J Mol Cell Cardio122, 555-563 (1990)
Oxygen Free Radical Producing Leukocytes Cause Functional Depression of Isolated Rat Hearts: Role of Leukotrienes A. G. Semb, J. Vaage’ and 0. D. Mjmrs Department of Physiology and Surgery’,
University of Tromse, Tronsa, Norway
(Received 29 June, accepted in revisedform 21 December,i989) A. G. SEMB, J. VAACE AND 0. D. MJEB. Oxygen Free Radical Producing Leukocytes Cause Functional Depressionof Isolated Rat Hearts: Role of Leukotrienes. Journal of Molecular and Cellular Cardiology (1990) 22, 555-563. Polymorphonuclear granulocytes (PMN) are suggested mediators of myocardial ischemia-reperfusion injury. We have previously shown that activated PMN producing oxygen free radicals (OFR) in the coronary circulation are cardiodepressive. OFR may induce lipid peroxidation and production of eicosanoids. We have investigated the influence of cycle-oxygenase and lipoxygenase inhibitors on the effects of activated, OFR producing PMN in the LangedortIrat heart model. Left ventricular developed pressure (LVDP) was measured by a balloon in the left ventricle. Human PMN and drugs were given into the aortic cannula for 10 min and the hearts were observed for 30 min thereafter. After infusion for 5 min OFR production in the cellular infusate was measured at the level of the aortic cannula by a chemiluminescence (CL) technique. Phorbol l2-myristate 13acetate (PMA)-activated PMN (n = 8), produced a CL response of27649 &- 11048 counts (mean + S.E.M.), and reduced coronary flow (CF) to 53 f 6% (mean + s.E.M.) and LVDP to 38 + 9% of baseline values at the end of the observation period. Ibuprofen (n = 6), a cyclooxygenase (CO) inhibitor, neither influenced the CL response (31915 k 7563) of activated PMN, nor the reduction of CF and LVDP at this time. Although both BW 755C (n = 7), a dual inhibitor of CO and lipoxygenase (LO) (CF:90 f 4%, LVDP:99 + 6%) and diethylcarbamazine (DCM) (n = 8), a LO inhibitor (CF:88 k ll%, LVDP:87 + 4%), significantly inhibited the cardiodepressive effects of activated PMN. BW 755C alone abolished the CL response (431 k 158 counts), whereas DCM had no effect on CL (30105 f. 1698 counts). In conclusion, the injurious effects of activated leukocytes in the coronary circulation seem to be mediated through LO products. KEY WORDS: Leukocytes; Oxygen free radicals; Leukotrienes; Ibuprofen; Dirthylcarbamazine; Myocardial injury; Myocardial depression; Rat heart.
Introduction Infiltration of leukocytes irzto infarcted myocardium has long been recognized [4, 421, where they take part in an inflammatory reaction [ff] and their function has been thought to “clear up” irreversibly damaged tissue [.%I. The presence of leukocytes in the myocardium has been thought to be a result of injury, rather than a cause of it. But recent studies indicate that leukocyte activation may occur within 15-20 min of coronary occlusion [43], that is before myocardial necrosis, which occurs after 30 min of ischemia [33]. Reperfusion accelerates myocardial trapping of polymorphonuclear granulocytes (PMN) [28, 401. Leukocytes may theoretically be cytotoxic and contribute to an inflammatory reaction during reperfusion of ischemic myocardium even before extravasation. This concept is based on
Eicosanoids;
BW
755C;
two different observations. Various pharmacologic modulations of neutrophil function attenuate myocardial ischemia-reperfusion injury [S, 27, 341. Additionally, reperfusion of ischemic myocardium with leukopenic blood has been shown to reduce infarct size [351, increase collateral flow, reduce myocardial edema and ventricular arrhythmias, as well as to inhibit the no-reflow phenomenon [7, 8, 91. The mechanism by which intravascular granulocytes cause injury to the reperfused myocardium is incompletely understood. Activated granulocytes produce and release hydrolytic enzymes [19], eicosanoids [32] and reactive oxygen intermediates, often termed oxygen free radicals (OFR) (superoxide, hydrogen peroxide, hydroxyl radical, singlet oxygen) [47J, and injure eudothelial cells [36, 371. Particular attention has been focused on
Please address all correspondence to: A. G. Semb, Binnevn. 6B, N-0387 Oslo 3, Norway. 0022-2828/90/050555 + 09 $03.00/O
0 1990 Academic Press Limited
A. G. Semb
556
the participation of OFR in reperfusion injury of ischemic myocardium [25,22] and we have shown that intracoronary infusion of activated leukocytes, producing OFR, depressed the function of isolated rat hearts [.%I. OFR are also able to activate phospholipases [4s] with subsequent production of cycle-oxygenase (CO) and lipoxygenase (LO) metabolites of arachidonic acid [31]. In the present investigation we studied the possible role of eicosanoids in the OFRmediated effects of activated leukocytes in the isoIated rat heart. This was done by evaluating the effects of CO and LO inhibition.
Materials
and Methods
Heart preparation
Male-Wistar rats (250-300 g) were anesthetized with diethyl ether. Heparin (200 IU) was injected into the femoral vein. Through a median sternotomy the hearts were rapidly excised and retrogradely perfused via the ascending aorta (Langendorff model) with Krebs-Henseleit solution (37°C) at constant, non-pulsatile perfusion pressure (80 mmHg) . Coronary flow was measured by timed collections of the efBuent. Left ventricular developed pressure (LVDP) and left ventricular end diastolic pressure (LVEDP) were measured by a latex balloon in the left ventricle, connected to a pressure transducer (Gould P23 ID). Heart rate was counted from the pressure curve. Leukocyte suspension
Human leukocytes were separated from freshly drawn, heparinized blood by a simple dextran sedimentation method. The cell suspension was evaluated by differential smear and found to contain l-3% lymphocytes (range), 82-86% polymorphonuclear granulocytes (PMN), and 12-16% monocytes. Since the majority of the cells (both PMN and monocytes) produce OFR and the PMN dominate, the suspension was called PMN for simplicity. The cell suspension was diluted with gassed (95% 02 + 5% COZ) Krebs-Henseleit solution at 37°C and pH 7.4 to obtain a concentration of 3 x IO6 cells/ml. It was this diluted suspension which was infused.
et al. Drugs and chemicals
In order to activate PMN, phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO., USA) was mixed with the cells at a ratio of 3 x 1O6 cells/ml : 10 ng PMA. Ibuprofen (IBU), a cycle-oxygenase inhibitor (Apothekernes Laboratorium, Oslo, Norway), was dissolved in NazCOs, pH adjusted to 7.58 with 1 N HCl. BW 755C (British Wellcome, Wellcome Foundation Ltd., Kent, UK) a dual inhibitor of cycle-oxygenase and lipoxygenase, was dissolved in distilled water. The lipoxygenase inhibitor diethylcarbamazine (DCM) (Sigma, St. Louis, MO., USA) was dissolved in phosphate-buffered saline. Each drug was added to the cell suspension at the same time as PMA. Chemiluminescence (CL)
A modified lumanol dependent CL technique [Z] was used to verify the generation of OFR by activated PMN over time, so the infusion of the activated cell suspension could be done when the leukocytes produced an abundance of OFR. Dose response curves were studied in vitro with PMN + PMA and with various doses of each of the three different drugs used. IBU (7.6 x lo-’ M, 7.6 x 10m4 M and 7.6 x 10-3M), DCM (2.5 x lO-3 M, 5 x lO-3 M, 1 x IO-’ M and 2 x 10e2 M) and BW 755C (2 x 1o-8 M 2 X lo-7 M, 2 X 1o-6 M, 2 x IO-’ M and 2 x low4 M) were added to
3 x 10e6 cells CL was performed on cells and drugs in vitro for 1 h. In the dose response studies, each series of experiments was started and ended with PMN + PMA to check the reliability of the system. OFR production was also measured in samples of the cell suspension taken from the aortic root after 5 min of infusion. For measurements of CL, samples of the cell suspension (200 ~1) were transferred to a cuvette in a photon counter (Biocounter, Model 2010, Lumac/SM, Schaesberg, The Netherlands). Lumanol (100 ~1, 10e4 M) was added automatically to initiate the CL. Experimental protocol
The hearts were stabilized for 10 min before the cell suspension was infused for 10 min at a
Cardiac
Injury
rate of 1 ml/min into the aortic cannula. The total amount of infused cells was 3 x 106. The cells were incubated with PMA for 10 min before the start of the infusion. The concentration of PMN in the coronary circulation was approximately 0.3 x lO’/ml. After the end of the infusion the hearts were observed for 30 min, (total observation time: 40 min after the start of the infusion). A total of 3 x lo6 leukocytes were infused into the coronary circulation. The following groups were studied: Group I (n = 8): PMN + PMA. Group II (n = 6): IBU was added to the PMA activated PMN at a concentration of 7.4 x low4 M. Group III ‘,“~~7); PMN + PMA+BW 755C (2 M m the cell suspension). Group IV (a = 8): PMN + PMA + DCM (2.5 x lo--’ M in the cell suspension). Group V (n = 6): IBU was dissolved in gassed Krebs-Henseleit and infused into the aortic cannula to obtain the same concentration as in Group II. Group VI (n = 6): BW 755C was infused into the coronary circulation to obtain the same concentration as in Group III. Group VII (n = 7): DCM was infused at the same concentration as in Group IV.
557
by Leukocytes
Time
(mini
FIGURE 1. Chemiiuminescence response of phorbol 12-my&ate 13-acetate (PMA) stimulated leukocytes (PMN) l --~ 0, and unstimulated PMN 0 - -- 0.
The CL responses of cell samples taken from the aortic root in Group I-IV after 5 min of infusion are shown in Table 1. CoronaryJIow (CF)
Coronary flow was 8-15 ml/min (range) during the period of stabilization. Infusion of PMA activated PMN reduced CF to 58 f 12% and 49 f 19% of the preinfusion value after 5 and 10 min respectively (Fig. 3).
Statistical analysis
Differences between groups were analysed by one way analysis of variance. Statistical comparisons were made using Qtest [48]. Differences were considered significant when P < 0.05. All results are expressed as mean + S.E.M.
t
140-
.
120-
.
E : IOO-
.
.
“0.
Results Cheniluminescence
A representative in vitro CL response of in vitro PMA activated leukocytes is shown in Fig. 1. Between 10 and 20 min incubation the production of OFR was high. Therefore we have period of chosen a 10 min incubation PMA + PMN before the cell suspension was infused into the isolated rat hearts. BW 7556 (Fig. 2) showed a dose-dependent inhibition of OFR production as measured by the CL response (Fig. \ - 2,, P < 0.0001, r = -0.86 in a 1 two-tailed test), whereas no significant inhibition of CL was found by IBU and DCM in the doses studied.
E x ’ .5
60
E
c
g 4O
5
2{i
1 2.10-4*
2.10.5M
? 2.10‘% [BW755c3
y 2.10.‘M
-I
2.10
FIGURE 2. Effect ofdifferent doses of BW 755C on the chemiluminescence response of phorbol 12-my&ate 13acetate stimulated leukocytes (0) after 15 min incubation at 37”C, and unstimulated leukocytes with BW r = -0.86. 755C (0 ) Correlation coefficient, P < 0.0001.
A.G.Sembctal.
558 TABLE
1. Chemiluminescence
of cells in the coronary
Groups I.
II. III. IV.
Counts
PMN PMN PMN PMN
+ + + +
PMA PMA PMA PMA
+ IBU + BW 755C + DCM
(n = 6) (n = 6) (n = 5) (n = 8)
infustate
(mean
f
27649 31915 431 30105
s.E.M.)
+ f + f
4510 3088 71 * 600
Samples were drawn from the aorta after 5 min of infusion of phorbol 12myristate 13-acetate activated cells (PMN + PMA) alone, and with the addition ofibuprofen (IBU), BW 755C or diethylcarbamazine (DCM). *P < 0.01 compared to PMN + PMA.
No recovery was observed 30 min after the end of infusion (56 f 6%). Although CF in Group II was higher than in Group I throughout the observation period, the effect of IBU was not significant at any time (88 + 14%, 55 + 15% and 66 f 11 at 5, 10 and 40 min respectively) (Fig. 3). However, BW 755C (Group III) significantly inhibited the decrease in CF induced by activated PMN (94 + 6%; 96 + 6% and 90 +_4% at 5, 10 and 40 min) (Fig. 3). DCM had an effect similar to that of BW 755C (94 f: 4%, 94 f 5% and 88 f 11y. at 5, 10 and 40 min) (Fig. 3).
PMN+PMA
I
I
I
0
5
IO
I
40
FIGURE 3. Coronary flow ofisolated rat hearts during and after infusion of leukocytes (PMN) stimulated by phorbol IL?-myristate 13-acetate (PMA) for 10 min, and the effect of ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM). *P < 0.01 compared to PMN + PMA. All values are mean + S.E.M.
Infusion of BW 755C or DCM alone for 10 min (Fig. 4) did not significantly influence CF. IBU significantly increased CF (130 f 2% and 129 + 5% after 5 and 10 min respectively). Left ventricular developed pressure
The left ventricular developed pressure was 90-150 mmHg during stabilization. Infusion of activated PMN decreased LVDP to 42 + 10% and 32 + 8% of the baseline value after 5 and 10 min respectively (Fig. 5). No recovery was observed 30 min after the end of infusion (38 + 9%). IBU attenuated this re-
I501
5ol
-. Min
FIGURE 4. The effect ofa 10 min infusion of ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM) on coronary flow in isolated, perfused rat hearts. *P < 0.01 compared to BW 755C and DCM. All values are mean * s.a.u.
Cardiac
Injury
559
by Leukocytes
aa z 73 b 3 75.; : 5
50-
, 0
I 40
, , 5 IO Min
3
Min
FIGURE 5. The effect of a 10 min infusion of leukocytes (PMN) stimulated with phorbol 12-myristate 13acetate (PMA) on left ventricular developed pressure, and with the addition of ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM) in isolated rat hearts. *P < 0.01 compared to PMN + PMA. All values are mean + s.E.M.
FIGURE 6. The effect ofinfusing ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM) for 10 min on left ventricular developed pressure (LVDP) in isolated, perfused rat hearts. *P < 0.01 compared to baselinr. illI values are mean + S.E.M.
Neither BW 755C nor DCM when given alone influenced LVDP whereas IBU initially increased LVDP (Fig. 6).
Left ventricular end diastolic pressure duction in LVDP (Fig. 5), but the difference was not significant at any time (71 + 12%, 49 + 15% and 62 + 8% at 5, 10 and 40 min). However, BW 755C significantly inhibited the decrease in LVDP (85 f 6%, 88 + 6% and 99 f 6% at 5, 10 and 40 min) (Fig. 5). DCM the decrease in LVDP also inhibited (95 f 5%, 91 + 5% and 87 f 4% at 5, 10 and 40 min) (Fig. 5).
TABLE 2.
No changes in LVEDP were observed group during the observation period.
Heart rate Baseline heart rate was 26&360 beats/min. Although all groups (I-VI) showed a tendency towards a decrease in heart rate, no or difference between significant change groups were observed (Table 21,
Heart rate Time after start of infusion:
Groups I. II.
III. IV. V. VI. VII.
in any
PMN + PMA PMN + PMA + IBU PMN + PMA + BW 755C PMN + PMA + DCM IBU BW 755C DCM
____-40 min
5 min
10 min
(n = 8) (n = 6) (n = 7)
100 f 5 86f 10 93 -t 3
95 + 5 77 * 1 92 f 3
89+ 10 88 f. 6 94 * 5
(n = 8)
94 f 2
92 f 3
95 f 3
(II = 6)
79f 14 94 + 3 95 * 3
76+15
93 f 3 98 + 3
86 * 7 93 * 3 102 f 5
Intrinsic, unpaced heart rate in a rat LangendorKmodeI during and after addition of Phorbol 12-acetate 13-myristatc activated cells (PMA + PMN) alone, or with the addition of ibuprofen (IBU), BW 755C or diethylcarmbamazine (DCM). The effect of the drugs given alone is also shown. All values are in percent of baseline values (mean + s.E.M.).
560
A.G.Sembetal. Discussion
Infusion of activated PMN into the coronary circulation may represent an enhancement of the initial effects of the early and immediate intravascular trapping and activation of granulocytes in the reperfused ischemic myocardium. Consequently, this model may be useful to elucidate the early cardiac effects of activated leukocytes. Unless the delayed inflammatory response by leukocytes is qualitatively different, it may also shed some light on the general mechanisms whereby leukocytes are able to injure myocardial cells, affect the coronary circulation, and depress cardiac function. Infusion of unactivated PMN into the coronary circulation of isolated rat heart did not affect CF, LVDP or heart rate, while activated PMN severely impaired heart function [39]. We have previously shown that scavengers of OFR inhibited the cardiodepressive effects of PMN activated by PMA, suggesting an important role of OFR as mediators [39]. This observation, however, does not exclude the possibility that other mediators are involved, either as prerequisite for OFR production, or as secondary mediators produced or released by the action of OFR. Reactive oxygen species induce lipid peroxidation. Recent studies suggest that freeradical triggered peroxidation of phospholipids renders them more susceptible to the action of phospholipases A or C [4sJ, and may initiate the arachidonic acid cascade [28].
Cyclo-oxygenase
inhibition
The doses of IBU used did not influence the CL response of activated PMN, although the concentration of IBU used (7.6 x 10m3 M) was similar to the plasma concentration in studies where IBU has been shown to reduce release from infarct size, reduce enzyme ischemic myocardium and diminish leukocyte accumulation in ischemic myocardium [1, 26, 171. That we did not observe an inhibition of OFR production as Flynn and coworkers did [IJI, might be due to differences in the PMNstimulating agents used, the methods for measuring OFR, or species differences. Theoretically, CO products might be involved in the cardiac effects of stimulated PMN
producing OFR. Rowe et al. [36] suggested that hydroxyl radical generation by the CO pathway was partly responsible for the in vitro depression of cardiac sarcoplasmic reticulum by PMA activated leukocytes. The various CO products have both inotropic and chronotropic effects and may also modulate coronary vascular tone [18, 291. This picture is complicated by species differences and antagonistic actions of the various CO products. For instance, thromboxane A2 has been suggested as a mediator of ischemic myocardial damage [14], whereas prostacyclin may have cardioprotective actions [3]. Th e present study shows that CO products are not important for the cardiodepressive effects of PMA activated PMN. In fact, the tendency towards decreasing inhibition of CF and LVDP by IBU might well be secondary to a direct effect of IBU which increased both CF and LVDP when infused alone. However, a possible beneficial effect of IBU might partly be counteracted by the enhancement of leukotriene production by CO inhibition [453.
Lipoxygenase
inhibition
Four structurally dissimilar LO inhibitors are able to reduce myocardial infarct size [12, 25, 26,301. In the present study, 2 structurally and pharmacologically different LO inhibitors abolished all the cardiodepressive effects of PMA activated PMN. BW 755C is’ a dual inhibitor of both CO and LO, as well as being an antioxidant. The concentration of BW 755C used in the isolated heart model was similar to those used in in vivo studies [26, 271. DCM is an antifilarial agent and a specific inhibitor of the enzymatic (5-lipoxygenase) conversion of 5-hydroperoxyeicosatetranoic acid (5-HPETE) into leukotriene A4 [21]. None of the doses of DCM mixed with the activated cell suspension reduced the CL response. Mathews et al. [.?I] showed that 250 PM DCM inhibited 90% of the production of LTB4 and LTC4 in calcium ionophore stimulated mastocytoma cells. Consequently this concentration of DCM was therefore added to the cell suspension infused into the coronary circulation. BW 755C either inhibited OFR production or acted as a scavenger of OFR in the present study, since it abolished the CL response by
Cardiac
Injury
561
by Leukocytes
LTD4 . LTA,
t LTC4
Endothelium
FIGURE 7. Proposed mechanism of interaction between stimulated granulocytes (PMN) and endothelium. Activated PMN release superoxide (0;) which may form H202 through a dismutation reaction. HsOs may react with myeloperoxidase (MPO) or lactoferrin (LF). From the latter reaction the oxidative and lipidsoluble hydroxyl radical (OH-) is formed, which may result in the activation of the arachidonic acid (AA) metabolism through the action of phospholipase As (PLAs) on phosphatidyl choline (PC), with the production of leukotriene & (LTA.+). Alternatively, endothelial cells may produce LTC4 and LTD4 from LTA4 released from activated PMN. LTC4 and LTD., cause vasoconstriction and decrease myocardial contractile power.
PMA activated PMN. DCM did not affect the CL response of PMA + PMN, and yet it gave equipotent inhibition of the cardiac effects. Thus, the beneficial effects of DCM and BW 7556 seem to be caused by their inhibition of the production of leukotrienes from arachidonic acid by inhibition of 5-lipoxygenase. Shak and coworkers [41] showed that 0.5 pg/ml PMA: IO6 PMN did not stimulate LTB4 production. We used a lower concentration of PMA (0.01 pg/ml: lo6 PMN), and it is not likely that LTB4 is formed by the cells in our model. But the peptidoleukotrienes LTC4 and LTD4 might be produced by endothelial cells through the direct action of OFR on the endothelial membranes as suggested in Fig. 7, or from the PMN-derived LTA4. This is in accordance with the suggestion of Feinmark et al. [II], that neutrophil derived LTA4 can be transformed to LTC4 by endothelial cells after donation of the LTA4 from the PMN. Since LTC4 and LTD4 are able to increase vascular permeability, induce coronary vasoconstriction and have negative inotropic effects [.5,20, 23, 24,30), they may be the final mediators of the cardiac depression induced by activated PMN. However, the direct evidence for this hypothesis can be strengthened by using specific peptide Ieukotriene receptor antagonists
and/or measurements of LTC4/LTD4 coronary effluent.
in the
Concept From our previous [39] and present findings we propose that OFR production is the primary event in the injury mechanism of activated PMN. Through peroxidation of cell membranes the arachidonic cascade is triggered. LO products are then released and may exacerbate leukocyte activation, induce coronary vasoconstriction, and impair cardiac thus serving as secondary contractility, mediators. It might be that the cardiac effects of OFR are mostly through their ability to trigger the LO cascade. Acknowledgements
A. G. Semb is a research fellow of the Norwegian Research Council for Science and the Humanities. Further support has been received from the Norwegian Council on Cardiovascular Disease, Thomas Fearnley Bidrags- og Gave Fond, Dagny og Kare Holts Legat, and the University of Tromsra, all of which is greatly acknowledged.
562
A. G. Scab
et al.
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PARKER, C. W. Lipid mediators produced through the lipoxygenase pathway. Ann Rev Immunol5,65-84 (19871. R. B. The wavefront phenomenon of ischemic cell death. I. Myocardial infarct vs REIMER, K. A., JENNINGS, duration of coronary occlusion. Circulation 56, 786794 (1977). ROMSON, J. L., JOLLY, S. R., LUCCHESI,B. R. Protection ofischemic myocardium by pharmacologic manipulation of leukocyte function. Cardiovasc Rev Rep 5, 690-709 (1984). ROMSON,R. L., KUNKEL, S. L., ABRAMS, G. D., SHORK, M. A., Lucn~ssx, B. R. Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation 67, 1016-1023 (1983). ROWE, G. T., MANSON, N. H., CAPLAN, M., HESS,M. L. Hydrogen peroxide and hydroxyl radical mediation 01 activated leukocyte depression of cardiac sarcolplasmatic reticulum. Circ Res 53, 584591 (1983). SACKS, T., MOLDOW, C. F., CRADDOCK, P. R., BOWERS, T. K., JACOB, H. S. Oxygen radicals mediate cndothrlial cell damage by complement-stimulated granulocytes. J Clin Invest 61, 1161-l 167 (1978). SCHMID-SCH~NBEIN,G. W., ENGLER, R. L. Granulocytes are active participants in acute myocardial ischemia and infarction. Am J Cardiovasc Path01 1, 15-30 (1986). SEMB, A. G., YTREHUS, K., VAAGE, J., MYKLEBUST, R., MJBS, 0. D. Functional impairment in isolated rat hearts induced by activated leukocytes: protective effect ofoxygen free radical scavengers. J Mol Cell Cardiol21,8777887 (1989). SEMB,A. G., VAAGE, J., LIE, M., SBRLIE, D., MJ~s, 0. D. Cardiac leukocyte sequestration and chemiluminescencr of leukocytes during cardiopulmonary bypass in man. Microcirculationm-an update, vol 1. Elscvier Srirncc Publishers B.V. M. Tsuchiya er al. (Eds), pp. 721-722 (1987). SHAK, S., GOLDSTEIN, I. M. The major pathway for leukotriene B, catabolism in human polymorphonuclear leukocytes involves omega-oxidation by a cytochrome P-450 enzyme. In: Prostaglandins. leukotrienes. and 1~poxin~. Bailey, J. M. (Ed.) New York, Plenum Press, pp. 97-l 10 (1985). SOMMERS, H. M., JENNINGS, R. B. Experimental acute myocardial infarction. Histologic and histochemiral studies of early myocardial infarcts induced by temporary or permanent occlusion of a coronary artery. Lab Invest 13, 1491ll503 (1964). TILLMANS, H., LEINBERGER, H., NEUMAN, N., ZIMMERMAN, R. Early microcirculatory changes during brief ischemia--primary events in ischemic myocardial injury? Circulation 70 [Suppl II], 88 (1984). VAAGE, J.. SEMB,A. G., Myocardial reperfusion injury: an inflammatory reaction? Biomed Biochim Acta 1989 [in press). WARD, P. A., SULAVIK, M. C., JOHNSON, K. J. Rat neutrophil activation and effects of lipoxygrnase and cyclooxygenase inhibitors. Am J Physiol 116, 223-233 (1984). WEGLICKI, W. B., Low, M. G. Phospholipases of the myocardium. In: Basic Researchofthe MQocardium. Stam. H.. Van der Vusse, G. S. (Eds) 82, 107-l 12 (1987). WEISS,S. L., LOBUGLIO, A. F. Phagocyte-generated oxygen mctabolite and cellular injury. Lab Invest 47. 5 18 (1982). WINER, B. J. Statistical principles in experimental design. McGraw-Hill Book Company, New York. pp 5 I5 545 (1977).
Oxygen Free Radical Producing Leukocytes Cause Functional Depression of Isolated Rat Hearts: Role of Leukotrienes A. G. Semb, J. Vaage’ and 0. D. Mjmrs Department of Physiology and Surgery’,
University of Tromse, Tronsa, Norway
(Received 29 June, accepted in revisedform 21 December,i989) A. G. SEMB, J. VAACE AND 0. D. MJEB. Oxygen Free Radical Producing Leukocytes Cause Functional Depressionof Isolated Rat Hearts: Role of Leukotrienes. Journal of Molecular and Cellular Cardiology (1990) 22, 555-563. Polymorphonuclear granulocytes (PMN) are suggested mediators of myocardial ischemia-reperfusion injury. We have previously shown that activated PMN producing oxygen free radicals (OFR) in the coronary circulation are cardiodepressive. OFR may induce lipid peroxidation and production of eicosanoids. We have investigated the influence of cycle-oxygenase and lipoxygenase inhibitors on the effects of activated, OFR producing PMN in the LangedortIrat heart model. Left ventricular developed pressure (LVDP) was measured by a balloon in the left ventricle. Human PMN and drugs were given into the aortic cannula for 10 min and the hearts were observed for 30 min thereafter. After infusion for 5 min OFR production in the cellular infusate was measured at the level of the aortic cannula by a chemiluminescence (CL) technique. Phorbol l2-myristate 13acetate (PMA)-activated PMN (n = 8), produced a CL response of27649 &- 11048 counts (mean + S.E.M.), and reduced coronary flow (CF) to 53 f 6% (mean + s.E.M.) and LVDP to 38 + 9% of baseline values at the end of the observation period. Ibuprofen (n = 6), a cyclooxygenase (CO) inhibitor, neither influenced the CL response (31915 k 7563) of activated PMN, nor the reduction of CF and LVDP at this time. Although both BW 755C (n = 7), a dual inhibitor of CO and lipoxygenase (LO) (CF:90 f 4%, LVDP:99 + 6%) and diethylcarbamazine (DCM) (n = 8), a LO inhibitor (CF:88 k ll%, LVDP:87 + 4%), significantly inhibited the cardiodepressive effects of activated PMN. BW 755C alone abolished the CL response (431 k 158 counts), whereas DCM had no effect on CL (30105 f. 1698 counts). In conclusion, the injurious effects of activated leukocytes in the coronary circulation seem to be mediated through LO products. KEY WORDS: Leukocytes; Oxygen free radicals; Leukotrienes; Ibuprofen; Dirthylcarbamazine; Myocardial injury; Myocardial depression; Rat heart.
Introduction Infiltration of leukocytes irzto infarcted myocardium has long been recognized [4, 421, where they take part in an inflammatory reaction [ff] and their function has been thought to “clear up” irreversibly damaged tissue [.%I. The presence of leukocytes in the myocardium has been thought to be a result of injury, rather than a cause of it. But recent studies indicate that leukocyte activation may occur within 15-20 min of coronary occlusion [43], that is before myocardial necrosis, which occurs after 30 min of ischemia [33]. Reperfusion accelerates myocardial trapping of polymorphonuclear granulocytes (PMN) [28, 401. Leukocytes may theoretically be cytotoxic and contribute to an inflammatory reaction during reperfusion of ischemic myocardium even before extravasation. This concept is based on
Eicosanoids;
BW
755C;
two different observations. Various pharmacologic modulations of neutrophil function attenuate myocardial ischemia-reperfusion injury [S, 27, 341. Additionally, reperfusion of ischemic myocardium with leukopenic blood has been shown to reduce infarct size [351, increase collateral flow, reduce myocardial edema and ventricular arrhythmias, as well as to inhibit the no-reflow phenomenon [7, 8, 91. The mechanism by which intravascular granulocytes cause injury to the reperfused myocardium is incompletely understood. Activated granulocytes produce and release hydrolytic enzymes [19], eicosanoids [32] and reactive oxygen intermediates, often termed oxygen free radicals (OFR) (superoxide, hydrogen peroxide, hydroxyl radical, singlet oxygen) [47J, and injure eudothelial cells [36, 371. Particular attention has been focused on
Please address all correspondence to: A. G. Semb, Binnevn. 6B, N-0387 Oslo 3, Norway. 0022-2828/90/050555 + 09 $03.00/O
0 1990 Academic Press Limited
A. G. Semb
556
the participation of OFR in reperfusion injury of ischemic myocardium [25,22] and we have shown that intracoronary infusion of activated leukocytes, producing OFR, depressed the function of isolated rat hearts [.%I. OFR are also able to activate phospholipases [4s] with subsequent production of cycle-oxygenase (CO) and lipoxygenase (LO) metabolites of arachidonic acid [31]. In the present investigation we studied the possible role of eicosanoids in the OFRmediated effects of activated leukocytes in the isoIated rat heart. This was done by evaluating the effects of CO and LO inhibition.
Materials
and Methods
Heart preparation
Male-Wistar rats (250-300 g) were anesthetized with diethyl ether. Heparin (200 IU) was injected into the femoral vein. Through a median sternotomy the hearts were rapidly excised and retrogradely perfused via the ascending aorta (Langendorff model) with Krebs-Henseleit solution (37°C) at constant, non-pulsatile perfusion pressure (80 mmHg) . Coronary flow was measured by timed collections of the efBuent. Left ventricular developed pressure (LVDP) and left ventricular end diastolic pressure (LVEDP) were measured by a latex balloon in the left ventricle, connected to a pressure transducer (Gould P23 ID). Heart rate was counted from the pressure curve. Leukocyte suspension
Human leukocytes were separated from freshly drawn, heparinized blood by a simple dextran sedimentation method. The cell suspension was evaluated by differential smear and found to contain l-3% lymphocytes (range), 82-86% polymorphonuclear granulocytes (PMN), and 12-16% monocytes. Since the majority of the cells (both PMN and monocytes) produce OFR and the PMN dominate, the suspension was called PMN for simplicity. The cell suspension was diluted with gassed (95% 02 + 5% COZ) Krebs-Henseleit solution at 37°C and pH 7.4 to obtain a concentration of 3 x IO6 cells/ml. It was this diluted suspension which was infused.
et al. Drugs and chemicals
In order to activate PMN, phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO., USA) was mixed with the cells at a ratio of 3 x 1O6 cells/ml : 10 ng PMA. Ibuprofen (IBU), a cycle-oxygenase inhibitor (Apothekernes Laboratorium, Oslo, Norway), was dissolved in NazCOs, pH adjusted to 7.58 with 1 N HCl. BW 755C (British Wellcome, Wellcome Foundation Ltd., Kent, UK) a dual inhibitor of cycle-oxygenase and lipoxygenase, was dissolved in distilled water. The lipoxygenase inhibitor diethylcarbamazine (DCM) (Sigma, St. Louis, MO., USA) was dissolved in phosphate-buffered saline. Each drug was added to the cell suspension at the same time as PMA. Chemiluminescence (CL)
A modified lumanol dependent CL technique [Z] was used to verify the generation of OFR by activated PMN over time, so the infusion of the activated cell suspension could be done when the leukocytes produced an abundance of OFR. Dose response curves were studied in vitro with PMN + PMA and with various doses of each of the three different drugs used. IBU (7.6 x lo-’ M, 7.6 x 10m4 M and 7.6 x 10-3M), DCM (2.5 x lO-3 M, 5 x lO-3 M, 1 x IO-’ M and 2 x 10e2 M) and BW 755C (2 x 1o-8 M 2 X lo-7 M, 2 X 1o-6 M, 2 x IO-’ M and 2 x low4 M) were added to
3 x 10e6 cells CL was performed on cells and drugs in vitro for 1 h. In the dose response studies, each series of experiments was started and ended with PMN + PMA to check the reliability of the system. OFR production was also measured in samples of the cell suspension taken from the aortic root after 5 min of infusion. For measurements of CL, samples of the cell suspension (200 ~1) were transferred to a cuvette in a photon counter (Biocounter, Model 2010, Lumac/SM, Schaesberg, The Netherlands). Lumanol (100 ~1, 10e4 M) was added automatically to initiate the CL. Experimental protocol
The hearts were stabilized for 10 min before the cell suspension was infused for 10 min at a
Cardiac
Injury
rate of 1 ml/min into the aortic cannula. The total amount of infused cells was 3 x 106. The cells were incubated with PMA for 10 min before the start of the infusion. The concentration of PMN in the coronary circulation was approximately 0.3 x lO’/ml. After the end of the infusion the hearts were observed for 30 min, (total observation time: 40 min after the start of the infusion). A total of 3 x lo6 leukocytes were infused into the coronary circulation. The following groups were studied: Group I (n = 8): PMN + PMA. Group II (n = 6): IBU was added to the PMA activated PMN at a concentration of 7.4 x low4 M. Group III ‘,“~~7); PMN + PMA+BW 755C (2 M m the cell suspension). Group IV (a = 8): PMN + PMA + DCM (2.5 x lo--’ M in the cell suspension). Group V (n = 6): IBU was dissolved in gassed Krebs-Henseleit and infused into the aortic cannula to obtain the same concentration as in Group II. Group VI (n = 6): BW 755C was infused into the coronary circulation to obtain the same concentration as in Group III. Group VII (n = 7): DCM was infused at the same concentration as in Group IV.
557
by Leukocytes
Time
(mini
FIGURE 1. Chemiiuminescence response of phorbol 12-my&ate 13-acetate (PMA) stimulated leukocytes (PMN) l --~ 0, and unstimulated PMN 0 - -- 0.
The CL responses of cell samples taken from the aortic root in Group I-IV after 5 min of infusion are shown in Table 1. CoronaryJIow (CF)
Coronary flow was 8-15 ml/min (range) during the period of stabilization. Infusion of PMA activated PMN reduced CF to 58 f 12% and 49 f 19% of the preinfusion value after 5 and 10 min respectively (Fig. 3).
Statistical analysis
Differences between groups were analysed by one way analysis of variance. Statistical comparisons were made using Qtest [48]. Differences were considered significant when P < 0.05. All results are expressed as mean + S.E.M.
t
140-
.
120-
.
E : IOO-
.
.
“0.
Results Cheniluminescence
A representative in vitro CL response of in vitro PMA activated leukocytes is shown in Fig. 1. Between 10 and 20 min incubation the production of OFR was high. Therefore we have period of chosen a 10 min incubation PMA + PMN before the cell suspension was infused into the isolated rat hearts. BW 7556 (Fig. 2) showed a dose-dependent inhibition of OFR production as measured by the CL response (Fig. \ - 2,, P < 0.0001, r = -0.86 in a 1 two-tailed test), whereas no significant inhibition of CL was found by IBU and DCM in the doses studied.
E x ’ .5
60
E
c
g 4O
5
2{i
1 2.10-4*
2.10.5M
? 2.10‘% [BW755c3
y 2.10.‘M
-I
2.10
FIGURE 2. Effect ofdifferent doses of BW 755C on the chemiluminescence response of phorbol 12-my&ate 13acetate stimulated leukocytes (0) after 15 min incubation at 37”C, and unstimulated leukocytes with BW r = -0.86. 755C (0 ) Correlation coefficient, P < 0.0001.
A.G.Sembctal.
558 TABLE
1. Chemiluminescence
of cells in the coronary
Groups I.
II. III. IV.
Counts
PMN PMN PMN PMN
+ + + +
PMA PMA PMA PMA
+ IBU + BW 755C + DCM
(n = 6) (n = 6) (n = 5) (n = 8)
infustate
(mean
f
27649 31915 431 30105
s.E.M.)
+ f + f
4510 3088 71 * 600
Samples were drawn from the aorta after 5 min of infusion of phorbol 12myristate 13-acetate activated cells (PMN + PMA) alone, and with the addition ofibuprofen (IBU), BW 755C or diethylcarbamazine (DCM). *P < 0.01 compared to PMN + PMA.
No recovery was observed 30 min after the end of infusion (56 f 6%). Although CF in Group II was higher than in Group I throughout the observation period, the effect of IBU was not significant at any time (88 + 14%, 55 + 15% and 66 f 11 at 5, 10 and 40 min respectively) (Fig. 3). However, BW 755C (Group III) significantly inhibited the decrease in CF induced by activated PMN (94 + 6%; 96 + 6% and 90 +_4% at 5, 10 and 40 min) (Fig. 3). DCM had an effect similar to that of BW 755C (94 f: 4%, 94 f 5% and 88 f 11y. at 5, 10 and 40 min) (Fig. 3).
PMN+PMA
I
I
I
0
5
IO
I
40
FIGURE 3. Coronary flow ofisolated rat hearts during and after infusion of leukocytes (PMN) stimulated by phorbol IL?-myristate 13-acetate (PMA) for 10 min, and the effect of ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM). *P < 0.01 compared to PMN + PMA. All values are mean + S.E.M.
Infusion of BW 755C or DCM alone for 10 min (Fig. 4) did not significantly influence CF. IBU significantly increased CF (130 f 2% and 129 + 5% after 5 and 10 min respectively). Left ventricular developed pressure
The left ventricular developed pressure was 90-150 mmHg during stabilization. Infusion of activated PMN decreased LVDP to 42 + 10% and 32 + 8% of the baseline value after 5 and 10 min respectively (Fig. 5). No recovery was observed 30 min after the end of infusion (38 + 9%). IBU attenuated this re-
I501
5ol
-. Min
FIGURE 4. The effect ofa 10 min infusion of ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM) on coronary flow in isolated, perfused rat hearts. *P < 0.01 compared to BW 755C and DCM. All values are mean * s.a.u.
Cardiac
Injury
559
by Leukocytes
aa z 73 b 3 75.; : 5
50-
, 0
I 40
, , 5 IO Min
3
Min
FIGURE 5. The effect of a 10 min infusion of leukocytes (PMN) stimulated with phorbol 12-myristate 13acetate (PMA) on left ventricular developed pressure, and with the addition of ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM) in isolated rat hearts. *P < 0.01 compared to PMN + PMA. All values are mean + s.E.M.
FIGURE 6. The effect ofinfusing ibuprofen (IBU), BW 755C, or diethylcarbamazine (DCM) for 10 min on left ventricular developed pressure (LVDP) in isolated, perfused rat hearts. *P < 0.01 compared to baselinr. illI values are mean + S.E.M.
Neither BW 755C nor DCM when given alone influenced LVDP whereas IBU initially increased LVDP (Fig. 6).
Left ventricular end diastolic pressure duction in LVDP (Fig. 5), but the difference was not significant at any time (71 + 12%, 49 + 15% and 62 + 8% at 5, 10 and 40 min). However, BW 755C significantly inhibited the decrease in LVDP (85 f 6%, 88 + 6% and 99 f 6% at 5, 10 and 40 min) (Fig. 5). DCM the decrease in LVDP also inhibited (95 f 5%, 91 + 5% and 87 f 4% at 5, 10 and 40 min) (Fig. 5).
TABLE 2.
No changes in LVEDP were observed group during the observation period.
Heart rate Baseline heart rate was 26&360 beats/min. Although all groups (I-VI) showed a tendency towards a decrease in heart rate, no or difference between significant change groups were observed (Table 21,
Heart rate Time after start of infusion:
Groups I. II.
III. IV. V. VI. VII.
in any
PMN + PMA PMN + PMA + IBU PMN + PMA + BW 755C PMN + PMA + DCM IBU BW 755C DCM
____-40 min
5 min
10 min
(n = 8) (n = 6) (n = 7)
100 f 5 86f 10 93 -t 3
95 + 5 77 * 1 92 f 3
89+ 10 88 f. 6 94 * 5
(n = 8)
94 f 2
92 f 3
95 f 3
(II = 6)
79f 14 94 + 3 95 * 3
76+15
93 f 3 98 + 3
86 * 7 93 * 3 102 f 5
Intrinsic, unpaced heart rate in a rat LangendorKmodeI during and after addition of Phorbol 12-acetate 13-myristatc activated cells (PMA + PMN) alone, or with the addition of ibuprofen (IBU), BW 755C or diethylcarmbamazine (DCM). The effect of the drugs given alone is also shown. All values are in percent of baseline values (mean + s.E.M.).
560
A.G.Sembetal. Discussion
Infusion of activated PMN into the coronary circulation may represent an enhancement of the initial effects of the early and immediate intravascular trapping and activation of granulocytes in the reperfused ischemic myocardium. Consequently, this model may be useful to elucidate the early cardiac effects of activated leukocytes. Unless the delayed inflammatory response by leukocytes is qualitatively different, it may also shed some light on the general mechanisms whereby leukocytes are able to injure myocardial cells, affect the coronary circulation, and depress cardiac function. Infusion of unactivated PMN into the coronary circulation of isolated rat heart did not affect CF, LVDP or heart rate, while activated PMN severely impaired heart function [39]. We have previously shown that scavengers of OFR inhibited the cardiodepressive effects of PMN activated by PMA, suggesting an important role of OFR as mediators [39]. This observation, however, does not exclude the possibility that other mediators are involved, either as prerequisite for OFR production, or as secondary mediators produced or released by the action of OFR. Reactive oxygen species induce lipid peroxidation. Recent studies suggest that freeradical triggered peroxidation of phospholipids renders them more susceptible to the action of phospholipases A or C [4sJ, and may initiate the arachidonic acid cascade [28].
Cyclo-oxygenase
inhibition
The doses of IBU used did not influence the CL response of activated PMN, although the concentration of IBU used (7.6 x 10m3 M) was similar to the plasma concentration in studies where IBU has been shown to reduce release from infarct size, reduce enzyme ischemic myocardium and diminish leukocyte accumulation in ischemic myocardium [1, 26, 171. That we did not observe an inhibition of OFR production as Flynn and coworkers did [IJI, might be due to differences in the PMNstimulating agents used, the methods for measuring OFR, or species differences. Theoretically, CO products might be involved in the cardiac effects of stimulated PMN
producing OFR. Rowe et al. [36] suggested that hydroxyl radical generation by the CO pathway was partly responsible for the in vitro depression of cardiac sarcoplasmic reticulum by PMA activated leukocytes. The various CO products have both inotropic and chronotropic effects and may also modulate coronary vascular tone [18, 291. This picture is complicated by species differences and antagonistic actions of the various CO products. For instance, thromboxane A2 has been suggested as a mediator of ischemic myocardial damage [14], whereas prostacyclin may have cardioprotective actions [3]. Th e present study shows that CO products are not important for the cardiodepressive effects of PMA activated PMN. In fact, the tendency towards decreasing inhibition of CF and LVDP by IBU might well be secondary to a direct effect of IBU which increased both CF and LVDP when infused alone. However, a possible beneficial effect of IBU might partly be counteracted by the enhancement of leukotriene production by CO inhibition [453.
Lipoxygenase
inhibition
Four structurally dissimilar LO inhibitors are able to reduce myocardial infarct size [12, 25, 26,301. In the present study, 2 structurally and pharmacologically different LO inhibitors abolished all the cardiodepressive effects of PMA activated PMN. BW 755C is’ a dual inhibitor of both CO and LO, as well as being an antioxidant. The concentration of BW 755C used in the isolated heart model was similar to those used in in vivo studies [26, 271. DCM is an antifilarial agent and a specific inhibitor of the enzymatic (5-lipoxygenase) conversion of 5-hydroperoxyeicosatetranoic acid (5-HPETE) into leukotriene A4 [21]. None of the doses of DCM mixed with the activated cell suspension reduced the CL response. Mathews et al. [.?I] showed that 250 PM DCM inhibited 90% of the production of LTB4 and LTC4 in calcium ionophore stimulated mastocytoma cells. Consequently this concentration of DCM was therefore added to the cell suspension infused into the coronary circulation. BW 755C either inhibited OFR production or acted as a scavenger of OFR in the present study, since it abolished the CL response by
Cardiac
Injury
561
by Leukocytes
LTD4 . LTA,
t LTC4
Endothelium
FIGURE 7. Proposed mechanism of interaction between stimulated granulocytes (PMN) and endothelium. Activated PMN release superoxide (0;) which may form H202 through a dismutation reaction. HsOs may react with myeloperoxidase (MPO) or lactoferrin (LF). From the latter reaction the oxidative and lipidsoluble hydroxyl radical (OH-) is formed, which may result in the activation of the arachidonic acid (AA) metabolism through the action of phospholipase As (PLAs) on phosphatidyl choline (PC), with the production of leukotriene & (LTA.+). Alternatively, endothelial cells may produce LTC4 and LTD4 from LTA4 released from activated PMN. LTC4 and LTD., cause vasoconstriction and decrease myocardial contractile power.
PMA activated PMN. DCM did not affect the CL response of PMA + PMN, and yet it gave equipotent inhibition of the cardiac effects. Thus, the beneficial effects of DCM and BW 7556 seem to be caused by their inhibition of the production of leukotrienes from arachidonic acid by inhibition of 5-lipoxygenase. Shak and coworkers [41] showed that 0.5 pg/ml PMA: IO6 PMN did not stimulate LTB4 production. We used a lower concentration of PMA (0.01 pg/ml: lo6 PMN), and it is not likely that LTB4 is formed by the cells in our model. But the peptidoleukotrienes LTC4 and LTD4 might be produced by endothelial cells through the direct action of OFR on the endothelial membranes as suggested in Fig. 7, or from the PMN-derived LTA4. This is in accordance with the suggestion of Feinmark et al. [II], that neutrophil derived LTA4 can be transformed to LTC4 by endothelial cells after donation of the LTA4 from the PMN. Since LTC4 and LTD4 are able to increase vascular permeability, induce coronary vasoconstriction and have negative inotropic effects [.5,20, 23, 24,30), they may be the final mediators of the cardiac depression induced by activated PMN. However, the direct evidence for this hypothesis can be strengthened by using specific peptide Ieukotriene receptor antagonists
and/or measurements of LTC4/LTD4 coronary effluent.
in the
Concept From our previous [39] and present findings we propose that OFR production is the primary event in the injury mechanism of activated PMN. Through peroxidation of cell membranes the arachidonic cascade is triggered. LO products are then released and may exacerbate leukocyte activation, induce coronary vasoconstriction, and impair cardiac thus serving as secondary contractility, mediators. It might be that the cardiac effects of OFR are mostly through their ability to trigger the LO cascade. Acknowledgements
A. G. Semb is a research fellow of the Norwegian Research Council for Science and the Humanities. Further support has been received from the Norwegian Council on Cardiovascular Disease, Thomas Fearnley Bidrags- og Gave Fond, Dagny og Kare Holts Legat, and the University of Tromsra, all of which is greatly acknowledged.
562
A. G. Scab
et al.
References 1 ALLAN, G., BHATTACHERJEE, P., BROOOK, C. D., READ, N. G.. PARKE, A. J. Myeloperoxidase activity ah ct the quantitative marker of polymorphonuclear leukocyte accumulation into an experimental myocardial infarct effect of ibuprofen on infarct size and polymorphonuclear leukocyte accumulation. J Cardiovasc Pharmacol 7. 11541160 (1985). chemiluminescence in rabbit alveolar and 2 ALLEN, E. C., LOOSE,L. D.. Phagocytic activation ofluminol-dependent peritoneal macrophages. Biochem Biophys Res Commun 69,245-252 (1976). 3 ARAKI, H., LEFER, A. M. Role of prostacyclin in preservation of ischemic myocardial tissue in the perfused cat heart. Circ Res 47, 745-763 (1980). 4 BAUMGARTEN,W. Infarction in the heart. Am J Physio12, 243-265 (1899). 5 BnaKE,J. A., Layt, R., GUI, Z., COREY,E. J. Leukotrienes C,, D, and E,: Effects on human and guinea pig cardiac preparation in vitro. J Pharmacol Exp Ther 221, 235-245 (1982). 6 ENGLER, R. 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