610
C E L L M O D E L S OF L I P I D M E D I A T O R P R O D U C T I O N
[66]
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.
[66]
P A F AND EICOSANOID STUDIES: ISOLATED HEART
611
TABLE I PHYSIOLOGICAL SALT SOLUTIONS C o n c e n t r a t i o n (g/liter)
Salt/Sugar
KrebsHenseleit
Tyrode
Ringer
NaC1 KCI MgCI2 • 6 H 2 0 MgSO4 • 7 H 2 0 CaCI2 • 2 H 2 0 NaH2PO4 • H 2 0 NaHCO3 Glucose
6.87 0.40 -0.30 0.36 0.16 2.10 2.00
8.00 0.20 0.21 -0.26 0.06 1.00 1.00
9.00 0.42 --0.32 -0.50 1.00
pulmonary or systemic circulation. Either blood or a variety of oxygenated physiological solutions is infused at constant flow or constant pressure into a cannula inserted in the ascending aorta. As the aortic valves remain closed, the perfusing fluid enters the coronary arteries and exits through the coronary sinus into the right atrium and from there to the exterior through the superior and inferior venae cavae; depending on the particular experimental protocol, the effluent can either be discarded or recirculated through the heart. Thus, in the "Langendorff heart," the ventricular and atrial chambers remain practically empty and, although they contract rhythmically, they do not pump fluid. In contrast, in the "working heart" version, the left ventricle performs pressure-volume work using a second or "systemic" circulation (vide infra). Most commonly, guinea pigs are chosen for this preparation, although rabbits and rats are also used. The animals are deprived of food, but not water, overnight; heparin (500 USP/100 g body weight, iv or ip) is administered to maintain patency of the coronary vasculature. Thirty minutes after heparinization, the animals are anesthetized with CO2 vapors or with sodium pentobarbital (30 mg/kg ip), killed by cervical dislocation, and exsanguinated. The aorta is cut as far cranially as possible and the heart is rapidly excised from the surrounding tissue (the procedure should take not more than 1 minute). The aorta is cannulated ensuring that the tip of the cannula is not placed beyond the semilunar valves and that it does not obstruct the ostia of the coronary arteries. 2,3 The composition of the most frequently used physiological solutions is shown in Table I. 2 G. Allan a n d R. Levi, J. Pharmacol. Exp. Ther. 214, 45 (1980). 3 R. Levi, J. A. Burke, Z.-G. Guo, Y. Hattori, C. M. H o p p e n s , L. M. M c M a n u s , D. J. H a n a h a n , a n d R. N. Pinckard, Circ. Res. 54, 117 (1984).
612
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[66]
In the constant-pressure version, the heart is perfused at a pressure closely resembling diastolic pressure in vioo, corrected to account for the lower viscosity of buffered solutions as compared with blood. In this model, increases or decreases in coronary vascular resistance translate into decreases or increases in coronary flow, respectively. The latter are assessed by measuring the volume of coronary venous effluent in a given time or by the use of a flowmeter. In the constant-flow version, increases or decreases in coronary vascular resistance translate into increases or decreases in perfusion pressure, respectively. These changes are monitored with a pressure transducer. 3 Left ventricular contractile force is measured most times isometrically by connecting the tip of the left ventricle to a force transducer via a thread or by monitoring left ventricular pressure (LVP) with a small balloon connected to a pressure transducer via a catheter inserted into the left ventricle through the left atrium and the mitral valve. For a more complete assessment of the changes in contractility, the derivative of LVP can be obtained (i.e., L V dp/dt). Spontaneous heart rate is best determined by recording the bipolar electrogram from the surface of the right atrium and left ventricle and then measuring the interval between two consecutive P or R waves. Further, the state of atrioventricular conduction can be assessed by measuring the duration of the P - R interval and by determining the number of P and R waves in a given time frame. Aside from atrioventricular conduction blocks of various degrees, the electrogram facilitates the recognition of idioventricular arrhythmias due to enhanced automaticity or reentry. Inasmuch as changes in rate will affect contractility, at times it may be necessary to pace the heart at constant rate with rectangular pulses generated by a stimulator and delivered by a pair of electrodes placed on the right atrium with the interposition of a stimulus isolation unit. In a more sophisticated version, the right atrium is opened to expose the atrioventricular node and additional recording electrodes are placed in the region of the bundle of His; stimulus-to-His bundle interval measurements are continuously monitored with the aid of a microcomputer. 4 This system allows for the most reliable assessment of atrioventricular conduction on a beat-to-beat basis. Hearts can also be isolated with an intact sympathetic innervationS; right and left sympathetic ganglia are stimulated with square pulses of appropriate intensity and duration. Overflow of endogenous norepinephrine is assayed in the coronary effluent in response to increasing frequencies of sympathetic nerve stimulation. 5 With this model, one can de4 j. R. Jenkins and L. Belardinelli, Circ. Res. 63, 97 (1988). 5 S. S. Gross, Z.-G. Guo, R. Levi, W. H. Bailey, and A. A. Chenouda, Circ. Res. 54, 516 (1984).
[66]
PAF ANDEICOSANOIDSTUDIES:ISOLATEDHEART
613
termine the modulatory effect of a variety of substances, including eicosanoids, on norepinephrine release from the heart. 6 Cardiac Effects of Prostaglandins The cardiac effects of prostaglandins (PG) have been studied in the isolated guinea pig heart perfused at constant pressure. 2 Arachidonic acid (AA; 0.1-10/xg), PGE2 (0.01-1/zg), and prostacyclin (PGI2; 0.01-10/xg) administered by intraaortic bolus injection elicit dose-dependent increases in coronary flow rate. Dose-dependent coronary vasodilatation also occurs when PGI2 or PGE2 (0.3 nM-0.3/zM) is infused in isolated guinea pig hearts perfused at constant flow. 7 In contrast, PGD2 (0.01-10 tzg), PGF2~ (1-100/zg), and the stable thromboxane (TxA2) analog compound U46,619 (0.01-1 tzg) cause dose-dependent decreases in coronary flow rate. 2 Indeed, TxA2 generated by conversion of PGH2 with platelet microsomes elicits a dose-dependent coronary-vasoconstricting effect,s Over the dose range studied, PGE2, AA, PGI2, and PGFz,~ increase, and PGD2 decreases, the spontaneous sinus rate of the isolated guinea pig heart, whereas U46,619 has no consistent chronotropic effects.2 AA, PGF2,~, and U46,619 cause a dose-dependent decrease in contractile force; however, the negative inotropic effect of U46,619 is probably secondary to its coronary-vasoconstricting action, s PGE2 has no effect on the contractility of the isolated guinea pig heart, whereas PGI2 elicits a modest positive inotropic response. 2 PGD2 has a primary positive inotropic action; on the other hand, because PGD2 is also a potent coronary vasoconstrictor, a secondary negative inotropic response predominates. 9 Cardiac Effects of Leukotrienes Coronary vasoconstriction and contractile failure are the hallmark of partially purified slow-reacting substance of anaphylaxis (SRS-A), HPLCpurified SRS, and synthetic leukotrienes (LT)C4 and D4 injected into isolated guinea pig hearts perfused at either constant flow or pressure. 9 Although the dose-dependent decrease in contractility parallels the decrease in coronary flow, the two phenomena are likely to be independent
6 ~,. Wennmalm, Acta Physiol. Scand. 105, 254 (1979). 7 K. Schrfr and S. Moncada, Prostaglandins 17, 367 (1979). s Z.-I. Terashita, H. Fukui, K. Nichikawa, M. Hirata, and S. Kikuchi, Eur. J. Pharmacol. 53, 49 (1978). 9 R. Levi, Y. Hattori, J. A. Burke, Z.-G. Guo, U. Hachfeld del Batzo, W. A. Scott, and C. A. Rouzer, in "Prostaglandins, Leukotrienes, and Lipoxins" (J. M. Bailey, ed.), p. 275. Plenum, New York, 1985.
614
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[66]
both in the Langendorff and working heart models. 9'1° Nevertheless, the possibility remains that the negative inotropic effect of leukotrienes may be amplified by local ischemia resulting from their concomitant coronaryconstricting effect. The rank order of potency for the negative inotropic and coronary-vasoconstricting effects of leukotrienes is LTD4 > LTC4 > LTE4 (LTD4,0.01-1 ng; LTC4,0.3-30 ng; LTE4, 100 ng-3/.tg). 9 Both the negative inotropic and coronary-vasoconstricting effects of leukotrienes are antagonized by specific leukotriene receptor antagonists, but are unaffected by cyclooxygenase inhibitors. 9 Cardiac Effects of Platelet-Activating Factor In the isolated guinea pig heart perfused at constant pressure, the intraaortic administration of platelet-activating factor (PAF) by bolus injection (0.01 pmol to 3 nmol) elicits dose-related decreases in left ventricular contractile force and coronary flow ( - 5 to -85%). 3 The negative inotropic effect of PAF persists also in hearts perfused at constant flow. 3 The deacetylated derivative of PAF has only minimal cardiac effects in doses as high as 10 nmol. 3 Neither the coronary vasoconstriction nor the decrease in contractility caused by PAF are affected by indomethacin or FPL 55172, indicating that although cyclooxygenase and lipoxygenase products may be released by PAF,11 they do not play a functional role in the cardiac response to P A F . 3'12 Indeed, the cardiac effects of PAF are specifically antagonized by pharmacological agents known to block the platelet-activating effects of PAF. 13 Platelet-Perfused
Hearts
For the investigation of platelet-vessel wall interactions in eicosanoid research, hearts of guinea pigs, 14 rats,15 o r r a b b i t s 16 have been perfused with buffer containing either human ~5 or rabbit 15a6 platelets. The lO O. G. Bj6rnsson, K. Kobayashi, and J. R. Williamson, Eur. J. Clin. Invest. 17, 146 (1987). 11 p. j. Piper and A. G. Stewart, Br. J. Pharmacol. 88, 595 (1986). 12 G. L. Stahl, D. J. Lefer, and A. M. Lefer, Arch. Pharmacol. 336, 459 (1987). ~3 R. Levi, T, Y. Shen, S. J. Yee, D. A. Robertson, A. Genovese, O. W. Isom, and K. H. Krieger, in " N e w Horizons in Platelet Activating Factor Research" (C. M. Winslow and M. L. Lee, eds.), p. 255. John Wiley & Sons, Chichester, England, 1987. 14 K. Schrtr, P. K6hler, M. Miiller, B. A. Peskar, and P. R6sen, Am. J. Physiol. 241, HI8 0981). 15 M. Purchase, G. J. Dusting, D. M. F. Li, and M. A. Read, Circ. Res. 58, 172 (1986). t6 G. Montrucchio, G. Alloatti, C. Tetta, R. DeLuca, R. N. Saunders, G. Emanuelli, and G. Camussi, Am. J. Physiol. 256, H1236 (1989).
[66]
P A F AND EICOSANOID STUDIES: ISOLATED HEART
615
"Langendorffheart" is prepared as described above. Washed platelets are prepared from platelet-rich plasma. The details of platelet preparation vary, with some investigators using PGE1 (Ref. 4) or PGI2 (Ref. 15) to limit platelet activation during the steps of centrifugation and suspension of the platelet pellet. The platelets are finally resuspended in an albumincontaining buffer lacking any PGE~ or PGI2. The platelets are infused into the aortic inflow tract just above the heart at a flow rate of 0.1 ml/min for guinea pig or rat hearts, or 0.1 to 1 ml/min in the rabbit heart, representing 1-3% of the total perfusate flow. The concentration of platelets used varies from - 1 × 106 tO 2 X 108 platelets per milliliter perfusate. The predominant use of this preparation has been in the study of interactions between PGI2 released from the heart, and TxA2 produced mainly by the platelets. The formation of these two mediators has been determined by bioassay where the coronary effluent continuously superfuses strips of vascular tissue (e.g., rabbit aorta, porcine or bovine coronary arteries; all three are contracted by TxA2, but only bovine coronary artery is relaxed by PGI2) or by radioimmunoassay of the stable hydrolysis products 6-keto-PGFj,~ and TxB2. Perfusion of hearts with a plateletcontaining buffer enhances the production of PGI2 and, to a lesser extent, TxA2, while the formation of both mediators can be increased substantially by the addition of arachidonic acid. ~4'15 Despite the formation of approximately three times m o r e T x A 2 than PGI2 in the presence of arachidonic acid, coronary vasodilatation predominates. Infusions of low concentrations of epinephrine (0.6-6 pmol/ml), that are insufficient to provoke platelet aggregation, potentiate platelet TxA2 production from arachidonic acid within the heart. ~5 It is proposed that perfused hearts produce a stable nonprostanoid factor that potentiates TxA2 release when incubated with platelets. ~5 Neutrophil-Perfused Hearts Rabbit hearts perfused in the Langendorffmode with rabbit neutrophils added to the perfusate have been used to study interactions between leukocytes and the heart. ~7'18 Neutrophils are prepared from citrated whole blood (3.8% citrate at ratio 1 : 10 citrate : blood), mixed with 6% dextran (molecular weight = 500,000; ratio 1:5) for sedimentation of erythrocytes. The supernatant is centrifuged (400 g for 5 min), contaminating erythrocytes are eliminated by hypotonic lysis, and the cells separated 17 M. N. Gillespie, S. Kojima, J. O. Owasoyo, H. H. Tai, and M. Jay, J. Pharmacol. Exp. Therap. 241, 812 (1987). t8 R. Kraemer and K. M. Mullane, J. Pharmacol. Exp. Therap. 251, 620 (1989).
616
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[66]
by Ficoll/Hypaque density gradient centrifugation (600 g for 20 rain). The pellet is resuspended in Hanks' balanced salt solution and is generally comprised of 90-95% neutrophils with cell viability close to 100%, as assessed by trypan blue exclusion. Neutrophils can also be radiolabeled with either indium-11117 or chromium-51,18 and radioactivity determined using a y-counter. The perfused heart preparation is essentially similar to that described above, except that the superior and inferior venae cavae and pulmonary veins are ligated, and the pulmonary artery cannulated to capture the coronary outflow and to perfuse the hearts in a recirculating mode. The reservoir volume is 400 ml, containing 5 × 103 neutrophils per milliliter in Krebs-Henseleit buffer with 1% BSA; it is gassed with 95%02 + 5%CO2 by either blowing the gas across the surface of the buffer in a revolving reservoir or by using a membrane lung system, to minimize "frothing" of the protein-rich buffer. The hearts are perfused at a constant flow of 25 to 30 ml/min at a left ventricular end-diastolic pressure (LVEDP) of 10 to 15 mm Hg and paced at a constant rate of 200 to 400 beats/min. Myocardial hypoxia is induced by gassing the perfusion fluid with either 12%O2 + 83%N2 + 5%CO2 or 100%N2 for 30 min and then reoxygenating with the original 95%02 + 5%CO2. Hypoxia and reoxygenation provoke the accumulation of radiolabeled neutrophils in the heart. 17'18 Hypoxia-induced neutrophil accumulation is attributed to the generation of LTB4 by the myocardium, as determined by increases in the tissue content of LTB4-1ike immunoreactivity and suppression of cell accumulation by diethylcarbamazine or nordihydroguaiaretic acid, purported lipoxygenase inhibitors. 17 Neutrophil sequestration is associated with a reduced recovery of function during reoxygenation. 18 Ischemia-Reperfusion
in Langendorff Hearts
Studies on the release and role of eicosanoids in myocardial ischemia and reperfusion have benefitted enormously from the isolated perfused heart technique. Hearts of rats, 19,20guinea pigs, 21 rabbits, 16'22 and cats 23 have been used in these studies. Generally, global ischemia is induced by decreasing the flow rate to < I ml/min for 30 to 120 min before restoring preischemic flow for a 15- to 30-rain reperfusion period. 16'19'2°'23 The degree of ischemia is more severe if the heart is paced at a fixed rate 19 M. Karmazyn, Am. J. Physiol. 251, H133 (1986). 2o W. G. Nayler, M. Purchase, and G. J. Dusting, Basic Res. Cardiol. 79, 125 (1984). 21 M. P. Mot'fat, J. Pharmacol. Exp. Ther. 242, 292 (1987). 22 A. Edlund, K. Sahlin, and A Wennmalm, J. Mol. Cell. Cardiol. 18, 1067 (1986). 23 H. Araki and A. M. Lefer, Circ. Res. 47, 757 (1980).
[66]
P A F AND EICOSANOID STUDIES: ISOLATED HEART
617
throughout the procedure; this enables the ischemic period to be abbreviated and thus to minimize time-related changes in cardiac function. An alternative procedure that has been used in the rabbit heart is to ligate the left anterior descending branch of the main coronary artery for 2 hr to study the effect of PGI2 on regional ischemia.22 Ischemia does not provoke prostaglandin production; however, reperfusion of the globally ischemic rat 19 or guinea pig21 heart is a potent stimulus for PGI2 synthesis: PGI2 release is increased from 0.01 to 0.3-0.4 ng/ml/min or from 0.2 to 1.5 ng/ml/min (measured as 6-keto-PGF1, by radioimmunoassay) for the rat and guinea pig, respectively. This release is sustained in the rat heart, but rapidly declines in the guinea pig preparation. The magnitude and duration of PGI2 release at reperfusion is independent of the duration of the preceding ischemic period when varied from 5 to 60 min. 19 The significance of the PGI2 production at reperfusion on the postischemic recovery of cardiac function is a subject of controversy. PGI2 is reported to be either beneficial,2s d e t r i m e n t a l , 19'21'24 o r without e f f e c t . 22 While this discrepancy may relate in part to PGI2 improving the rate, but not the magnitude, of functional recovery,2° it appears that the discrepancy may also result from different concentration-dependent effects of PGI2. Small PGI2 concentrations that mimic the endogenously released material (-0.5-1 ng/ml) impair recovery of contractile function, whereas larger concentrations of PGI2 may be cardioprotective. 19,20,23,24However, the true effects of endogenous PGI2 produced by ischemia and reperfusion remain to be elucidated. Ischemia and reperfusion of the Langendorff rabbit heart is also a stimulus for transient PAF release. PAF is undetectable during control perfusion and ischemia (
C E L L M O D E L S OF L I P I D M E D I A T O R P R O D U C T I O N
[66]
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.
[66]
P A F AND EICOSANOID STUDIES: ISOLATED HEART
611
TABLE I PHYSIOLOGICAL SALT SOLUTIONS C o n c e n t r a t i o n (g/liter)
Salt/Sugar
KrebsHenseleit
Tyrode
Ringer
NaC1 KCI MgCI2 • 6 H 2 0 MgSO4 • 7 H 2 0 CaCI2 • 2 H 2 0 NaH2PO4 • H 2 0 NaHCO3 Glucose
6.87 0.40 -0.30 0.36 0.16 2.10 2.00
8.00 0.20 0.21 -0.26 0.06 1.00 1.00
9.00 0.42 --0.32 -0.50 1.00
pulmonary or systemic circulation. Either blood or a variety of oxygenated physiological solutions is infused at constant flow or constant pressure into a cannula inserted in the ascending aorta. As the aortic valves remain closed, the perfusing fluid enters the coronary arteries and exits through the coronary sinus into the right atrium and from there to the exterior through the superior and inferior venae cavae; depending on the particular experimental protocol, the effluent can either be discarded or recirculated through the heart. Thus, in the "Langendorff heart," the ventricular and atrial chambers remain practically empty and, although they contract rhythmically, they do not pump fluid. In contrast, in the "working heart" version, the left ventricle performs pressure-volume work using a second or "systemic" circulation (vide infra). Most commonly, guinea pigs are chosen for this preparation, although rabbits and rats are also used. The animals are deprived of food, but not water, overnight; heparin (500 USP/100 g body weight, iv or ip) is administered to maintain patency of the coronary vasculature. Thirty minutes after heparinization, the animals are anesthetized with CO2 vapors or with sodium pentobarbital (30 mg/kg ip), killed by cervical dislocation, and exsanguinated. The aorta is cut as far cranially as possible and the heart is rapidly excised from the surrounding tissue (the procedure should take not more than 1 minute). The aorta is cannulated ensuring that the tip of the cannula is not placed beyond the semilunar valves and that it does not obstruct the ostia of the coronary arteries. 2,3 The composition of the most frequently used physiological solutions is shown in Table I. 2 G. Allan a n d R. Levi, J. Pharmacol. Exp. Ther. 214, 45 (1980). 3 R. Levi, J. A. Burke, Z.-G. Guo, Y. Hattori, C. M. H o p p e n s , L. M. M c M a n u s , D. J. H a n a h a n , a n d R. N. Pinckard, Circ. Res. 54, 117 (1984).
612
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[66]
In the constant-pressure version, the heart is perfused at a pressure closely resembling diastolic pressure in vioo, corrected to account for the lower viscosity of buffered solutions as compared with blood. In this model, increases or decreases in coronary vascular resistance translate into decreases or increases in coronary flow, respectively. The latter are assessed by measuring the volume of coronary venous effluent in a given time or by the use of a flowmeter. In the constant-flow version, increases or decreases in coronary vascular resistance translate into increases or decreases in perfusion pressure, respectively. These changes are monitored with a pressure transducer. 3 Left ventricular contractile force is measured most times isometrically by connecting the tip of the left ventricle to a force transducer via a thread or by monitoring left ventricular pressure (LVP) with a small balloon connected to a pressure transducer via a catheter inserted into the left ventricle through the left atrium and the mitral valve. For a more complete assessment of the changes in contractility, the derivative of LVP can be obtained (i.e., L V dp/dt). Spontaneous heart rate is best determined by recording the bipolar electrogram from the surface of the right atrium and left ventricle and then measuring the interval between two consecutive P or R waves. Further, the state of atrioventricular conduction can be assessed by measuring the duration of the P - R interval and by determining the number of P and R waves in a given time frame. Aside from atrioventricular conduction blocks of various degrees, the electrogram facilitates the recognition of idioventricular arrhythmias due to enhanced automaticity or reentry. Inasmuch as changes in rate will affect contractility, at times it may be necessary to pace the heart at constant rate with rectangular pulses generated by a stimulator and delivered by a pair of electrodes placed on the right atrium with the interposition of a stimulus isolation unit. In a more sophisticated version, the right atrium is opened to expose the atrioventricular node and additional recording electrodes are placed in the region of the bundle of His; stimulus-to-His bundle interval measurements are continuously monitored with the aid of a microcomputer. 4 This system allows for the most reliable assessment of atrioventricular conduction on a beat-to-beat basis. Hearts can also be isolated with an intact sympathetic innervationS; right and left sympathetic ganglia are stimulated with square pulses of appropriate intensity and duration. Overflow of endogenous norepinephrine is assayed in the coronary effluent in response to increasing frequencies of sympathetic nerve stimulation. 5 With this model, one can de4 j. R. Jenkins and L. Belardinelli, Circ. Res. 63, 97 (1988). 5 S. S. Gross, Z.-G. Guo, R. Levi, W. H. Bailey, and A. A. Chenouda, Circ. Res. 54, 516 (1984).
[66]
PAF ANDEICOSANOIDSTUDIES:ISOLATEDHEART
613
termine the modulatory effect of a variety of substances, including eicosanoids, on norepinephrine release from the heart. 6 Cardiac Effects of Prostaglandins The cardiac effects of prostaglandins (PG) have been studied in the isolated guinea pig heart perfused at constant pressure. 2 Arachidonic acid (AA; 0.1-10/xg), PGE2 (0.01-1/zg), and prostacyclin (PGI2; 0.01-10/xg) administered by intraaortic bolus injection elicit dose-dependent increases in coronary flow rate. Dose-dependent coronary vasodilatation also occurs when PGI2 or PGE2 (0.3 nM-0.3/zM) is infused in isolated guinea pig hearts perfused at constant flow. 7 In contrast, PGD2 (0.01-10 tzg), PGF2~ (1-100/zg), and the stable thromboxane (TxA2) analog compound U46,619 (0.01-1 tzg) cause dose-dependent decreases in coronary flow rate. 2 Indeed, TxA2 generated by conversion of PGH2 with platelet microsomes elicits a dose-dependent coronary-vasoconstricting effect,s Over the dose range studied, PGE2, AA, PGI2, and PGFz,~ increase, and PGD2 decreases, the spontaneous sinus rate of the isolated guinea pig heart, whereas U46,619 has no consistent chronotropic effects.2 AA, PGF2,~, and U46,619 cause a dose-dependent decrease in contractile force; however, the negative inotropic effect of U46,619 is probably secondary to its coronary-vasoconstricting action, s PGE2 has no effect on the contractility of the isolated guinea pig heart, whereas PGI2 elicits a modest positive inotropic response. 2 PGD2 has a primary positive inotropic action; on the other hand, because PGD2 is also a potent coronary vasoconstrictor, a secondary negative inotropic response predominates. 9 Cardiac Effects of Leukotrienes Coronary vasoconstriction and contractile failure are the hallmark of partially purified slow-reacting substance of anaphylaxis (SRS-A), HPLCpurified SRS, and synthetic leukotrienes (LT)C4 and D4 injected into isolated guinea pig hearts perfused at either constant flow or pressure. 9 Although the dose-dependent decrease in contractility parallels the decrease in coronary flow, the two phenomena are likely to be independent
6 ~,. Wennmalm, Acta Physiol. Scand. 105, 254 (1979). 7 K. Schrfr and S. Moncada, Prostaglandins 17, 367 (1979). s Z.-I. Terashita, H. Fukui, K. Nichikawa, M. Hirata, and S. Kikuchi, Eur. J. Pharmacol. 53, 49 (1978). 9 R. Levi, Y. Hattori, J. A. Burke, Z.-G. Guo, U. Hachfeld del Batzo, W. A. Scott, and C. A. Rouzer, in "Prostaglandins, Leukotrienes, and Lipoxins" (J. M. Bailey, ed.), p. 275. Plenum, New York, 1985.
614
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[66]
both in the Langendorff and working heart models. 9'1° Nevertheless, the possibility remains that the negative inotropic effect of leukotrienes may be amplified by local ischemia resulting from their concomitant coronaryconstricting effect. The rank order of potency for the negative inotropic and coronary-vasoconstricting effects of leukotrienes is LTD4 > LTC4 > LTE4 (LTD4,0.01-1 ng; LTC4,0.3-30 ng; LTE4, 100 ng-3/.tg). 9 Both the negative inotropic and coronary-vasoconstricting effects of leukotrienes are antagonized by specific leukotriene receptor antagonists, but are unaffected by cyclooxygenase inhibitors. 9 Cardiac Effects of Platelet-Activating Factor In the isolated guinea pig heart perfused at constant pressure, the intraaortic administration of platelet-activating factor (PAF) by bolus injection (0.01 pmol to 3 nmol) elicits dose-related decreases in left ventricular contractile force and coronary flow ( - 5 to -85%). 3 The negative inotropic effect of PAF persists also in hearts perfused at constant flow. 3 The deacetylated derivative of PAF has only minimal cardiac effects in doses as high as 10 nmol. 3 Neither the coronary vasoconstriction nor the decrease in contractility caused by PAF are affected by indomethacin or FPL 55172, indicating that although cyclooxygenase and lipoxygenase products may be released by PAF,11 they do not play a functional role in the cardiac response to P A F . 3'12 Indeed, the cardiac effects of PAF are specifically antagonized by pharmacological agents known to block the platelet-activating effects of PAF. 13 Platelet-Perfused
Hearts
For the investigation of platelet-vessel wall interactions in eicosanoid research, hearts of guinea pigs, 14 rats,15 o r r a b b i t s 16 have been perfused with buffer containing either human ~5 or rabbit 15a6 platelets. The lO O. G. Bj6rnsson, K. Kobayashi, and J. R. Williamson, Eur. J. Clin. Invest. 17, 146 (1987). 11 p. j. Piper and A. G. Stewart, Br. J. Pharmacol. 88, 595 (1986). 12 G. L. Stahl, D. J. Lefer, and A. M. Lefer, Arch. Pharmacol. 336, 459 (1987). ~3 R. Levi, T, Y. Shen, S. J. Yee, D. A. Robertson, A. Genovese, O. W. Isom, and K. H. Krieger, in " N e w Horizons in Platelet Activating Factor Research" (C. M. Winslow and M. L. Lee, eds.), p. 255. John Wiley & Sons, Chichester, England, 1987. 14 K. Schrtr, P. K6hler, M. Miiller, B. A. Peskar, and P. R6sen, Am. J. Physiol. 241, HI8 0981). 15 M. Purchase, G. J. Dusting, D. M. F. Li, and M. A. Read, Circ. Res. 58, 172 (1986). t6 G. Montrucchio, G. Alloatti, C. Tetta, R. DeLuca, R. N. Saunders, G. Emanuelli, and G. Camussi, Am. J. Physiol. 256, H1236 (1989).
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"Langendorffheart" is prepared as described above. Washed platelets are prepared from platelet-rich plasma. The details of platelet preparation vary, with some investigators using PGE1 (Ref. 4) or PGI2 (Ref. 15) to limit platelet activation during the steps of centrifugation and suspension of the platelet pellet. The platelets are finally resuspended in an albumincontaining buffer lacking any PGE~ or PGI2. The platelets are infused into the aortic inflow tract just above the heart at a flow rate of 0.1 ml/min for guinea pig or rat hearts, or 0.1 to 1 ml/min in the rabbit heart, representing 1-3% of the total perfusate flow. The concentration of platelets used varies from - 1 × 106 tO 2 X 108 platelets per milliliter perfusate. The predominant use of this preparation has been in the study of interactions between PGI2 released from the heart, and TxA2 produced mainly by the platelets. The formation of these two mediators has been determined by bioassay where the coronary effluent continuously superfuses strips of vascular tissue (e.g., rabbit aorta, porcine or bovine coronary arteries; all three are contracted by TxA2, but only bovine coronary artery is relaxed by PGI2) or by radioimmunoassay of the stable hydrolysis products 6-keto-PGFj,~ and TxB2. Perfusion of hearts with a plateletcontaining buffer enhances the production of PGI2 and, to a lesser extent, TxA2, while the formation of both mediators can be increased substantially by the addition of arachidonic acid. ~4'15 Despite the formation of approximately three times m o r e T x A 2 than PGI2 in the presence of arachidonic acid, coronary vasodilatation predominates. Infusions of low concentrations of epinephrine (0.6-6 pmol/ml), that are insufficient to provoke platelet aggregation, potentiate platelet TxA2 production from arachidonic acid within the heart. ~5 It is proposed that perfused hearts produce a stable nonprostanoid factor that potentiates TxA2 release when incubated with platelets. ~5 Neutrophil-Perfused Hearts Rabbit hearts perfused in the Langendorffmode with rabbit neutrophils added to the perfusate have been used to study interactions between leukocytes and the heart. ~7'18 Neutrophils are prepared from citrated whole blood (3.8% citrate at ratio 1 : 10 citrate : blood), mixed with 6% dextran (molecular weight = 500,000; ratio 1:5) for sedimentation of erythrocytes. The supernatant is centrifuged (400 g for 5 min), contaminating erythrocytes are eliminated by hypotonic lysis, and the cells separated 17 M. N. Gillespie, S. Kojima, J. O. Owasoyo, H. H. Tai, and M. Jay, J. Pharmacol. Exp. Therap. 241, 812 (1987). t8 R. Kraemer and K. M. Mullane, J. Pharmacol. Exp. Therap. 251, 620 (1989).
616
CELL MODELS OF LIPID MEDIATOR PRODUCTION
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by Ficoll/Hypaque density gradient centrifugation (600 g for 20 rain). The pellet is resuspended in Hanks' balanced salt solution and is generally comprised of 90-95% neutrophils with cell viability close to 100%, as assessed by trypan blue exclusion. Neutrophils can also be radiolabeled with either indium-11117 or chromium-51,18 and radioactivity determined using a y-counter. The perfused heart preparation is essentially similar to that described above, except that the superior and inferior venae cavae and pulmonary veins are ligated, and the pulmonary artery cannulated to capture the coronary outflow and to perfuse the hearts in a recirculating mode. The reservoir volume is 400 ml, containing 5 × 103 neutrophils per milliliter in Krebs-Henseleit buffer with 1% BSA; it is gassed with 95%02 + 5%CO2 by either blowing the gas across the surface of the buffer in a revolving reservoir or by using a membrane lung system, to minimize "frothing" of the protein-rich buffer. The hearts are perfused at a constant flow of 25 to 30 ml/min at a left ventricular end-diastolic pressure (LVEDP) of 10 to 15 mm Hg and paced at a constant rate of 200 to 400 beats/min. Myocardial hypoxia is induced by gassing the perfusion fluid with either 12%O2 + 83%N2 + 5%CO2 or 100%N2 for 30 min and then reoxygenating with the original 95%02 + 5%CO2. Hypoxia and reoxygenation provoke the accumulation of radiolabeled neutrophils in the heart. 17'18 Hypoxia-induced neutrophil accumulation is attributed to the generation of LTB4 by the myocardium, as determined by increases in the tissue content of LTB4-1ike immunoreactivity and suppression of cell accumulation by diethylcarbamazine or nordihydroguaiaretic acid, purported lipoxygenase inhibitors. 17 Neutrophil sequestration is associated with a reduced recovery of function during reoxygenation. 18 Ischemia-Reperfusion
in Langendorff Hearts
Studies on the release and role of eicosanoids in myocardial ischemia and reperfusion have benefitted enormously from the isolated perfused heart technique. Hearts of rats, 19,20guinea pigs, 21 rabbits, 16'22 and cats 23 have been used in these studies. Generally, global ischemia is induced by decreasing the flow rate to < I ml/min for 30 to 120 min before restoring preischemic flow for a 15- to 30-rain reperfusion period. 16'19'2°'23 The degree of ischemia is more severe if the heart is paced at a fixed rate 19 M. Karmazyn, Am. J. Physiol. 251, H133 (1986). 2o W. G. Nayler, M. Purchase, and G. J. Dusting, Basic Res. Cardiol. 79, 125 (1984). 21 M. P. Mot'fat, J. Pharmacol. Exp. Ther. 242, 292 (1987). 22 A. Edlund, K. Sahlin, and A Wennmalm, J. Mol. Cell. Cardiol. 18, 1067 (1986). 23 H. Araki and A. M. Lefer, Circ. Res. 47, 757 (1980).
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throughout the procedure; this enables the ischemic period to be abbreviated and thus to minimize time-related changes in cardiac function. An alternative procedure that has been used in the rabbit heart is to ligate the left anterior descending branch of the main coronary artery for 2 hr to study the effect of PGI2 on regional ischemia.22 Ischemia does not provoke prostaglandin production; however, reperfusion of the globally ischemic rat 19 or guinea pig21 heart is a potent stimulus for PGI2 synthesis: PGI2 release is increased from 0.01 to 0.3-0.4 ng/ml/min or from 0.2 to 1.5 ng/ml/min (measured as 6-keto-PGF1, by radioimmunoassay) for the rat and guinea pig, respectively. This release is sustained in the rat heart, but rapidly declines in the guinea pig preparation. The magnitude and duration of PGI2 release at reperfusion is independent of the duration of the preceding ischemic period when varied from 5 to 60 min. 19 The significance of the PGI2 production at reperfusion on the postischemic recovery of cardiac function is a subject of controversy. PGI2 is reported to be either beneficial,2s d e t r i m e n t a l , 19'21'24 o r without e f f e c t . 22 While this discrepancy may relate in part to PGI2 improving the rate, but not the magnitude, of functional recovery,2° it appears that the discrepancy may also result from different concentration-dependent effects of PGI2. Small PGI2 concentrations that mimic the endogenously released material (-0.5-1 ng/ml) impair recovery of contractile function, whereas larger concentrations of PGI2 may be cardioprotective. 19,20,23,24However, the true effects of endogenous PGI2 produced by ischemia and reperfusion remain to be elucidated. Ischemia and reperfusion of the Langendorff rabbit heart is also a stimulus for transient PAF release. PAF is undetectable during control perfusion and ischemia (
