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Leukotriene D4 reduces coronary blood flow in the anesthetized dog
PROSTAGLANDINS
LEUKOTRIENE D4 REDUCES CORONARY BLOOD FLOW IN THE ANESTHETIZED DOG Maret J. Panzenbeck and Gabor Kaley Department of Physiology New York Medical College Valhalla. New York 10595 ABSTRACT We studied the effects of intracoronary administration of leukotriene (LT)D4 on coronary blood flow and myocardial function in chloralose anesthetized dogs. For comparison, the effects of injections of U-46619 were examined in the same dogs. Both LTD4 and U-46619 decreased coronary blood flow, left ventricular dP/dt and cardiac output. LTD4 was ten times more potent than U-46619 in decreasing coronary blood flow. The effects of neither drug were different after indomethacin administration. INTRODUCTION The leukotrienes,LTC,, and LTD4, are recently discovered metabolites of the lipoxygenase pathway of arachidonic acid metabolism (1, 2, 3) and are most likely responsible for the immediate hypersensitivity reactions that were thought to be mediated by "slow reacting substance of anaphylaxis" (SRS-A) (4). Both LTC4 and LTD4 are potent arteriolar constrictors in the circulation of the hamster cheek pouch (5). Because LTC$ and LTD are known to be formed in lung tissue during anaphylaxis (6) an ti because decreases in coronary blood flow and arrhythmias are known to occur following severe immediate hypersensitivity reactions (71, it is of interest to determine the possible effects of the leukotrienes on myocardial performance and the coronary circulation. Previous workers have shown that LTC4 and LTD are potent coronary vasoconstrictors in Langendorff heart preparazions of the guinea pig, rat and rabbit (8). It has also been reported recently that LTD causes a decrease in coronary blood flow and a reduction of regiona 4 wall shortening in anesthetized sheep (9). The present study was undertaken to investigate the effects of LTD4 on the coronary circulation and myocardial function of the anesthetized dog. Since many species differences exist with regard to the effects of other arachidonic acid metabolites. it seemed important to establish the effects of leukotrienes in the dog, an animal that is used extensively in cardiovascular studies.
MAY 1983 VOL. 25 NO. 5
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METHODS Animal Preparation: Eight male mongrel dogs with a mean weight of 28.8 Kg (S.~.=4.8) were used in this study. Anesthesia was induced with sodium thiopental (25 mglkg, IV), followed by The level of anesthesia was assessed chloralose (60 mg/kg, IV). periodically and was supplemented by administering a-chloralose at a minimum dose of 10 mg/kg/hr or more as needed. Dogs were intubated and the intubation tube was attached to a respiration pump (Harvard) for the maintenance of ventilation. The femoral vein and arterywere cannulated for the administration of anesthesia and the recording of arterial pressure (Narco Telecare transducer LDI-5). The dog was placed on its right side and an incision was made in the fifth left intercostal space. Just distal to the aortic arch the descending aorta was cleaned of surrounding connective tissue for a short distance and an electromagnetic flow probe (Carolina Medical Electronics 440 C) was placed around the aorta for the measurement of descending aortic flow, for an approximation of cardiac output. Following this procedure, the pericardium was incised parallel to the phrenic nerve and sewn to the chest wall, thus forming a pericardial cradle for support of the heart. A cannula connected to an external pressure transducer, for the measurement of left ventricular pressure, was introduced into the left ventricle through a stab wound in the apex of the heart and was held in place with a purse-string suture. In two dogs, left ventricular pressure was measured by advancing a catheter-tipped pressure transducer (Millar) into the left ventricle via the left femoral artery. The left circumflex coronary artery was carefully freed of fat and connective tissue for a short distance, approximately 2 cm distal to the origin of the left anterior descending coronary artery. A flow probe, of appropriate size (8 or 9 mm in circumference), was placed on the circumflex artery and a 23-gauge needle connected to a cannula for the injection of drugs was inserted into the artery just distal to the probe. All recordings were made using a direct writing oscillograph (Narco Biosystems). Heart rate was determined electronically with a cardiotachometer that was triggered by the blood pressure channel amplifier. Left ventricu!ar a measure of myocardial contractility, was determined by dP/dt feedi$pChe output of the left ventricular pressure channel amplifier through a differentiator coupler. All flow probes were calibrated and checked for zero flow by momentarily occluding the vessels distal to the probes. Preparation of Drugs: Leukotrienes were dissolved in distilled water at aconcentration of20 pglml and 1 ml aliquots were kept frozen at -70°C until the aay of the experiment. During each experiment LTD was kept on ice and diluted to the desired concentration with 0.'40 h saline just prior to injection into the circumflex artery. The endoperoxide analog, U-46619[(15S)-hydroxy-llc,9e(epoxymethano)prosta-52,13E-dienoic acid, Upjohn],was dissolved at a concentration of 5 mglml in distilled water containing 3 mg/ml sodium bicarbonate. This stock solution was kept refrigerated at
MAY
1983VOL.25NO.S
PROSTAGLANDINS 4OC and was diluted with 0.9% saline just prior to injection. Indomethacin was prepared fresh each day by dissolving the drug at a concentration of 50 mg/ml in l.OM TRIS buffer, pH 8.4. Mean arterial blood pressure was Calculations and Statistics: calculated as one-third of the sum of the systolic pressure and twice the diastolic pressure. All responses before and after the administration of indomethacin were analyzed for possible differences using a two-tailed Student's t-test. A probability of less than 0.05 was taken to indicate a statistically significant difference. An analysis ofcovariance for two groups was used to compare the doseresponse curves of LTD4 and U-46619 (10). Experimental Protocol: Following all surgical procedures, one hour was allowed for stabilization of cardiovascular function. Leukotriene D4, (50 ng-10 vg) and U-46619 (100 ng-50pg) were given as 1 ml bolus injections into the circumflex coronary artery.followed by a 2 ml 0.9% saline flush. Injections of vehicle (3 ml of 0.9% saline) resulted in small transient increases in coronary blood flow At least 5 but had no effect on other cardiovascular parameters. min were allowed after each injection for the return of all parameters to baseline. After a dose-response relationship had been established for each drug, indomethacin (5 mg/kg) was administered intravenously. Thirty minutes after the administration of indomethacin each drug was again injected into the circumflex artery at the same dosesas it had been given previously. A test dose of sodium arachidonate (500 pg) was injected into the coronary artery prior to and thirty minutes after the administration of indomethacin in order to establish the adequacy of cyclooxygenase inhibition. RESULTS Effects of LTD4: Injection of LTD4 into the coronary artery caused decreases in coronary blood flow, cardiac output and left ventricular dP/dt (Figure 1). The threshold dose of LTD needed to cause a decrease in coronary blood flow was 100 ng (2 x 40-l" mole). This dose caused a small, but statistically significant (p < .05) decrease in circumflex artery blood flow of -3.8+0.85 ml/min (n=6). The dose response curves for the effects of LTD4 on coronary blood flow are shown in Figure 2. The decrease in coronary blood flow to injections of LTD was characterized as being rapid in onset and 4 relatively long in duration. The time for coronary blood flow to return half way to its initial baseline value (t$) after injection of LTD4 ranged from 1.45kO.54 min at lug to 3.01*0.43 min at 5 ,,gand 2.5+0.68 min at 10 ug. At most doses, LTD4 had no significant effect on either left ventricular pressure. arterial pressure or heart rate (Figure 1). At a dose of 10 pig the administration of LTD caused a small rise in mean arterial pressure of 622.9 mm Hg (n-Y), although in one dog mean arterial pressure actually decreased by 5 mm Hg. The increased driving pressure is probably responsible for the apparent decrease in the ti of the flow response at this dose of LTD4. Often, arrhythmias were apparent in the pressure tracings following injections of LTD4. Increasing doses of LTD4 in-
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1983 VOL. 25 NO. 5
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BP (mmlig)
HR (beats.min-‘1
170l50-
120-T LVP (mmHg)
CX Flow (ml .min-‘1
100 50O-
co (L.min-‘1
LV dP/d (mmHg.sec-‘1
t LTD4 (lOp_g)
2 min
Figure 1. Reproduction of an actual record showing the effects of 10 ug of LTD4 injected into the left circumflex coronary artery of a dog. BP: pulsatile arterial blood pressure. HR: heart rate. LVP: left ventricular pressure. CX Flow: circumflex coronary blood flow. CO: cardiac output. LV dP/dt: left ventricular dP/dt. LTD caused a significant reduction in CX Flow, cardiac output and LV d# /dt.
664
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Figure 2. Effects of intra-coronary LTD4 and U-46619 on circumflex coronary blood flow. Mean baseline coronary blood flow of all dogs studied was 65k9.9 ml/min. Points represent the actual changes from baseline. With the exception of the changes induced by .05 ug of LTD4 and 1 pg of U-46619. all changes in coronary blood flow are significantly different from control. Standard errors and number of dogs used are also shown.
jetted into the circumflex coronary artery also reduced significantly cardiac output(Figure 3) and left ventricular dP/dt (Figure 4). Both of these parameters seemed to return towards normal at a rate similar to that of coronary blood flow. Effects of U-46619: Intra-coronary artery injections of U46619 caused dose-dependent decreases in coronary blood flow (Figure 2). The threshold dose of U-46619 needed to cause a fall in coronary blood flow was on the order of lug, however, not all dogs responded to this dose. The mean change in coronary blood flow to 1 ug of U-46619 was-3.0k1.44 ml/min (n=4). U-46619 atdosesof 5,10, and 50 ug caused reductions in left ventricular dP/dt of -250265 (n=4), -175263 (n=4) and -367267 (n=3) mm Hg/sec, and decreases in cardiac outputof -16Ok50 (n-5),-2902121 (n=b)and -102Ok153 (n=5) ml/ min, respectively. At these doses U-46619 had variable effects on heart rate and arterial blood pressure. The absence of an actual fall in blood pressure in the face of a significant decrease in cardiac output, especially following the 50 pg dose of U-46619, suggests that this drug caused peripheral vasoconstriction. The
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t$ of the blood flow response to injections of 5, 10 and 50 ug of U-46619 was 0.75iO.27. 1.07+0.24 and 2.21+ 0.69 min respectively. Effects of Indomethacin: Indomethacin had no effect on coronarv blood flow but increased resting mean arterial oressure (from 123k6.1 to 13gk3.8 mm Hg) and reduced heart rate (from 146fll.4 to 12Ok8.4 beats/min) significantly. Indomethacin totally abolished the pronounced increase in coronary blood flow to the injection of 5OOpg of arachidonic acid. On the other hand, responses to LTD4 or U-46619 were not affected by cyclooxygenase blockade. Dose response curves for the decrease in coronary blood flow in response to these two drugs were virtually superimposable on those obtained before the administration of indomethacin (not shown). Similarly, the effects of LTD4 on cardiac output (Figure 3) and left ventricular dP/dt (Figure 4) were not changed after indomethacin. In contrast, the small increase in arterial blood pressure seen after the injection of 10 ug of LTD was either attenuated or reverted to a fall in blood pressure. Ihe ta i of the blood flow responses to LTD4 or U-46619 were not significantly different after indomethacin administration. In one dog, with heart rate kept constant by pacing at 172 beatslmin, the intra-coronary actions of both LTC4 and LTD were tested following indomethacin administration. The effects o r these agents on coronary blood flow and cardiac output were virtually identical to each other throughout the full dose range tested.
0.1
0.5
I
5
1.0
5.0
IO.Okg LTDq
I
-100
6 .;
-200
6
2 -300 z a
6
-400 -
-500
N 1
Control
Fssl After
lndomethacin 6
Figure 3. The effects of various doses of intra-coronary LTD on cardiac output (CO) in ml/min, before and after indomethacin 't 5 mg/kg). Bars represent the actual changes from baseline. Standard errors and number of dogsused are also shown. Only dogs in which LTD4 was given before and after indomethacin are included in this figure. Mean baseline cardiac output was 2.8f.22 before and 2.8c.16 L/min after indomethacin.
MAY 1983 VOL. 25 NO. 5
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On the other hand, injections of up to 50 ug of LTD4 into the circumflex coronary artery of three dogs, both before and after the administration of
‘04
-100
-600
_
NC
ontrol
&! After
4
lndomethacin
4. The effects of various doses of LTD,,, injected into the Bars circumflex coronary artery on left ventricular'(LV) dP/dt represent the actual changes from baseline. Standard err%%'and number of dogs used are also shown. Only dogs in which LTD4 was given before and after indomethacin are included in this figure. Mean baseline LV dP/dtmax was 26932147 before and 2857k300 mmHgl set after indomethacin.
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DISCUSSION We have demonstrated that LTD4 causes dose-related reductions in coronary blood flow, cardiac output and left ventircular dP/dt in open-chest anesthetized dogs. These effects are unrelated to the release of cyclooxygenase products as indomethacin had no effect on the responses. Leukotriene D4 is by an order of magnitude more potent than U-46619, a thromboxane A2-like agonist (11). in decreasing coronary blood flow. These results parallel the findings of other workers who studied the effects of LTD on the isolated 4 hearts of the guinea pig, rat and rabbit (8)and on the intact heart of anesthetized sheep (9). The decrease in coronary blood flow seen after injection of LTD4 is most likely due to vasoconstriction of the resistance vessels of the coronary vascular bed. Arterial driving pressure was not significantly changed at mostdoses of LTD4,but calculated coronary vascular resistance increased in all instances. It is also possible that the observed decrease in flow was caused by compression of the resistance vessels, secondary to plasma leakage into the tissue spaces surrounding the capillaries. Leukotrienes have been shown previously to cause extravasation of macromolecules and plasma leakage in the microcirculation of the hamster cheek pouch (5). The rapid onset of the decrease in blood flow, however, makes this mechanism unlikely as a contributory cause. In the present study. the decrease in myocardial performance, as measured by the changes in cardiac output and left ventricular dP/dt.might not be a direct effect of LTD . The observed negative inotropic effect may be secondary to the r all in the coronary blood flow and hence a decrease in oxygen delivery to the myocardium. However, Michelassi et al. (9) have shown that in sheep the depressed regional shortening of the ventricular wall produced by LTD4 was not abolished by FPL 55712, whereas the decrease in blood flow was inhibited. Thus, LTD may have myocardial depressant activity, independent of its erfeet on coronary blood flow. The possible pathophysiologicalroles of LTD4 in mediating the myocardial ischemia and heart failure are apparent. Levi et al. (12) have recently demonstrated that in guinea pigs, leukotrienes play a significant role in the decreased coronary blood flow and cardiac dysfunction seen following immediate hypersensitivity reactions. Additionally, in myocardial infarction followed by leukocyte infiltration of the damaged myocardium (13), release of leukotrienes could serve to further depress blood flow to the area at risk, thus setting off a vicious cycle of cell damage. It is also interesting to speculate whether the paradoxical "ischemic" vasoconstriction recently described could not be related to the synthesis of leukotrienes in the myocardium (14). In summary, our studies support the suggestion that leukotrienes might have an important role in a variety of conditions related to myocardial dysfunction.
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ACKNOWLEDGMENTS
We thank Dr. J. Pike,Upjohn Company (Kalamazoo, Michigan) for supplying U-46619; Dr. J. Rokach, Merck-Frosst Laboratories (Pointe Claire, Quebec, Canada) for synthetic leukotrienes and Dr. F. Kuehl, Merck, Inc. (Rahway, New Jersey) for indomethacin. Also, we gratefully acknowledge Arsenio Baez for his excellent technical assistance and Ms. Kathleen Vagi for her expert help in preparing the manuscript. This work was supported in part by USPHS grant HL 24934 and a grant from the Whitehall Foundation.
REFERENCES 1.
Samuelsson, B., P. Borgeat, S. Hammarstrgm and R. C. Murphy. Introduction of a nomenclature: leukotrienes. Prostaglandins -17:785. 1979. Morris, H-R., G.W. Taylor, P.J. Piper,M.N. Samhoun and J.R. Tippins. Slow reacting substances (SRSs); the structure identification of SRSs from rat basophil leukaemia (RBL-I) cells. Prostaglandins-19:185. 1980.
3.
Samuelsson, B. and S. HammarstrEm. Nomenclaturefor leukotrienes. Prostaglandins. -19:645. 1980.
4.
Orange, R. P. and K. F. Austen. Slow-reacting substance of anaphylaxis. Ad. Immunology. -10:105. 1969.
5.
Dahlen, S-E., J. Bjork, P. Hedqvist, K.-E. Arfors, S. HammarstrSm, J.-A. Lindgren and B. Samuelsson. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: In vivo effects with relevance to the acute inflammatory response. Proc. Natl. Acad. SC. USA 78~3887. 1981. Lewis, R.A. and K.F. Austen, Mediation of local homeostasis and inflammation by leukotrienes and other mast cell-dependent compounds. Nature. 293:1o3. 1981. Feigen, G.A. and D.J. Prager. Experimental cardiac anaphylaxis. Physiologic, pharmacologic and biochemical aspects of immune reactions in the isolated heart. Amer. J. Cardiology. 243474. 1969.
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Piper, P-J., L.G. Letts, M.N. Samhoun, J.R. Tippins and M.A. Palmer. In: Advances in Prostaglandin, Thromboxane and Leukotriez Research. Vol. 9 (B.Samuelssonand R. Paoletti, eds.) Raven Press, New York, 1982. p. 169.
9.
Michelassi, F., L. Landa, R.D. Hill, E. Lowenstein, W.D. Watkins, A.J. Petkau and W.M. Zapol. Leukotriene D4: a potent coronary artery vasoconstrictor associated with im1982. paired ventricular contraction. Science 217:841.
10.
Armitage, P. Statistical Methods in Medical Research. Blackwell Scientific Publications, Oxford, 1971. p. 288.
11.
Coleman, R.A., P.P.A. Humphrey, I. Kennedy, G.P. Levy and P. Lumley. U-46619, a selective thromboxane A2-like agonist? Br. J. Pharmacol. -68:127P. 1980.
12.
Levi, El.,J.A. Burke andE.J. Corey. In: Advances in Prostaglandin, Thromboxane and Leukotxene Research, Vol. 9 (B. Samuelsson and R. Paoletti, eds. ) Raven Press, New York, 1982, p. 215.
13.
Romson, J., B. Hook, S. Kunkel, G. Abrams and B.R. Lucchesi. Reduction in myocardial infarct size by neutrophil depletion in the dog. Circulation -66 (Supp. II): n-85. 1982.
14.
Sparks, H.V., and M.W. Gorman. In: Vasodilation (P.M. Vanhoutte and I. Leusen, eds.) zven Press, New York, 1981 p. 193.
Editor: Peter W. Ramwell
670
Received: l-21-83
Accepted: 4-5-83
MAY 1983 VOL. 25 NO. 5
LEUKOTRIENE D4 REDUCES CORONARY BLOOD FLOW IN THE ANESTHETIZED DOG Maret J. Panzenbeck and Gabor Kaley Department of Physiology New York Medical College Valhalla. New York 10595 ABSTRACT We studied the effects of intracoronary administration of leukotriene (LT)D4 on coronary blood flow and myocardial function in chloralose anesthetized dogs. For comparison, the effects of injections of U-46619 were examined in the same dogs. Both LTD4 and U-46619 decreased coronary blood flow, left ventricular dP/dt and cardiac output. LTD4 was ten times more potent than U-46619 in decreasing coronary blood flow. The effects of neither drug were different after indomethacin administration. INTRODUCTION The leukotrienes,LTC,, and LTD4, are recently discovered metabolites of the lipoxygenase pathway of arachidonic acid metabolism (1, 2, 3) and are most likely responsible for the immediate hypersensitivity reactions that were thought to be mediated by "slow reacting substance of anaphylaxis" (SRS-A) (4). Both LTC4 and LTD4 are potent arteriolar constrictors in the circulation of the hamster cheek pouch (5). Because LTC$ and LTD are known to be formed in lung tissue during anaphylaxis (6) an ti because decreases in coronary blood flow and arrhythmias are known to occur following severe immediate hypersensitivity reactions (71, it is of interest to determine the possible effects of the leukotrienes on myocardial performance and the coronary circulation. Previous workers have shown that LTC4 and LTD are potent coronary vasoconstrictors in Langendorff heart preparazions of the guinea pig, rat and rabbit (8). It has also been reported recently that LTD causes a decrease in coronary blood flow and a reduction of regiona 4 wall shortening in anesthetized sheep (9). The present study was undertaken to investigate the effects of LTD4 on the coronary circulation and myocardial function of the anesthetized dog. Since many species differences exist with regard to the effects of other arachidonic acid metabolites. it seemed important to establish the effects of leukotrienes in the dog, an animal that is used extensively in cardiovascular studies.
MAY 1983 VOL. 25 NO. 5
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PROSTAGLANDINS
METHODS Animal Preparation: Eight male mongrel dogs with a mean weight of 28.8 Kg (S.~.=4.8) were used in this study. Anesthesia was induced with sodium thiopental (25 mglkg, IV), followed by The level of anesthesia was assessed chloralose (60 mg/kg, IV). periodically and was supplemented by administering a-chloralose at a minimum dose of 10 mg/kg/hr or more as needed. Dogs were intubated and the intubation tube was attached to a respiration pump (Harvard) for the maintenance of ventilation. The femoral vein and arterywere cannulated for the administration of anesthesia and the recording of arterial pressure (Narco Telecare transducer LDI-5). The dog was placed on its right side and an incision was made in the fifth left intercostal space. Just distal to the aortic arch the descending aorta was cleaned of surrounding connective tissue for a short distance and an electromagnetic flow probe (Carolina Medical Electronics 440 C) was placed around the aorta for the measurement of descending aortic flow, for an approximation of cardiac output. Following this procedure, the pericardium was incised parallel to the phrenic nerve and sewn to the chest wall, thus forming a pericardial cradle for support of the heart. A cannula connected to an external pressure transducer, for the measurement of left ventricular pressure, was introduced into the left ventricle through a stab wound in the apex of the heart and was held in place with a purse-string suture. In two dogs, left ventricular pressure was measured by advancing a catheter-tipped pressure transducer (Millar) into the left ventricle via the left femoral artery. The left circumflex coronary artery was carefully freed of fat and connective tissue for a short distance, approximately 2 cm distal to the origin of the left anterior descending coronary artery. A flow probe, of appropriate size (8 or 9 mm in circumference), was placed on the circumflex artery and a 23-gauge needle connected to a cannula for the injection of drugs was inserted into the artery just distal to the probe. All recordings were made using a direct writing oscillograph (Narco Biosystems). Heart rate was determined electronically with a cardiotachometer that was triggered by the blood pressure channel amplifier. Left ventricu!ar a measure of myocardial contractility, was determined by dP/dt feedi$pChe output of the left ventricular pressure channel amplifier through a differentiator coupler. All flow probes were calibrated and checked for zero flow by momentarily occluding the vessels distal to the probes. Preparation of Drugs: Leukotrienes were dissolved in distilled water at aconcentration of20 pglml and 1 ml aliquots were kept frozen at -70°C until the aay of the experiment. During each experiment LTD was kept on ice and diluted to the desired concentration with 0.'40 h saline just prior to injection into the circumflex artery. The endoperoxide analog, U-46619[(15S)-hydroxy-llc,9e(epoxymethano)prosta-52,13E-dienoic acid, Upjohn],was dissolved at a concentration of 5 mglml in distilled water containing 3 mg/ml sodium bicarbonate. This stock solution was kept refrigerated at
MAY
1983VOL.25NO.S
PROSTAGLANDINS 4OC and was diluted with 0.9% saline just prior to injection. Indomethacin was prepared fresh each day by dissolving the drug at a concentration of 50 mg/ml in l.OM TRIS buffer, pH 8.4. Mean arterial blood pressure was Calculations and Statistics: calculated as one-third of the sum of the systolic pressure and twice the diastolic pressure. All responses before and after the administration of indomethacin were analyzed for possible differences using a two-tailed Student's t-test. A probability of less than 0.05 was taken to indicate a statistically significant difference. An analysis ofcovariance for two groups was used to compare the doseresponse curves of LTD4 and U-46619 (10). Experimental Protocol: Following all surgical procedures, one hour was allowed for stabilization of cardiovascular function. Leukotriene D4, (50 ng-10 vg) and U-46619 (100 ng-50pg) were given as 1 ml bolus injections into the circumflex coronary artery.followed by a 2 ml 0.9% saline flush. Injections of vehicle (3 ml of 0.9% saline) resulted in small transient increases in coronary blood flow At least 5 but had no effect on other cardiovascular parameters. min were allowed after each injection for the return of all parameters to baseline. After a dose-response relationship had been established for each drug, indomethacin (5 mg/kg) was administered intravenously. Thirty minutes after the administration of indomethacin each drug was again injected into the circumflex artery at the same dosesas it had been given previously. A test dose of sodium arachidonate (500 pg) was injected into the coronary artery prior to and thirty minutes after the administration of indomethacin in order to establish the adequacy of cyclooxygenase inhibition. RESULTS Effects of LTD4: Injection of LTD4 into the coronary artery caused decreases in coronary blood flow, cardiac output and left ventricular dP/dt (Figure 1). The threshold dose of LTD needed to cause a decrease in coronary blood flow was 100 ng (2 x 40-l" mole). This dose caused a small, but statistically significant (p < .05) decrease in circumflex artery blood flow of -3.8+0.85 ml/min (n=6). The dose response curves for the effects of LTD4 on coronary blood flow are shown in Figure 2. The decrease in coronary blood flow to injections of LTD was characterized as being rapid in onset and 4 relatively long in duration. The time for coronary blood flow to return half way to its initial baseline value (t$) after injection of LTD4 ranged from 1.45kO.54 min at lug to 3.01*0.43 min at 5 ,,gand 2.5+0.68 min at 10 ug. At most doses, LTD4 had no significant effect on either left ventricular pressure. arterial pressure or heart rate (Figure 1). At a dose of 10 pig the administration of LTD caused a small rise in mean arterial pressure of 622.9 mm Hg (n-Y), although in one dog mean arterial pressure actually decreased by 5 mm Hg. The increased driving pressure is probably responsible for the apparent decrease in the ti of the flow response at this dose of LTD4. Often, arrhythmias were apparent in the pressure tracings following injections of LTD4. Increasing doses of LTD4 in-
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1983 VOL. 25 NO. 5
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BP (mmlig)
HR (beats.min-‘1
170l50-
120-T LVP (mmHg)
CX Flow (ml .min-‘1
100 50O-
co (L.min-‘1
LV dP/d (mmHg.sec-‘1
t LTD4 (lOp_g)
2 min
Figure 1. Reproduction of an actual record showing the effects of 10 ug of LTD4 injected into the left circumflex coronary artery of a dog. BP: pulsatile arterial blood pressure. HR: heart rate. LVP: left ventricular pressure. CX Flow: circumflex coronary blood flow. CO: cardiac output. LV dP/dt: left ventricular dP/dt. LTD caused a significant reduction in CX Flow, cardiac output and LV d# /dt.
664
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Figure 2. Effects of intra-coronary LTD4 and U-46619 on circumflex coronary blood flow. Mean baseline coronary blood flow of all dogs studied was 65k9.9 ml/min. Points represent the actual changes from baseline. With the exception of the changes induced by .05 ug of LTD4 and 1 pg of U-46619. all changes in coronary blood flow are significantly different from control. Standard errors and number of dogs used are also shown.
jetted into the circumflex coronary artery also reduced significantly cardiac output(Figure 3) and left ventricular dP/dt (Figure 4). Both of these parameters seemed to return towards normal at a rate similar to that of coronary blood flow. Effects of U-46619: Intra-coronary artery injections of U46619 caused dose-dependent decreases in coronary blood flow (Figure 2). The threshold dose of U-46619 needed to cause a fall in coronary blood flow was on the order of lug, however, not all dogs responded to this dose. The mean change in coronary blood flow to 1 ug of U-46619 was-3.0k1.44 ml/min (n=4). U-46619 atdosesof 5,10, and 50 ug caused reductions in left ventricular dP/dt of -250265 (n=4), -175263 (n=4) and -367267 (n=3) mm Hg/sec, and decreases in cardiac outputof -16Ok50 (n-5),-2902121 (n=b)and -102Ok153 (n=5) ml/ min, respectively. At these doses U-46619 had variable effects on heart rate and arterial blood pressure. The absence of an actual fall in blood pressure in the face of a significant decrease in cardiac output, especially following the 50 pg dose of U-46619, suggests that this drug caused peripheral vasoconstriction. The
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1983 VOL. 25 NO. 5
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t$ of the blood flow response to injections of 5, 10 and 50 ug of U-46619 was 0.75iO.27. 1.07+0.24 and 2.21+ 0.69 min respectively. Effects of Indomethacin: Indomethacin had no effect on coronarv blood flow but increased resting mean arterial oressure (from 123k6.1 to 13gk3.8 mm Hg) and reduced heart rate (from 146fll.4 to 12Ok8.4 beats/min) significantly. Indomethacin totally abolished the pronounced increase in coronary blood flow to the injection of 5OOpg of arachidonic acid. On the other hand, responses to LTD4 or U-46619 were not affected by cyclooxygenase blockade. Dose response curves for the decrease in coronary blood flow in response to these two drugs were virtually superimposable on those obtained before the administration of indomethacin (not shown). Similarly, the effects of LTD4 on cardiac output (Figure 3) and left ventricular dP/dt (Figure 4) were not changed after indomethacin. In contrast, the small increase in arterial blood pressure seen after the injection of 10 ug of LTD was either attenuated or reverted to a fall in blood pressure. Ihe ta i of the blood flow responses to LTD4 or U-46619 were not significantly different after indomethacin administration. In one dog, with heart rate kept constant by pacing at 172 beatslmin, the intra-coronary actions of both LTC4 and LTD were tested following indomethacin administration. The effects o r these agents on coronary blood flow and cardiac output were virtually identical to each other throughout the full dose range tested.
0.1
0.5
I
5
1.0
5.0
IO.Okg LTDq
I
-100
6 .;
-200
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N 1
Control
Fssl After
lndomethacin 6
Figure 3. The effects of various doses of intra-coronary LTD on cardiac output (CO) in ml/min, before and after indomethacin 't 5 mg/kg). Bars represent the actual changes from baseline. Standard errors and number of dogsused are also shown. Only dogs in which LTD4 was given before and after indomethacin are included in this figure. Mean baseline cardiac output was 2.8f.22 before and 2.8c.16 L/min after indomethacin.
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On the other hand, injections of up to 50 ug of LTD4 into the circumflex coronary artery of three dogs, both before and after the administration of
‘04
-100
-600
_
NC
ontrol
&! After
4
lndomethacin
4. The effects of various doses of LTD,,, injected into the Bars circumflex coronary artery on left ventricular'(LV) dP/dt represent the actual changes from baseline. Standard err%%'and number of dogs used are also shown. Only dogs in which LTD4 was given before and after indomethacin are included in this figure. Mean baseline LV dP/dtmax was 26932147 before and 2857k300 mmHgl set after indomethacin.
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DISCUSSION We have demonstrated that LTD4 causes dose-related reductions in coronary blood flow, cardiac output and left ventircular dP/dt in open-chest anesthetized dogs. These effects are unrelated to the release of cyclooxygenase products as indomethacin had no effect on the responses. Leukotriene D4 is by an order of magnitude more potent than U-46619, a thromboxane A2-like agonist (11). in decreasing coronary blood flow. These results parallel the findings of other workers who studied the effects of LTD on the isolated 4 hearts of the guinea pig, rat and rabbit (8)and on the intact heart of anesthetized sheep (9). The decrease in coronary blood flow seen after injection of LTD4 is most likely due to vasoconstriction of the resistance vessels of the coronary vascular bed. Arterial driving pressure was not significantly changed at mostdoses of LTD4,but calculated coronary vascular resistance increased in all instances. It is also possible that the observed decrease in flow was caused by compression of the resistance vessels, secondary to plasma leakage into the tissue spaces surrounding the capillaries. Leukotrienes have been shown previously to cause extravasation of macromolecules and plasma leakage in the microcirculation of the hamster cheek pouch (5). The rapid onset of the decrease in blood flow, however, makes this mechanism unlikely as a contributory cause. In the present study. the decrease in myocardial performance, as measured by the changes in cardiac output and left ventricular dP/dt.might not be a direct effect of LTD . The observed negative inotropic effect may be secondary to the r all in the coronary blood flow and hence a decrease in oxygen delivery to the myocardium. However, Michelassi et al. (9) have shown that in sheep the depressed regional shortening of the ventricular wall produced by LTD4 was not abolished by FPL 55712, whereas the decrease in blood flow was inhibited. Thus, LTD may have myocardial depressant activity, independent of its erfeet on coronary blood flow. The possible pathophysiologicalroles of LTD4 in mediating the myocardial ischemia and heart failure are apparent. Levi et al. (12) have recently demonstrated that in guinea pigs, leukotrienes play a significant role in the decreased coronary blood flow and cardiac dysfunction seen following immediate hypersensitivity reactions. Additionally, in myocardial infarction followed by leukocyte infiltration of the damaged myocardium (13), release of leukotrienes could serve to further depress blood flow to the area at risk, thus setting off a vicious cycle of cell damage. It is also interesting to speculate whether the paradoxical "ischemic" vasoconstriction recently described could not be related to the synthesis of leukotrienes in the myocardium (14). In summary, our studies support the suggestion that leukotrienes might have an important role in a variety of conditions related to myocardial dysfunction.
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ACKNOWLEDGMENTS
We thank Dr. J. Pike,Upjohn Company (Kalamazoo, Michigan) for supplying U-46619; Dr. J. Rokach, Merck-Frosst Laboratories (Pointe Claire, Quebec, Canada) for synthetic leukotrienes and Dr. F. Kuehl, Merck, Inc. (Rahway, New Jersey) for indomethacin. Also, we gratefully acknowledge Arsenio Baez for his excellent technical assistance and Ms. Kathleen Vagi for her expert help in preparing the manuscript. This work was supported in part by USPHS grant HL 24934 and a grant from the Whitehall Foundation.
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Samuelsson, B., P. Borgeat, S. Hammarstrgm and R. C. Murphy. Introduction of a nomenclature: leukotrienes. Prostaglandins -17:785. 1979. Morris, H-R., G.W. Taylor, P.J. Piper,M.N. Samhoun and J.R. Tippins. Slow reacting substances (SRSs); the structure identification of SRSs from rat basophil leukaemia (RBL-I) cells. Prostaglandins-19:185. 1980.
3.
Samuelsson, B. and S. HammarstrEm. Nomenclaturefor leukotrienes. Prostaglandins. -19:645. 1980.
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Orange, R. P. and K. F. Austen. Slow-reacting substance of anaphylaxis. Ad. Immunology. -10:105. 1969.
5.
Dahlen, S-E., J. Bjork, P. Hedqvist, K.-E. Arfors, S. HammarstrSm, J.-A. Lindgren and B. Samuelsson. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: In vivo effects with relevance to the acute inflammatory response. Proc. Natl. Acad. SC. USA 78~3887. 1981. Lewis, R.A. and K.F. Austen, Mediation of local homeostasis and inflammation by leukotrienes and other mast cell-dependent compounds. Nature. 293:1o3. 1981. Feigen, G.A. and D.J. Prager. Experimental cardiac anaphylaxis. Physiologic, pharmacologic and biochemical aspects of immune reactions in the isolated heart. Amer. J. Cardiology. 243474. 1969.
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Piper, P-J., L.G. Letts, M.N. Samhoun, J.R. Tippins and M.A. Palmer. In: Advances in Prostaglandin, Thromboxane and Leukotriez Research. Vol. 9 (B.Samuelssonand R. Paoletti, eds.) Raven Press, New York, 1982. p. 169.
9.
Michelassi, F., L. Landa, R.D. Hill, E. Lowenstein, W.D. Watkins, A.J. Petkau and W.M. Zapol. Leukotriene D4: a potent coronary artery vasoconstrictor associated with im1982. paired ventricular contraction. Science 217:841.
10.
Armitage, P. Statistical Methods in Medical Research. Blackwell Scientific Publications, Oxford, 1971. p. 288.
11.
Coleman, R.A., P.P.A. Humphrey, I. Kennedy, G.P. Levy and P. Lumley. U-46619, a selective thromboxane A2-like agonist? Br. J. Pharmacol. -68:127P. 1980.
12.
Levi, El.,J.A. Burke andE.J. Corey. In: Advances in Prostaglandin, Thromboxane and Leukotxene Research, Vol. 9 (B. Samuelsson and R. Paoletti, eds. ) Raven Press, New York, 1982, p. 215.
13.
Romson, J., B. Hook, S. Kunkel, G. Abrams and B.R. Lucchesi. Reduction in myocardial infarct size by neutrophil depletion in the dog. Circulation -66 (Supp. II): n-85. 1982.
14.
Sparks, H.V., and M.W. Gorman. In: Vasodilation (P.M. Vanhoutte and I. Leusen, eds.) zven Press, New York, 1981 p. 193.
Editor: Peter W. Ramwell
670
Received: l-21-83
Accepted: 4-5-83
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