wet-to-dry ratio calculated as an index of lung edema. Assessment of lung mechanics and airway reactivity using a heated pneumotachograph and airway pressure transducer in isolated perfused rat lungs have also been reported. 6 Assessment of lung vascular permeability can be done by measuring either capillary filtration coefficient7 or the extravascular leakage of radiolabeled protein. We prefer the later method and use a technique modified from Kern et al.S After an equilibration period of 20 min, during which time the lungs are in an isogravimetric state, approximately 1/zCi of I25I-labeled albumin (specific activity 1.06 mCi/mg; ICN radiochemicals, Irvine, CA; greater than 99.9% binding to albumin) is added to the perfusate and allowed to recirculate for exactly 3 min. After 1 ml of the perfusate is collected, the lungs are perfused with a fresh, nonradioactive perfusate for the additional 3 min without recirculation to remove intravascular I25Ilabeled albumin. The lungs are dissected, weighed, and their radioactivity determined in an auto-y-scintillation spectrometer. The lung permeability-surface area product (PS) can be calculated as follows: Lung PS = total lung 125Iactivity/(I25I activity in 1.0 g of initial perfusate x 3 min) 4 M. I. Townsley, R. J. Korthius, B. Rippe, J. C. Parker, and A. E. Taylor, J. Appl. Physiol. 61, 127 (1986). 5 A. Sakai, S. Chang, and N. F. Voelkel, J. Appl. Physiol., in press (1989). 6 K. B. Nolop, J. H. Newman, J. R. Sheller, and K. C. Brigham, Am. Rev. Respir. Dis. 129, A231 (1984). 7 K. A. Gaar, Jr., A. E. Taylor, L. J. Owens, and A. C. Guyton, Am. J. Physiol. 213, 910 (1967). s D. F. Kern, D. Levitt, and D. Wangensteen, Am. J. Physiol. 245, H229 (1983).
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While lung PS can be affected by capillary recruitment (by altering perfused surface area available for macromolecular exchange), this effect is relatively small and changes in PS of greater than 50% are likely due to changes in vascular permeability.
Advantages and Disadvantages The isolated perfused rat lung allows the studies of an intact, physiologically stable organ preparation in which influences from plasma-borne mediators, hormones, and circulating blood cells can be excluded. The intact pulmonary circulation is under investigation and is capable of vasoreactivity and local regulation, yet the investigator has control over blood flow and vascular pressures. In addition, the effects of varying gas tensions on metabolism and vascular responses are easily studied. All relevant cell types intrinsic to the lung are present and the cell-cell relationships are preserved in their normal state. With care, this preparation remains stable and metabolically active for up to 4 hr. Concerns which limit the use of the isolated perfused rat lung are" (1) long-term studies are not feasible; (2) the lung is composed of a mixture of many cell types, and, therefore, it is difficult to attribute mediator synthesis or metabolism to a specific cell type; finally (3), one needs to take into account the possibility of activation of certain synthetic and metabolic pathways by the lung isolation and perfusion procedures. Use of Isolated Perfused Rat Lung in Arachidonate Studies
Isolated Perfused Rat Lung as Bioassay Organ for Assessment of Eicosanoid Action Infusion of arachidonic acid into the pulmonary artery of isolated perfused rat lungs results in vascular effects attributable to both cyclooxygenase and lipoxygenase metabolites. 9 The acute pulmonary vasoconstrictive effect of arachidonic acid is inhibited by indomethacin and probably due to thromboxane A2. In the presence of indomethacin, arachidonic acid causes a delayed pressure rise and lung edema that can be attributed to the actions of leukotrienes since both can be inhibited by 5-1ipoxygenase inhibitors. Furthermore, injections of individual purified lipoxygenase metabolites showed that LTC4 is the most potent vasoconstrictor in the rat lung, while both LTC4 and LTB4 cause lung edema. 9 9 N. F. Voelkei, K. R. Stenmark, J. T. Reeves, M. M. Mathias, and R. C. Murphy, J. Appl. Physiol. 57, 860 (1984).
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Subsequently, LTE4 was also found to be a pulmonary vasoconstrictor in the rat lung. 10An important concept from this and other studies 9' 1l, 12is the marked attenuation of the vascular activity of eicosanoids in the presence of albumin perfusate, presumably due to binding of the mediators to albumin. Avid binding of these lipid mediators by plasma proteins may serve to localize their vasoactive effects to near sites of biosynthesis. Injection of another lipid mediator, platelet-activating factor (PAF), into isolated rat lung causes pulmonary vasoconstriction and lung edema. This effect is, in a large part, mediated by the production of LTC4 by the lung tissue. 13 We have recently found that injection of both PAF and LTC4 cause significant increase in Pmv, indicating a significant degree of pulmonary venoconstriction. Furthermore, inhibition of the vascular pressure changes using dibutyryl-cAMP completely blocked LTC4-induced lung edema and albumin leak in perfused lungs: Thus, pulmonary venoconstriction is an important mechanism in lipid mediator-induced lung edema. Another important concept in regards to the vasoactivity of lipid mediators in the pulmonary circulation is that the observed response is often dependent on both the dose of the mediator as well as the tone of the pulmonary circulation. Under basal conditions, the pulmonary circulation is nearly maximally vasodilated. Thus, it is difficult to demonstrate vasodilatory responses unless the tone is first increased with vasoconstrictors or exposure to alveolar hypoxia (hypoxic pulmonary vasoconstriction). For example, a low dose (10 ng) of PAF has minimal effect during basal condition but causes vasodilation during hypoxic vasoconstriction; a higher dose of PAF (1/zg) causes vasoconstriction during basal condition but vasodilation followed by vasoconstriction during hypoxic vasoconstriction. Both the vasodilatory and vasoconstrictive actions of PAF are receptor-mediated 14 and can be inhibited by receptor blockers such as CV3988 or WEB 2086. The vasodilatory effect is likely due to the release of endothelium-dependent relaxing factor, while the vasoconstrictive effect may be due to the release of leukotriene C4. 1o C. O. Feddersen, M. Mathias, R. C. Murphy, J. T. Reeves, and N. F. Voelkel, Prostaglandins 26, 869 (1983). 11 V. J. Iacopino, S. Compton, T. Fitzpatrick, P. Rarnwell, J. Rose, and P. Kott, J. Pharmacol. Exp. Ther. 229, 654 (1984). 12 T. C. Noonan, W. M. Selig, K. E. Burhop, C. A. Burgess, and A. B. Malik, J. Appl. Physiol. 64, 1989 (1988). 13 N. F. Voelkel, S. Worthen, J. T. Reeves, P. M. Henson, and R. C. Murphy, Science 218, 286 (1982). 14 N. F. Voelkel, S. Chang, K. Pfeffer, S. G. Worthen, I. F. McMurtry, and P. M. Henson, Prostaglandins 32, 359 (1986).
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Injection of arachidonic acid during hypoxic vasoconstriction in PSSFicoll perfused rat lungs also causes a biphasic response with acute, transient vasoconstriction followed by a prolonged vasodilatory effect. In the presence of cyclooxygenase inhibition, only the acute vasodilation is evident.15 Recent results from our laboratory suggest that this cyclooxygenase-independent pulmonary vasodilatory effect of arachidonic acid is, in part, mediated by cytochrome P-450-dependent metabolites.
Role of Eicosanoids in Pulmonary Vasoregulation Cyclooxygenase metabolites are generated by respiratory movement ~6 as well as during blood flow alterations. 17Inhibition of the cyclooxygenase pathway enhances and preserves vascular reactivity in isolated rat lungs, suggesting that endogenous vasodilatory prostaglandins such as PGI2 modulate vascular responses in this preparation. This activation of vasodilatory prostaglandins is markedly amplified during lung infection or vascular injury and studies using diverse experimental preparations have suggested an important role for PGI2 in the depression of hypoxic pulmonary vasoconstriction observed in these settings. 18 Thromboxane A2, a potent vasoconstrictor, is also released following activation of the cyclooxygenase pathway. In species such as guinea pig and sheep, large amounts of thromboxane A2 are released in response to injury and play an important role in the observed pulmonary hypertension. In the rat lung, however, the amount of thromboxane A2 synthesized is modest compared with PGI2 and thromboxane A2 plays a minor role in mediating the vascular response to injury. 5-Lipoxygenase metabolites are clearly activated during acute lung injury. LTB4 is a potent chemoattractant for neutrophils, but whether it has a direct effect on vascular permeability is unclear. 12 L T C 4 and other peptidoleukotrienes are markedly increased in lung tissue after endotoxin-, a-staphylotoxin-, and protamine-induced lung injury. At least in the latter two injury models, the production of L T C 4 appears to promote lung edema by enhancing pulmonary venoconstriction.19 Whether L T C 4 plays a role in the physiological regulation of the normal pulmonary circulation is less clear. Although earlier studies from this laboratory suggested 15 C. O. Feddersen, S. Chang, J. Czartoloma, and N. F. Voelkel, J. Appl. Physiol., submitted. 16 R. Korbut, J. Boyd, and T. Eling, Prostaglandins 21, 491 (1981). 17 A. van Grondelle, G. S. Worthen, D. Ellis, M. M. Mathias, R. C. Murphy, R. J. Strife, J. T. Reeves, and N. F. Voelkel, J. Appl. Physiol. 57, 388 (1984). 18 S. Chang and N. F. Voelkel, in "Eicosanoids in Cardiovascular and Renal Systems" (P. V. Halushka and D. E. Mais, eds.), p. 62. MTP Press, Lancaster, 1988. ~9 S. Chang, J. Czartolomna, and N. F. Voelkel, unpublished observations (1989).
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a potential role for leukotrienes as mediator of hypoxic pulmonary vasoconstriction, 2° recent results have not supported this hypothesis. 21 There are currently no experimental data to support or refute a vasoregulatory role for other arachidonic acid metabolites, such as lipoxins and epoxyeicosatrienoic acids, in the normal pulmonary circulation. Eicosanoid Metabolism in Isolated Perfused Rat Lung
The lung is a metabolic organ and is capable of synthesis as well as metabolism of a large number of eicosanoids. Using the isolated perfused lung technique, Westcott et al. 22 observed marked species dependence in lung eicosanoid synthesis. When the perfused rat lung is treated with the calcium ionophore A23187, a large amount of PGI2 (as 6-keto-PGFl~), LTB4, and L T C 4 and lesser amounts of thromboxane A2 (as TxB2), PGE2, and LTD4 are synthesized. 22 Significant amounts of 6-keto-PGFl~ TxB2 and LTB4 are released into the lung perfusate. In guinea pig lungs, the cyclooxygenase metabolites, especially thromboxane A2, is the predominant eicosanoid product after A23187 stimulation, and very little 5-1ipoxygenase metabolites can be measured. In the ferret lung, the lipoxygenase products, especially LTB4, are prominent. In rabbit and hamster lungs, roughly equal amounts of cyclooxygenase and lipoxygenase products are synthesized, after A23187 stimulation. 22 Physiological factors that are important in stimulation of lung eicosanoid synthesis include respiratory motion, 16 and alterations in the hydrodynamic forces in the circulation.I7 Indeed, we find that the "baseline" levels of eicosanoids in isolated perfused lungs are generally severalfolds higher than those in lungs removed from anesthetized rats and immediately homogenized in methanol. This is particularly true for the PGI2 metabolite 6-keto-PGFi~ which averages around 15 ng/g wet lung in nonperfused lungs and may reach 340 ng/g in lungs perfused with PSS-Ficoll for 1 hr (Table I). Thus, PGI2 synthesis appears to be stimulated in the perfused lungs and likely contributes to the impaired vasoreactivity observed after prolonged perfusion. It is not surprising that cyclooxygenase inhibitors enhance and prolong vascular reactivity in isolated perfused rat lung. 20 M. L. Morganroth, J. T. Reeves, R. C. Murphy, and N. F. Voelkel, J. Appl. Physiol. 56, 1340 (1984). Zl A. J. Lorigro, R. S. Sprague, A. H. Stephenson, and T. E. Dahnms, J. Appl. Physiol. 64, 2538 (1988). 22 j. y . Westcott, T. J. McDonnell, P. Bostwick, and N. F. Voelkel, Am. Rev. Respir. Dis. 138, 895 (1988).
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TABLE I COMPARISON OF LUNG TISSUE EICOSANOID LEVELS IN CONTROL RATS BEFORE AND AFTER LUNG PEP,FUSION
Lung tissue
Thromboxane B2" (ng/g)
6-keto-PGF~ (ng/g)
LTC4 (ng/g)
Unperfusedb (11)d Peffusedc (6)d
6 -+ 1 15 -+ 3
15 -+ 4 338 -+ 55
1.5 -+ 0.3 5.0 -+ 1.2
a Data are mean + SEM and expressed as nanogram per gram wet lung weight. b Lung are removed from anesthetized rats and immediately homogenized in methanol. Thrombo×ane B2 and 6-keto-PGF~ are measured by enzyme-linked immunoassay. LTC4 is measured by enzyme-linked immunoassay after HPLC purification. Methods are as described by J. Y. Westcott et al. [Prostaglandins 32, 857 (1986)]. c Lungs are isolated and perfused in a physiological salt solution containing Ficoll for 60 min prior to lung homogenization and eicosanoid measurements. a Number of experiments.
Harper e t al. 23 have also used isolated perfused rat lung to study the metabolism of LTB4 and LTC4 and found that LTC4 is rapidly metabolized to LTC4 sulfoxide, LTD4, and LTE4. In contrast, LTB4 appears not to be significantly metabolized by the rat lung, either when added to the lung perfusate or when instilled into the airspace. In a subsequent paper, the time course of metabolism and transfer of eicosanoids instilled into the alveolar space was further characterized by Westcott e t al. 24 They confirmed the rapid metabolism of instilled LTC4 to LTD4 to LTE4, but found that LTE4 was further metabolized by the lung to N-acetyl-LTE4. Significantly, a large portion of peptidoleukotrienes instilled into the alveolar space was retained in the air space or lung tissue 2-15 minutes later, while much of instilled PGD2, PGE2, thromboxane B2, LTB4, and 5-HETE were quickly removed from the l u n g . 24 An important concept derived from these studies is that in the isolated lung three distinct compartments can be studied: the air space, the interstitium, and the vascular space. The synthetic and metabolic capabilities for eicosanoids are markedly different for each of these compartments-probably because of the differences in the metabolic pathways of the predominant cell types. Moreover, the ability to transfer from one compartment to another may differ between different eicosanoids. 24 It is therefore not surprising that lung lavage levels of 6-keto-PGFl~ and PAF do not reflect lung tissue levels in rats injured by systemic bacterial endotoxin. ~8 23 T. W. Harper, J. Y. Westcott, N. F. Voelkel, and R. C. Murphy, J. Biol. Chem. 259, 14437 (1984). 24 j. y . Westcott, T. J. McDonnell, and N. F. Voelkel, Am. Rev. Respir. Dis. 139, 80 (1989).
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Localization o f Synthesis~Metabolism
While it is difficult to be certain of the exact cell types involved in a certain metabolic process defined for the intact lung, Haroldsen et al. 25 have used autoradiographic analysis in isolated perfused rat lung to infer the cell types involved in mediator metabolism. They found rapid metabolism of [3H]PAF instilled into the airspace of isolated perfused rat lung. With autoradiography, they localized PAF uptake to the type II cell and Clara cell in the lung. This technique can be useful for localization of eicosanoid metabolism in the intact lung. Concluding Remarks We have described a method for isolated perfused rat lung studies and have given examples in which useful information on eicosanoid action and metabolism have been obtained in this preparation. Similar techniques can be used for lungs from different species of animals. We believe that studies in intact lungs can yield information relevant to the in vivo state and complement results from studies in isolated cells in culture. Acknowledgments This work was supported by NIH Program Project Grants HL-14985 and HL-07171 and Clinical Investigator Award HL-01966 and by the American Lung Association. We thank Marcia Brassor for her excellent secretarial assistance. 25 p. E. Haroldsen, N. F. Voelkel, J. E. Henson, P. M. Henson, and R. C. Murphy, J. Clin. Invest. 79, 1860 (1987).
[66] I s o l a t e d C o r o n a r y - P e r f u s e d M a m m a l i a n H e a r t : Assessment of Eicosanoid and Platelet-Activating Factor Release and Effects By ROBERTO LEVI a n d KEVIN M. MULLANE
Langendorff Heart
The isolated coronary-perfused whole-heart preparation was originally devised by Langendorff. 1In the "Langendorff heart" the coronary vascular bed is perfused by retrograde flow from the aorta in the absence of i O. Langendorff, Pfliigers Arch. Ges. Physiol. 61, 291 (1895).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All fights of reproduction in any form reserved.
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5 min (4°) and the pellets discarded. Acetone (1.5 volumes) is added to the supernatants which contain 12-HETE-1,20-dioic acid. Protein precipitation, acidification, and lipid extraction are carried out as described above. In this instance, 12-HETE-1,20-dioic acid is purified by HPLC on the txBondapak C1s column at 25° with a solvent system consisting of methanol/water/acetic acid (65 : 35 : 0.01, v/v/v), pH 6, at 0.42 ml/min. It is of critical importance to realize that 12-HETE-1,20-dioic acid undergoes spectral alterations if maintained and/or dried from the above acidic eluting solvent. Spectral integrity is best preserved by adding NH4OH to an approximate concentration of 0.4 mM after collection. Drying is then accomplished by rapid, direct flow of argon into the sample tube. The compound is then stored at - 7 0 ° in the dry state under argon. 8 Acknowledgments I would like to acknowledge the research and intellectual collaboration of the following colleagues: Lenore B. Sailer, Harris L. Ullman, Naziba Islam, M. Johan Broekman, M. Teresa Santos, and Juana Valles. I would also like to acknowledge the expertise provided by Ms. Evelyn M. Ludwig with regard to preparation of this manuscript. Research work mentioned in this chapter was supported by grants from the Veterans Administration, National Institutes of Health Grant HL-18828-14 SCOR, the Edward Gruenstein Fund, the Sallie Wichman Fund, and the S.M. Louis Fund.
[65] I s o l a t e d
P e r f u s e d R a t L u n g in A r a c h i d o n a t e S t u d i e s
By SHIn-WEN CHANG and NO~ERT F. VOELKEL
The isolated perfused rat lung preparation has been used for the past 20 years by both physiologists and pharmacologists interested in the circulatory, biochemical, and metabolic aspects of this complex organ. In particular, studies of arachidonate metabolism in isolated perfused rat lungs have yielded important information regarding the significant in vivo metabolic pathways of arachidonic acid metabolites, the action of various eicosanoids in the physiological regulation of the pulmonary circulation. In this section, we will detail the method of isolated lung perfusion currently used in our laboratory. We will also highlight some important results related to arachidonate metabolites obtained using this preparation. METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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General Methodology Lung Isolation and Perfusion
Pathogen-free, young adult Sprague-Dawley rats with body weight from 250 to 350 g are anesthetized with an ip injection of pentobarbital sodium (70 mg/kg). After removal of the overlying skin, a median incision is made in the center of the neck and the trachea exposed by blunt dissection. The trachea is partially transected and a 15-gauge blunt metal cannula is inserted and secured with a 0-silk suture. Following tracheal cannulation, ventilation is initiated with a small animal ventilator using a humidified gas mixture containing 21% 02, 5% CO2, and 74% nitrogen. The ventilator is set at a rate of 55 breaths per minute, using either a pressurelimited ventilator with a maximal inspiratory pressure of 8 cm water, or a volume-limited ventilator with a tidal volume of 2 to 4 ml/breath (approximately I ml/100 g body weight). We do not impose a positive endexpiratory pressure (PEEP) initially to allow partial lung collapse and to minimize the chance of nicking the lung during chest opening and lung removal. The sternum is split at the midline using a blunt-tipped scissors, taking care to avoid the ventilated lungs. The chest is then opened with a rib spreader and 100 units of heparin (in 0.5 ml saline) are immediately injected into the right ventricle to prevent intravascular clotting. Using a small, curved-tip hemostatic forceps, a suture is placed around the outflow tracts of both aorta and pulmonary artery and secured with a loose knot. At this time, it is prudent to be sure that the perfusion circuit is filled with the perfusate solution and that no air bubbles remain in the tubing. A small nick is made in the right ventricular-free wall and the pulmonary artery cannula (mounted at the end of the perfusion tubing) is introduced into the pulmonary artery through the preexisting ligature. The aorta and pulmonary artery are then tightly ligated. Another incision is made at the cardiac apex to allow insertion of a second cannula into the left ventricule to collect effluent perfusate coming from the lungs. The trachea, lungs, and the heart are removed en bloc from the chest cavity and suspended by the tracheal tubing in a constant-temperature, humidified chamber (Fig. 1). Perfusion is begun with a peristaltic Holter pump, starting at a slow rate, and gradually increasing to a final rate of 0.03 ml/g body weight/min. If blood-free perfusion is desired, the initial 50 ml of the perfusate containing residual plasma and blood cells are discarded prior to initiation of recirculation. When blood is used to perfuse the lungs, recirculation of the perfusate can be established from the beginning. Pulmonary artery perfusion pressure is measured (via a side arm of the inflow perfusion tubing) with a Statham P23AA transducer, the signal is amplified, and continu-
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ARACHIDONATE STUDIES; PERFUSED RAT LUNG
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• L • Weight
~
. . . . . . . . . . . . . . . . . . . . .
~
c
Ventilator
!
FIG. 1. Isolated peffused rat lung. The lungs are suspended on a weight transducer within
a humidified chamber, and ventilated with a small animal ventilator. The perfusate is recirculated using a Holter peristaltic pump. PEEP, Positive end-expiratory pressure; PPA, pulmonary arterial pressure.
ously recorded. Since perfusion rate is constant, changes in perfusion pressure reflect changes in pulmonary vascular resistance. At this time, the lungs are hyperinflated to remove any residual atelectasis and PEEP is initiated at 2 to 3 cm water. In a long perfusion protocol, periodic (every 30-40 min) hyperinflation of the lungs may be necessary to prevent atelectasis. Throughout the experiment, the lungs and the perfusate are maintained at 37° to 38° by recirculation of warmed water through waterjacketed chambers around the lungs, the humidification unit, and the perfusate reservoir.
Choice of Perfusate Depending on the experimental design, the lungs can be perfused with whole blood, plasma, or a blood- and plasma-free physiological salt solution (PSS). Initial perfusion pressure averages around 12-16 mmHg with blood perfusate and 5-7 mmHg with PSS, using the ventilation and perfusion parameters outlined above. Blood-perfused lungs closely resemble
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the in vivo state, maintain excellent vascular reactivity, and are often used in the studies of hypoxic pulmonary vasoconstriction. Ether-anesthetized, retired breeder rats are used as blood donors. Approximately 8-10 ml of whole blood may be removed from each donor rat via percutaneous cardiac aspiration. In general, 30 ml of whole blood are sufficient for each perfused lung experiment. However, in studies assessing the vascular action of lipid mediators or measuring lung metabolism of eicosanoids, binding of the mediator or metabolites to plasma proteins and metabolism by blood cells may complicate the interpretation of results. For these studies, perfusion with PSS containing either albumin or Ficoll is recommended. We use a modified Krebs-Henseleit PSS containing: 119 mM NaCI, 4.7 mM KCI, 1.17 mM MgSO4, 19.1 mM HaHCO3, 1.18 mM KH2PO4, 1.6 mM CaC12, and 5.5 mM glucose. Immediately prior to use, the solution is equilibrated with a gas mixture containing 95% 02 and 5% CO2 and Ficoll 70 (a synthetic copolymer of sucrose and epichlorohydrin with a molecular weight of 70,000) is dissolved to 40 g/liter. In experiments where bovine serum albumin is used in place of Ficoll, the PSS is modified to 22.6 mM NaHCO3 and 3.2 mM CaCI2. The use of PSS-Ficoll as perfusate is appropriate for short-term experiments in which mediator binding to albumin is a concern. Lungs effused with albumin-free perfusate (i.e., PSS-FicoI1) are more susceptible to the development of edema, especially with perfusion periods greater than 2-3 hr.
Stability of Preparation The major factors that affect the performance and stability of isolated perfused lung preparations are: (1) health status of the experimental animal, (2) technical expertise and care during lung isolation and removal, (3) perfusate composition, and (4) length ofperfusion time. Viral infections are not infrequent in rat colonies and superimposed bacterial pneumonitis can follow and complicate the viral respiratory tract infection. Lungs with macroscopic consolidation or scarring should be discarded as they develop edema easily and may have elevated baseline levels of eicosanoids. Extreme care must be taken not to traumatize the lung tissue during the isolation procedure. Some degree of flow interruption is unavoidable while the lung is removed from the chest cavity but, with practice, this ischemic period can be as short as 3-4 minutes; continued ventilation during this period of time should prevent any ischemia reperfusion-related problems. Some laboratories have reported rapid development of spontaneous edema in isolated lungs and the question is raised whether isolated lungs are unavoidably injured. In our experience, however, spontaneous edema
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is rare when healthy lung donor rats are used and stable and reproducible lung preparations are consistantly obtained. Moreover, we have recently found comparable values for lung permeability-surface area product between isolated rat lungs perfused with either blood or PSS-albumin and intact rats. i Blood-perfused lungs often maintain excellent vascular reactivity and remain edema free for up to 4 hr of perfusion. Even in lungs perfused with PSS-albumin, significant edema is not observed up to 4 hr of perfusion. 2 Although lungs perfused with PSS-Ficoll are somewhat more susceptible to edema than those perfused with PSS-albumin or blood, they should remain stable for up to 2 hr of perfusion. An important complication is the occasional development of ventricular spasm, which is observed most commonly between 5 and 20 rain after the initiation of lung perfusion and account for most cases of "spontaneous" edema that we have observed. Presumably, postmortem spasm in the heart muscle partially or completely interrupts the flow of effluent perfusate out of the left atrium, resulting in increased pulmonary venous and microvascular pressures. If uncorrected, the lungs rapidly become edematous. When ventricular spasm is suspected, the ventricle should be firmly squeezed with a tweezer and the left ventricular cannula repositioned. This should be followed by a rapid decrease in the pulmonary arterial pressure and a marked decrease in the size of the congested left atrial appendage. This complication can often be prevented by vigorously disrupting the mitral valve apparatus during the insertion of the left ventricular cannula and by a close monitoring of the lung preparation.
Physiological Parameters Mean pulmonary artery pressure is continuously measured and recorded on a strip chart recorder. Left atrial pressure is typically set at 0 cm water but can be varied by simply raising the platform supporting the perfusate reservoir. In some experiments, setting the left atrial pressure at 5 cm water may be desirable to fully recruit the rat lung vasculature. Since perfusion rate and left atrial pressure are fixed, changes in the pulmonary artery pressure reflect changes in total pulmonary vascular resistance. The total vascular resistance can be further partitioned to pre- and postcapillary sites by measuring the pulmonary microvascular pressure (Pmv). This is done by using the double occlusion technique initially described by Dawson and colleagues 3 and recently validated against the traditional t j. Czartolmna, N. F. Voelkel, and S. Chang, F A S E B J . 3, All40. A. B. Fisher, C. Dodia, and J. Linask, Exp. Lung Res. 1, 13 (1980). 3 C. A. Dawson, J. H. Linehan, and D. A. Rickaby, Ann. N. Y. Acad. Sci. 384, 90 (1982).
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gravimetric technique by Townsley et al. 4 The inflow and outflow tubings are simultaneously clamped for a period of 20 sec and the difference between this equilibration pressure and a stop-flow pressure is taken to be Pmv. This pressure averages around 2-2.5 mmHg in PSS-Ficoll perfused lungs and increases to 4.6 -+ 0.4 mm Hg after a bolus injection of LTC4 (2/~g) into the pulmonary artery.5 In experiments investigating mediator effects on lung edema, the isolated lungs can be suspended from a force-displacement transducer for continuous measurement of lung weight. Alternatively, the perfusate reservoir can be placed on a scale and changes in lung weight can be inferred by the loss of perfusate in a recirculating system. At the end of the experimental protocol, the lungs are dissected from the mediastinal tissues, weighed, and the wet lung-to-body weight ratio can be taken as a rough estimate of fluid accumulation. If lung tissues are not used for biochemical analysis, the lungs can be dried to constant weight and the lung wet-to-dry ratio calculated as an index of lung edema. Assessment of lung mechanics and airway reactivity using a heated pneumotachograph and airway pressure transducer in isolated perfused rat lungs have also been reported. 6 Assessment of lung vascular permeability can be done by measuring either capillary filtration coefficient7 or the extravascular leakage of radiolabeled protein. We prefer the later method and use a technique modified from Kern et al.S After an equilibration period of 20 min, during which time the lungs are in an isogravimetric state, approximately 1/zCi of I25I-labeled albumin (specific activity 1.06 mCi/mg; ICN radiochemicals, Irvine, CA; greater than 99.9% binding to albumin) is added to the perfusate and allowed to recirculate for exactly 3 min. After 1 ml of the perfusate is collected, the lungs are perfused with a fresh, nonradioactive perfusate for the additional 3 min without recirculation to remove intravascular I25Ilabeled albumin. The lungs are dissected, weighed, and their radioactivity determined in an auto-y-scintillation spectrometer. The lung permeability-surface area product (PS) can be calculated as follows: Lung PS = total lung 125Iactivity/(I25I activity in 1.0 g of initial perfusate x 3 min) 4 M. I. Townsley, R. J. Korthius, B. Rippe, J. C. Parker, and A. E. Taylor, J. Appl. Physiol. 61, 127 (1986). 5 A. Sakai, S. Chang, and N. F. Voelkel, J. Appl. Physiol., in press (1989). 6 K. B. Nolop, J. H. Newman, J. R. Sheller, and K. C. Brigham, Am. Rev. Respir. Dis. 129, A231 (1984). 7 K. A. Gaar, Jr., A. E. Taylor, L. J. Owens, and A. C. Guyton, Am. J. Physiol. 213, 910 (1967). s D. F. Kern, D. Levitt, and D. Wangensteen, Am. J. Physiol. 245, H229 (1983).
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While lung PS can be affected by capillary recruitment (by altering perfused surface area available for macromolecular exchange), this effect is relatively small and changes in PS of greater than 50% are likely due to changes in vascular permeability.
Advantages and Disadvantages The isolated perfused rat lung allows the studies of an intact, physiologically stable organ preparation in which influences from plasma-borne mediators, hormones, and circulating blood cells can be excluded. The intact pulmonary circulation is under investigation and is capable of vasoreactivity and local regulation, yet the investigator has control over blood flow and vascular pressures. In addition, the effects of varying gas tensions on metabolism and vascular responses are easily studied. All relevant cell types intrinsic to the lung are present and the cell-cell relationships are preserved in their normal state. With care, this preparation remains stable and metabolically active for up to 4 hr. Concerns which limit the use of the isolated perfused rat lung are" (1) long-term studies are not feasible; (2) the lung is composed of a mixture of many cell types, and, therefore, it is difficult to attribute mediator synthesis or metabolism to a specific cell type; finally (3), one needs to take into account the possibility of activation of certain synthetic and metabolic pathways by the lung isolation and perfusion procedures. Use of Isolated Perfused Rat Lung in Arachidonate Studies
Isolated Perfused Rat Lung as Bioassay Organ for Assessment of Eicosanoid Action Infusion of arachidonic acid into the pulmonary artery of isolated perfused rat lungs results in vascular effects attributable to both cyclooxygenase and lipoxygenase metabolites. 9 The acute pulmonary vasoconstrictive effect of arachidonic acid is inhibited by indomethacin and probably due to thromboxane A2. In the presence of indomethacin, arachidonic acid causes a delayed pressure rise and lung edema that can be attributed to the actions of leukotrienes since both can be inhibited by 5-1ipoxygenase inhibitors. Furthermore, injections of individual purified lipoxygenase metabolites showed that LTC4 is the most potent vasoconstrictor in the rat lung, while both LTC4 and LTB4 cause lung edema. 9 9 N. F. Voelkei, K. R. Stenmark, J. T. Reeves, M. M. Mathias, and R. C. Murphy, J. Appl. Physiol. 57, 860 (1984).
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Subsequently, LTE4 was also found to be a pulmonary vasoconstrictor in the rat lung. 10An important concept from this and other studies 9' 1l, 12is the marked attenuation of the vascular activity of eicosanoids in the presence of albumin perfusate, presumably due to binding of the mediators to albumin. Avid binding of these lipid mediators by plasma proteins may serve to localize their vasoactive effects to near sites of biosynthesis. Injection of another lipid mediator, platelet-activating factor (PAF), into isolated rat lung causes pulmonary vasoconstriction and lung edema. This effect is, in a large part, mediated by the production of LTC4 by the lung tissue. 13 We have recently found that injection of both PAF and LTC4 cause significant increase in Pmv, indicating a significant degree of pulmonary venoconstriction. Furthermore, inhibition of the vascular pressure changes using dibutyryl-cAMP completely blocked LTC4-induced lung edema and albumin leak in perfused lungs: Thus, pulmonary venoconstriction is an important mechanism in lipid mediator-induced lung edema. Another important concept in regards to the vasoactivity of lipid mediators in the pulmonary circulation is that the observed response is often dependent on both the dose of the mediator as well as the tone of the pulmonary circulation. Under basal conditions, the pulmonary circulation is nearly maximally vasodilated. Thus, it is difficult to demonstrate vasodilatory responses unless the tone is first increased with vasoconstrictors or exposure to alveolar hypoxia (hypoxic pulmonary vasoconstriction). For example, a low dose (10 ng) of PAF has minimal effect during basal condition but causes vasodilation during hypoxic vasoconstriction; a higher dose of PAF (1/zg) causes vasoconstriction during basal condition but vasodilation followed by vasoconstriction during hypoxic vasoconstriction. Both the vasodilatory and vasoconstrictive actions of PAF are receptor-mediated 14 and can be inhibited by receptor blockers such as CV3988 or WEB 2086. The vasodilatory effect is likely due to the release of endothelium-dependent relaxing factor, while the vasoconstrictive effect may be due to the release of leukotriene C4. 1o C. O. Feddersen, M. Mathias, R. C. Murphy, J. T. Reeves, and N. F. Voelkel, Prostaglandins 26, 869 (1983). 11 V. J. Iacopino, S. Compton, T. Fitzpatrick, P. Rarnwell, J. Rose, and P. Kott, J. Pharmacol. Exp. Ther. 229, 654 (1984). 12 T. C. Noonan, W. M. Selig, K. E. Burhop, C. A. Burgess, and A. B. Malik, J. Appl. Physiol. 64, 1989 (1988). 13 N. F. Voelkel, S. Worthen, J. T. Reeves, P. M. Henson, and R. C. Murphy, Science 218, 286 (1982). 14 N. F. Voelkel, S. Chang, K. Pfeffer, S. G. Worthen, I. F. McMurtry, and P. M. Henson, Prostaglandins 32, 359 (1986).
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Injection of arachidonic acid during hypoxic vasoconstriction in PSSFicoll perfused rat lungs also causes a biphasic response with acute, transient vasoconstriction followed by a prolonged vasodilatory effect. In the presence of cyclooxygenase inhibition, only the acute vasodilation is evident.15 Recent results from our laboratory suggest that this cyclooxygenase-independent pulmonary vasodilatory effect of arachidonic acid is, in part, mediated by cytochrome P-450-dependent metabolites.
Role of Eicosanoids in Pulmonary Vasoregulation Cyclooxygenase metabolites are generated by respiratory movement ~6 as well as during blood flow alterations. 17Inhibition of the cyclooxygenase pathway enhances and preserves vascular reactivity in isolated rat lungs, suggesting that endogenous vasodilatory prostaglandins such as PGI2 modulate vascular responses in this preparation. This activation of vasodilatory prostaglandins is markedly amplified during lung infection or vascular injury and studies using diverse experimental preparations have suggested an important role for PGI2 in the depression of hypoxic pulmonary vasoconstriction observed in these settings. 18 Thromboxane A2, a potent vasoconstrictor, is also released following activation of the cyclooxygenase pathway. In species such as guinea pig and sheep, large amounts of thromboxane A2 are released in response to injury and play an important role in the observed pulmonary hypertension. In the rat lung, however, the amount of thromboxane A2 synthesized is modest compared with PGI2 and thromboxane A2 plays a minor role in mediating the vascular response to injury. 5-Lipoxygenase metabolites are clearly activated during acute lung injury. LTB4 is a potent chemoattractant for neutrophils, but whether it has a direct effect on vascular permeability is unclear. 12 L T C 4 and other peptidoleukotrienes are markedly increased in lung tissue after endotoxin-, a-staphylotoxin-, and protamine-induced lung injury. At least in the latter two injury models, the production of L T C 4 appears to promote lung edema by enhancing pulmonary venoconstriction.19 Whether L T C 4 plays a role in the physiological regulation of the normal pulmonary circulation is less clear. Although earlier studies from this laboratory suggested 15 C. O. Feddersen, S. Chang, J. Czartoloma, and N. F. Voelkel, J. Appl. Physiol., submitted. 16 R. Korbut, J. Boyd, and T. Eling, Prostaglandins 21, 491 (1981). 17 A. van Grondelle, G. S. Worthen, D. Ellis, M. M. Mathias, R. C. Murphy, R. J. Strife, J. T. Reeves, and N. F. Voelkel, J. Appl. Physiol. 57, 388 (1984). 18 S. Chang and N. F. Voelkel, in "Eicosanoids in Cardiovascular and Renal Systems" (P. V. Halushka and D. E. Mais, eds.), p. 62. MTP Press, Lancaster, 1988. ~9 S. Chang, J. Czartolomna, and N. F. Voelkel, unpublished observations (1989).
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a potential role for leukotrienes as mediator of hypoxic pulmonary vasoconstriction, 2° recent results have not supported this hypothesis. 21 There are currently no experimental data to support or refute a vasoregulatory role for other arachidonic acid metabolites, such as lipoxins and epoxyeicosatrienoic acids, in the normal pulmonary circulation. Eicosanoid Metabolism in Isolated Perfused Rat Lung
The lung is a metabolic organ and is capable of synthesis as well as metabolism of a large number of eicosanoids. Using the isolated perfused lung technique, Westcott et al. 22 observed marked species dependence in lung eicosanoid synthesis. When the perfused rat lung is treated with the calcium ionophore A23187, a large amount of PGI2 (as 6-keto-PGFl~), LTB4, and L T C 4 and lesser amounts of thromboxane A2 (as TxB2), PGE2, and LTD4 are synthesized. 22 Significant amounts of 6-keto-PGFl~ TxB2 and LTB4 are released into the lung perfusate. In guinea pig lungs, the cyclooxygenase metabolites, especially thromboxane A2, is the predominant eicosanoid product after A23187 stimulation, and very little 5-1ipoxygenase metabolites can be measured. In the ferret lung, the lipoxygenase products, especially LTB4, are prominent. In rabbit and hamster lungs, roughly equal amounts of cyclooxygenase and lipoxygenase products are synthesized, after A23187 stimulation. 22 Physiological factors that are important in stimulation of lung eicosanoid synthesis include respiratory motion, 16 and alterations in the hydrodynamic forces in the circulation.I7 Indeed, we find that the "baseline" levels of eicosanoids in isolated perfused lungs are generally severalfolds higher than those in lungs removed from anesthetized rats and immediately homogenized in methanol. This is particularly true for the PGI2 metabolite 6-keto-PGFi~ which averages around 15 ng/g wet lung in nonperfused lungs and may reach 340 ng/g in lungs perfused with PSS-Ficoll for 1 hr (Table I). Thus, PGI2 synthesis appears to be stimulated in the perfused lungs and likely contributes to the impaired vasoreactivity observed after prolonged perfusion. It is not surprising that cyclooxygenase inhibitors enhance and prolong vascular reactivity in isolated perfused rat lung. 20 M. L. Morganroth, J. T. Reeves, R. C. Murphy, and N. F. Voelkel, J. Appl. Physiol. 56, 1340 (1984). Zl A. J. Lorigro, R. S. Sprague, A. H. Stephenson, and T. E. Dahnms, J. Appl. Physiol. 64, 2538 (1988). 22 j. y . Westcott, T. J. McDonnell, P. Bostwick, and N. F. Voelkel, Am. Rev. Respir. Dis. 138, 895 (1988).
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TABLE I COMPARISON OF LUNG TISSUE EICOSANOID LEVELS IN CONTROL RATS BEFORE AND AFTER LUNG PEP,FUSION
Lung tissue
Thromboxane B2" (ng/g)
6-keto-PGF~ (ng/g)
LTC4 (ng/g)
Unperfusedb (11)d Peffusedc (6)d
6 -+ 1 15 -+ 3
15 -+ 4 338 -+ 55
1.5 -+ 0.3 5.0 -+ 1.2
a Data are mean + SEM and expressed as nanogram per gram wet lung weight. b Lung are removed from anesthetized rats and immediately homogenized in methanol. Thrombo×ane B2 and 6-keto-PGF~ are measured by enzyme-linked immunoassay. LTC4 is measured by enzyme-linked immunoassay after HPLC purification. Methods are as described by J. Y. Westcott et al. [Prostaglandins 32, 857 (1986)]. c Lungs are isolated and perfused in a physiological salt solution containing Ficoll for 60 min prior to lung homogenization and eicosanoid measurements. a Number of experiments.
Harper e t al. 23 have also used isolated perfused rat lung to study the metabolism of LTB4 and LTC4 and found that LTC4 is rapidly metabolized to LTC4 sulfoxide, LTD4, and LTE4. In contrast, LTB4 appears not to be significantly metabolized by the rat lung, either when added to the lung perfusate or when instilled into the airspace. In a subsequent paper, the time course of metabolism and transfer of eicosanoids instilled into the alveolar space was further characterized by Westcott e t al. 24 They confirmed the rapid metabolism of instilled LTC4 to LTD4 to LTE4, but found that LTE4 was further metabolized by the lung to N-acetyl-LTE4. Significantly, a large portion of peptidoleukotrienes instilled into the alveolar space was retained in the air space or lung tissue 2-15 minutes later, while much of instilled PGD2, PGE2, thromboxane B2, LTB4, and 5-HETE were quickly removed from the l u n g . 24 An important concept derived from these studies is that in the isolated lung three distinct compartments can be studied: the air space, the interstitium, and the vascular space. The synthetic and metabolic capabilities for eicosanoids are markedly different for each of these compartments-probably because of the differences in the metabolic pathways of the predominant cell types. Moreover, the ability to transfer from one compartment to another may differ between different eicosanoids. 24 It is therefore not surprising that lung lavage levels of 6-keto-PGFl~ and PAF do not reflect lung tissue levels in rats injured by systemic bacterial endotoxin. ~8 23 T. W. Harper, J. Y. Westcott, N. F. Voelkel, and R. C. Murphy, J. Biol. Chem. 259, 14437 (1984). 24 j. y . Westcott, T. J. McDonnell, and N. F. Voelkel, Am. Rev. Respir. Dis. 139, 80 (1989).
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Localization o f Synthesis~Metabolism
While it is difficult to be certain of the exact cell types involved in a certain metabolic process defined for the intact lung, Haroldsen et al. 25 have used autoradiographic analysis in isolated perfused rat lung to infer the cell types involved in mediator metabolism. They found rapid metabolism of [3H]PAF instilled into the airspace of isolated perfused rat lung. With autoradiography, they localized PAF uptake to the type II cell and Clara cell in the lung. This technique can be useful for localization of eicosanoid metabolism in the intact lung. Concluding Remarks We have described a method for isolated perfused rat lung studies and have given examples in which useful information on eicosanoid action and metabolism have been obtained in this preparation. Similar techniques can be used for lungs from different species of animals. We believe that studies in intact lungs can yield information relevant to the in vivo state and complement results from studies in isolated cells in culture. Acknowledgments This work was supported by NIH Program Project Grants HL-14985 and HL-07171 and Clinical Investigator Award HL-01966 and by the American Lung Association. We thank Marcia Brassor for her excellent secretarial assistance. 25 p. E. Haroldsen, N. F. Voelkel, J. E. Henson, P. M. Henson, and R. C. Murphy, J. Clin. Invest. 79, 1860 (1987).
[66] I s o l a t e d C o r o n a r y - P e r f u s e d M a m m a l i a n H e a r t : Assessment of Eicosanoid and Platelet-Activating Factor Release and Effects By ROBERTO LEVI a n d KEVIN M. MULLANE
Langendorff Heart
The isolated coronary-perfused whole-heart preparation was originally devised by Langendorff. 1In the "Langendorff heart" the coronary vascular bed is perfused by retrograde flow from the aorta in the absence of i O. Langendorff, Pfliigers Arch. Ges. Physiol. 61, 291 (1895).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All fights of reproduction in any form reserved.
