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Coronary vasoconstriction and PGI2 release by leukotrienes in isolated guinea pig hearts
357
European Journal of Pharmacology, 73 (1981) 357-361 Elsevier/North-Holland Biomedical Press
Short communication
CORONARY VASOCONSTRICTION AND PGI 2 RELEASE BY LEUKOTRIENES IN ISOLATED GUINEA PIG HEARTS ZEN-ICHI TERASHITA, HIROSHI FUKUI, MINORU HIRATA, SHINJO TERAO, SHIGENORI OHKAWA, KOKEI NISHIKAWA and SHINTARO KIKUCHI Central Research Division, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka 532, Japan Received 19 June 1981, accepted 23 June 1981
Z. TERASHITA, H. FUKUI, M. HIRATA, S. TERAO, S. OHKAWA, K. NISHIKAWA and S. KIKUCHI, Coronary vasoconstriction and PGI2 release by leukotrienes in isolated guinea pig hearts, European J. Pharmacol. 73 (1981) 357-361. In isolated, perfused guinea pig hearts, leukotrienes C4 and D 4 (0.1-lO3 ng) dose-dependently decreased coronary flow rate and left ventricular systolic pressure. The leukotrienes (10-103 ng) released prostacyclin, but not thromboxane A2, from the heart. In isolated atria, they (10-s_ 10-6M) showed slight positive inotropism. Thus, it is concluded that leukotrienes C4 and D4 are potent coronary vasoconstrictors, and might play a role in coronary vasospasm during cardiac anaphylaxis. Coronary vasoconstriction
Leukotrienes
Prostanoid release
I. Introduction
2. Materials and methods
Coronary vasoconstriction and arrhythmia have been observed in cardiac anaphylaxis induced by an antigen (Liebig et al., 1975; Anhut et al., 1977). The chemical mediators of these effects have been the subject of extensive investigations, and slow reacting substance of anaphylaxis (SRS-A), thromboxane A 2 (TXA2) and other vasoactive substances have been detected in coronary perfusate (Liebig et al., 1975; Anhut et al., 1977). Recently, Lewis et al. (1980) suggested that SRS-A was composed of leukotrienes C4 and D 4 (LTC4 and LTD4). Although potent contractile activity of LTC 4 and LTD4 on several smooth muscles has been reported (Drazen et al., 1980), the effects of the leukotrienes on the heart, a target organ in systemic anaphylaxis (Capurro and Levi, 1975), have not been clarified. In this report, we describe the effects of LTC 4 and LTD4 on coronary flow rate, heart rate, left ventricular systolic pressure and its dp/dt, and release of prostanoids in isolated guinea pig hearts.
2.1. Materials [ 3 H]6- keto- Prostaglandin F la ([ 3H]6 - ketoPGFI, ) (100 Ci/mmol, New England Nuclear, USA), [3H]TXB2 (150 Ci/mmol, N e w England Nuclear, USA), antisera to 6-keto-PGFl~ and TXB2 (prepared by us), 6-keto-PGFl~, and TXB2 (synthesized by mr. Kato in this Chemistry Laboratory) were used.
2.2. Isolated heart preparation Heparinized male guinea pigs (about 250 g) were killed by a blow on the head, and the heart was excised. A polyethylene cannula for perfusion was inserted retrogradely into the aorta, and the heart was perfused according to the method of Langendorff (Terashita et al., 1978). The heart was suspended in a chamber maintained at 37°C, and perfused at a constant pressure of 750 mm H20 with a modified Krebs-Ringer bicarbonate solu-
0014-2999/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
358
tion composed of (raM) 127 NaC1, 4.7 KC1, 2.5 CaC12, 1.2 KH2PO 4, 25 NaHCO 3, 2.0 sodium pyruvate and 5.5 glucose, warmed to 37°C and bubbled with 3% CO 2 in 02. Coronary flow rate was measured with a cannulating-type flow probe of an electromagnetic flowmeter (Nihon Kohden, MF-26) placed just above the aortic cannula. A urethane polymer balloon connected to a glass tube was inserted into the left ventricular cavity through an incision in the left auricle. Left ventricular isovolumetric pressure was recorded with a pressure transducer (Nihon Kohden, MPU-0.5) connected to the intraventricular balloon filled with physiological saline. The heart rate was recorded from left ventricular pressure pulses with a cardiotachometer (Nihon Kohden, RT-2). Experiments were started after a 20-30 min adaptation period. The basal coronary flow rate, left ventricular systolic pressure and its dp/dt, and heart rate (the mean_S.E.M.) were 8.0___0.4 ml/min, 82.7___3.8 mm Hg, 1657___109 mm Hg/sec and 245-+-8 beats/min (n=12), respectively. Only one dose-response relation for each leukotriene was determined in each preparation. LTC4 and LTD 4 were dissolved in saline (200/~1), and injected in a bolus into the coronary perfusion system. The perfused hearts weighed 1.4-1.5 g.
2.3. Isolated atrial preparation The isolated atrial preparation was suspended with a diastolic tension of 0.5 g in a 40 ml organ bath containing Krebs-Henseleit solution, bubbled with 3% CO 2 in 02 at 30°C. The right atrium was allowed to beat spontaneously to measure the beating rate, and the left atrium was driven electrically by square wave pulses (5 V, 1 msec, 1 Hz) to measure the contractile force. Only one cumulative dose-response relation for each leukotriene was obtained in each preparation. Each dose was applied at intervals between 5-10 min after each plateau level of response was attained. The basal contractile force and beating rate were 971 ___88 mg and 129___6 beats/rain (n--6), respectively.
2.4. Radioimmunoassay of 6-keto-PGFla and TXB: in the perfusate The perfusate was collected serially o( intermittenly for 1 min just before and from 0 to 10 min after the injection of the test agents. Fifty/~1 of the perfusate were used to determine 6,keto-PGFj~ and TXB2 by a radioimmunoassay technique (Shibouta et al., 1981). The assay technique enabled us to detect 10-1000 pg of both prostanoids. Cross-reactivities of 6-keto-PGF~, antiserum with TXB 2 and other prostaglandins were less than 0.1% for PGD 2 and TXB2, 1.8% for PGE l, 1.3% for PGE 2 and 4.5% for PGF2a. Those of the TXB 2 antiserum were less than 1.0% for PGE1; PGE 2, PGF2~ and 6-keto-PGFla and 1.1% for PGD 2. LTC 4 and LTD 4, up to 100 ng, showed no crossreaction with either of the antisera.
2.5. Synthesis of L T C 4 and L T D 4 LTA 4 methyleser was prepared in three steps by using the (5S,6S)-methyl-5,6-epoxy-7hydroxyheptanoate derived from D-araboascorbic acid (Cohen et al., 1980, Gleason et al., 1980). LTC 4 and LTD 4 as potassium salts were obtained by the coupling reactions of LTA 4 methylester with the corresponding peptide components followed by alkaline hydrolysis. Purification was carried out by reverse phase high performance liquid chromatography. They were stored as a methanol solution of their potassium salts below - 2 0 ° C until used. The dose of leukotrienes is presented as their potassium salts.
3. Results
3.1. Coronary and cardiac actions of LTC 4 and L T D 4 in isolated guinea pig hearts The administration of both leukotrienes (0.1100 ng) rapidly decreased the coronary flow rate, and depressed the left ventricular systolic pressure and its d p / d t (fig. 1). Fig. 1A shows typical tracings of these experiments at 100 ng of LTC 4 and LTD4. At the highest dose (100 ng), LTC4 was more potent and had a longer lasting effect than
359
B
A (7 CA F (mb,n~wn)
5' 20'
I0 r
0L ~
0
,~
o.,--o LTC4
10
~
LTD 4
2O (ram I..~)
•
L v c~o,tl20001:
m"
(~mHo~) 0" HR
L.
300[
~
(b,,,mr:,~) 2ooL
50 60
ff CAF (ml/n~n)
5' Iff
I0 0E
LVP (,~ ~) ~
o_
0
>
_.J
19
"
20
Lv a ~
HR
2501" _ j
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0.i
200'-
i
10
100
Dose (ng)
~oong
Fig. 1. (A) Effects of 100 ng of LTC+ (upper) and LTD 4 (lower) on coronary flow rate and cardiac parameters. (B) Dose-dependent effects of leukotrienes on coronary flow rate and left ventricular systolic pressure. Values are the m e a n _ S . E . M . (n=6). CAF: coronary arterial flow; LVP, left ventricular pressure; L V d p / d t : left ventricular d p / d t ; HR: heart rate.
2.0 A
LTC4
LT D/~
1.5
~: 1.0
.~ 0.5
02/.6810
~
0246810
I()0
0 2 4 6 8 10 10 ng
0 2 4 6 8 10 {rain) 100 ng
Fig. 2. Effects of LTC 4 and LTD 4 on 6-keto-POFl~, release in isolated guinea pig hearts. Values are the m e a n ± S . E . M . (n=6).
360
LTD 4 in decreasing coronary flow rate. At the lower doses (0.1, 1 and 10 ng), the coronary action of LTC4 and LTD 4 was almost as potent (fig. 1B). Almost the same description can be applied to the depression in left ventricular systolic pressure and its dp/dt. At the highest dose (100 ng) this negative inotropic effect of LTC 4 was more potent and longer lasting than that of LTD 4. On the other hand, at lower doses (0.1-10 ng), the effect of LTC 4 was similar to or less potent than that of LTD 4. Neither of the leukotrienes disturbed cardiac rhythm (fig. 1A), although small, inconsistent changes in heart rate were observed; percent changes induced by 100 ng of leukotrienes were 0.8___2.4 (variations: - 4 . 3 to 8.3) for LTC 4 and 0.8___2.3 ( - 6 . 8 to 9.3) for LTD 4. 3.2. Effects of LTC 4 and LTD 4 on isolated guinea pig atria Both LTC 4 and LTD 4, at concentrations of 10-8, 10-7 and 10-6 M, increased the contractile force of electrically paced left atria in a dosedependent manner by 4.2___2.1, 11.8___5.4 and 21.0±7.2% for LTC 4, and 6.1___1.9, 17.2___2.3 and 25.4___2.7% for LTD4, respectively. On the other hand, they showed no significant effect on the spontaneously beating rate of the right atria. 3.3. Release of prostanoids by isolated guinea pig hearts
LTC 4
and
LTD 4
in
Fig. 2 shows the time course of the release of 6-keto-PGFl, into the perfusate by LTC4 and LTD 4. Both leukotrienes released 6-keto-PGF~ dose-dependently at higher doses (10 and 100 ng), but not at lower doses (0.1 and 1 ng); the release by LTC4 was slightly higher than that by LTD 4. Neither leukotriene released a detectable amount of TXB2. Administration of saline containing K + at a dose corresponding to the highest dose of leukotrienes did not affect the coronary flow rate and cardiac parameters in isolated heart and atrial preparations, or the release of prostanoids in the isolated heart preparation.
4. Discussion
Anhut et al. (1977) noted two phases of decrease in the coronary flow rate in sensitized guinea pig hearts challenged with an antigen. They also demonstrated that the early phase of a short lasting decrease in coronary flow rate was mediated by TXA 2. However, mediator(s) of the later phase of a long lasting decrease in coronary flow rate have not been found. In the present study, both LTC4 and LTD4, major components of SRS-A (Lewis et al., 1980), were found to be potent coronary vasospastics in isolated perfused guinea pig hearts. Both leukotrienes also depressed left ventricular systolic pressure and its dp/dt; the time course and magnitude of the depression paralleled those of the decrease in coronary flow rate. In isolated guinea pig atria, however, the leukotrienes showed a slight positive inotropic effect. Therefore, the depression of left ventricular systolic pressure and its d p / d t might result from the decrease in coronary flow rate. With respect to coronary and cardiac actions, LTC4 was equal to or more potent than LTD 4 (fig. 1). In contrast, Drazen et al. (1980) reported that LTD 4 was much more potent than LTC 4 in contracting a parenchymal strip of guinea pig lung. This difference might be attributed to organ specificity. Although the arrhythmia was observed in cardiac anaphylaxis in isolated perfused guinea pig heart (Liebig et al., 1975), the leukotrienes used in the present experiments did not disturb the regular sinus rhythm in isolated heart and atria. Thus, arrhythmia during cardiac anaphylaxis might be mediated through substance(s) other than the leukotrienes. SRS-A is reported to release TXA 2 in isolated perfused guinea pig lung (Berti et al., 1980). The present study demonstrated that, at higher doses, LTC4 and LTD 4 dose-dependently increased the release of 6-keto-PGFl~, but not TXB2, in the perfusate (fig. 2), indicating that these leukotrienes release prostacyclin (PGI2). These findings are interesting, because the leukotrienes are among the arachidonic acid cascade metabolites (Lewis et al., 1980), although the significance and mechanisms of PGI 2 release are unclear. As to the mechanisms
361
of PGI 2 release, the following possibilities might be put forward; first, the release might be due to hypoxia caused by a decrease in coronary flow, as hypoxia causes PG release from perfused heart (Wennmalm et al., 1974). Second, vasoconstriction by the leukotrines per se might be a stimulus of PG release, although there is no supporting evidence for it. Third, the leukotrinenes might stimulate the arachidonic acid cascade directly. In conclusion, LTC 4 and LTD 4 decrease coronary flow rate and depress left ventricular systolic pressure and its dp/dt in isolated perfused guinea pig hearts. The leukotrienes also release prostacyclin (PGI2) , but not TXA 2. These findings suggest that leukotrienes might play a role in coronary vasospasm during cardiac anaphylaxis induced by an antigen.
Acknowledgements We thank Dr. K. Kikuchi (Biology Laboratories) for critical discussion in preparing the manuscript, and Mr. K. Kato for kindly supplying 6-keto-PGFt~ and TXB2.
References Anhut, H., W. Bemaur and B.A. Peskar, 1977, Radioimmunological determination of thromboxane release in cardiac anaphylaxis, European J. Pharmacol. 44, 85. Berti, F., G.C. Folco, A. Giachetti, S. Malandrino, C. Omini
and T. Vigano, 1980, Atropine inhibits thromboxane A 2 generation in isolated lungs of the guinea pigs, Br. I. Pharmacol. 68, 467. Capurro, N. and R. Levi, 1975, The heart as a target organ in systemic allergic reactions. Comparison of cardiac anaphylaxis in vivo and in vitro, Circ. Res. 36, 520. Cohen, N., B.L. Banner and R.J. Lopresti, 1980, Synthesis of optically active leukotriene (SRS-A) intermediates, Tetrahe&on Letters 24, 4163. Drazen, J.M., K.F. Austen, R.A. Lewis, D.A. Clark, G. Goto, A. Marfat and E.J. Corey, 1980, Comparatve airway and vascular activities of leukotrienes C-I and D in vivo and in vitro, Proc. Nat. Acad. Sci. U.S.A. 77, 4354. Gleason, J.G., D.B. Bryan and C.M. Kinzig, 1980, Convergent synthesis of ieukotriene A methyl ester, Tetrahedron Letters 21, 1129. Liebig, R., W. Bernaur and B.A. Peskar, 1975, Prostaglandin, slow-reacting substance, and histamine release from anaphylactic guinea-pig hearts, and its pharmacological modification, Naunyn-Schmiedeb. Arch. Pharmacol. 289, 65. Lewis, R.A., K.F. Austen, LM. Drazen, D.A. Clark, A. Marfat and E.J. Corey, 1980, Slow reacting substances of anaphylaxis: Identification of leukotrienes C-! and D from human and rat sources, Proc. Natl. Acad. Sci. U.S.A. 77, 3710. Shibouta, Y., Z. Terashita, Y. Inada, K. Nishikawa and S. K.ikuchi, 1981, Enhanced thromboxane A 2 biosynthesis in the kidney of sponaneously hypertensive rats during development of hypertension, European L Pharmacol. 70, 247. Terashita, Z., H. Fukui, K. Nishikawa, M. Hirata and S. Kikuchi, 1978, Coronary vasospastic action of thromboxane A 2 in isolated, working guinea pig hearts, European J. Pharmacol. 53, 49. Wennmalm, A., Pham-Huu-Chanh and M. Junstad, 1974, Hypoxia causes prostaglandin release from perfused rabbit hearts, Acta Phsiol. Scand. 91,133.
European Journal of Pharmacology, 73 (1981) 357-361 Elsevier/North-Holland Biomedical Press
Short communication
CORONARY VASOCONSTRICTION AND PGI 2 RELEASE BY LEUKOTRIENES IN ISOLATED GUINEA PIG HEARTS ZEN-ICHI TERASHITA, HIROSHI FUKUI, MINORU HIRATA, SHINJO TERAO, SHIGENORI OHKAWA, KOKEI NISHIKAWA and SHINTARO KIKUCHI Central Research Division, Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka 532, Japan Received 19 June 1981, accepted 23 June 1981
Z. TERASHITA, H. FUKUI, M. HIRATA, S. TERAO, S. OHKAWA, K. NISHIKAWA and S. KIKUCHI, Coronary vasoconstriction and PGI2 release by leukotrienes in isolated guinea pig hearts, European J. Pharmacol. 73 (1981) 357-361. In isolated, perfused guinea pig hearts, leukotrienes C4 and D 4 (0.1-lO3 ng) dose-dependently decreased coronary flow rate and left ventricular systolic pressure. The leukotrienes (10-103 ng) released prostacyclin, but not thromboxane A2, from the heart. In isolated atria, they (10-s_ 10-6M) showed slight positive inotropism. Thus, it is concluded that leukotrienes C4 and D4 are potent coronary vasoconstrictors, and might play a role in coronary vasospasm during cardiac anaphylaxis. Coronary vasoconstriction
Leukotrienes
Prostanoid release
I. Introduction
2. Materials and methods
Coronary vasoconstriction and arrhythmia have been observed in cardiac anaphylaxis induced by an antigen (Liebig et al., 1975; Anhut et al., 1977). The chemical mediators of these effects have been the subject of extensive investigations, and slow reacting substance of anaphylaxis (SRS-A), thromboxane A 2 (TXA2) and other vasoactive substances have been detected in coronary perfusate (Liebig et al., 1975; Anhut et al., 1977). Recently, Lewis et al. (1980) suggested that SRS-A was composed of leukotrienes C4 and D 4 (LTC4 and LTD4). Although potent contractile activity of LTC 4 and LTD4 on several smooth muscles has been reported (Drazen et al., 1980), the effects of the leukotrienes on the heart, a target organ in systemic anaphylaxis (Capurro and Levi, 1975), have not been clarified. In this report, we describe the effects of LTC 4 and LTD4 on coronary flow rate, heart rate, left ventricular systolic pressure and its dp/dt, and release of prostanoids in isolated guinea pig hearts.
2.1. Materials [ 3 H]6- keto- Prostaglandin F la ([ 3H]6 - ketoPGFI, ) (100 Ci/mmol, New England Nuclear, USA), [3H]TXB2 (150 Ci/mmol, N e w England Nuclear, USA), antisera to 6-keto-PGFl~ and TXB2 (prepared by us), 6-keto-PGFl~, and TXB2 (synthesized by mr. Kato in this Chemistry Laboratory) were used.
2.2. Isolated heart preparation Heparinized male guinea pigs (about 250 g) were killed by a blow on the head, and the heart was excised. A polyethylene cannula for perfusion was inserted retrogradely into the aorta, and the heart was perfused according to the method of Langendorff (Terashita et al., 1978). The heart was suspended in a chamber maintained at 37°C, and perfused at a constant pressure of 750 mm H20 with a modified Krebs-Ringer bicarbonate solu-
0014-2999/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
358
tion composed of (raM) 127 NaC1, 4.7 KC1, 2.5 CaC12, 1.2 KH2PO 4, 25 NaHCO 3, 2.0 sodium pyruvate and 5.5 glucose, warmed to 37°C and bubbled with 3% CO 2 in 02. Coronary flow rate was measured with a cannulating-type flow probe of an electromagnetic flowmeter (Nihon Kohden, MF-26) placed just above the aortic cannula. A urethane polymer balloon connected to a glass tube was inserted into the left ventricular cavity through an incision in the left auricle. Left ventricular isovolumetric pressure was recorded with a pressure transducer (Nihon Kohden, MPU-0.5) connected to the intraventricular balloon filled with physiological saline. The heart rate was recorded from left ventricular pressure pulses with a cardiotachometer (Nihon Kohden, RT-2). Experiments were started after a 20-30 min adaptation period. The basal coronary flow rate, left ventricular systolic pressure and its dp/dt, and heart rate (the mean_S.E.M.) were 8.0___0.4 ml/min, 82.7___3.8 mm Hg, 1657___109 mm Hg/sec and 245-+-8 beats/min (n=12), respectively. Only one dose-response relation for each leukotriene was determined in each preparation. LTC4 and LTD 4 were dissolved in saline (200/~1), and injected in a bolus into the coronary perfusion system. The perfused hearts weighed 1.4-1.5 g.
2.3. Isolated atrial preparation The isolated atrial preparation was suspended with a diastolic tension of 0.5 g in a 40 ml organ bath containing Krebs-Henseleit solution, bubbled with 3% CO 2 in 02 at 30°C. The right atrium was allowed to beat spontaneously to measure the beating rate, and the left atrium was driven electrically by square wave pulses (5 V, 1 msec, 1 Hz) to measure the contractile force. Only one cumulative dose-response relation for each leukotriene was obtained in each preparation. Each dose was applied at intervals between 5-10 min after each plateau level of response was attained. The basal contractile force and beating rate were 971 ___88 mg and 129___6 beats/rain (n--6), respectively.
2.4. Radioimmunoassay of 6-keto-PGFla and TXB: in the perfusate The perfusate was collected serially o( intermittenly for 1 min just before and from 0 to 10 min after the injection of the test agents. Fifty/~1 of the perfusate were used to determine 6,keto-PGFj~ and TXB2 by a radioimmunoassay technique (Shibouta et al., 1981). The assay technique enabled us to detect 10-1000 pg of both prostanoids. Cross-reactivities of 6-keto-PGF~, antiserum with TXB 2 and other prostaglandins were less than 0.1% for PGD 2 and TXB2, 1.8% for PGE l, 1.3% for PGE 2 and 4.5% for PGF2a. Those of the TXB 2 antiserum were less than 1.0% for PGE1; PGE 2, PGF2~ and 6-keto-PGFla and 1.1% for PGD 2. LTC 4 and LTD 4, up to 100 ng, showed no crossreaction with either of the antisera.
2.5. Synthesis of L T C 4 and L T D 4 LTA 4 methyleser was prepared in three steps by using the (5S,6S)-methyl-5,6-epoxy-7hydroxyheptanoate derived from D-araboascorbic acid (Cohen et al., 1980, Gleason et al., 1980). LTC 4 and LTD 4 as potassium salts were obtained by the coupling reactions of LTA 4 methylester with the corresponding peptide components followed by alkaline hydrolysis. Purification was carried out by reverse phase high performance liquid chromatography. They were stored as a methanol solution of their potassium salts below - 2 0 ° C until used. The dose of leukotrienes is presented as their potassium salts.
3. Results
3.1. Coronary and cardiac actions of LTC 4 and L T D 4 in isolated guinea pig hearts The administration of both leukotrienes (0.1100 ng) rapidly decreased the coronary flow rate, and depressed the left ventricular systolic pressure and its d p / d t (fig. 1). Fig. 1A shows typical tracings of these experiments at 100 ng of LTC 4 and LTD4. At the highest dose (100 ng), LTC4 was more potent and had a longer lasting effect than
359
B
A (7 CA F (mb,n~wn)
5' 20'
I0 r
0L ~
0
,~
o.,--o LTC4
10
~
LTD 4
2O (ram I..~)
•
L v c~o,tl20001:
m"
(~mHo~) 0" HR
L.
300[
~
(b,,,mr:,~) 2ooL
50 60
ff CAF (ml/n~n)
5' Iff
I0 0E
LVP (,~ ~) ~
o_
0
>
_.J
19
"
20
Lv a ~
HR
2501" _ j
( ~ )
0.i
200'-
i
10
100
Dose (ng)
~oong
Fig. 1. (A) Effects of 100 ng of LTC+ (upper) and LTD 4 (lower) on coronary flow rate and cardiac parameters. (B) Dose-dependent effects of leukotrienes on coronary flow rate and left ventricular systolic pressure. Values are the m e a n _ S . E . M . (n=6). CAF: coronary arterial flow; LVP, left ventricular pressure; L V d p / d t : left ventricular d p / d t ; HR: heart rate.
2.0 A
LTC4
LT D/~
1.5
~: 1.0
.~ 0.5
02/.6810
~
0246810
I()0
0 2 4 6 8 10 10 ng
0 2 4 6 8 10 {rain) 100 ng
Fig. 2. Effects of LTC 4 and LTD 4 on 6-keto-POFl~, release in isolated guinea pig hearts. Values are the m e a n ± S . E . M . (n=6).
360
LTD 4 in decreasing coronary flow rate. At the lower doses (0.1, 1 and 10 ng), the coronary action of LTC4 and LTD 4 was almost as potent (fig. 1B). Almost the same description can be applied to the depression in left ventricular systolic pressure and its dp/dt. At the highest dose (100 ng) this negative inotropic effect of LTC 4 was more potent and longer lasting than that of LTD 4. On the other hand, at lower doses (0.1-10 ng), the effect of LTC 4 was similar to or less potent than that of LTD 4. Neither of the leukotrienes disturbed cardiac rhythm (fig. 1A), although small, inconsistent changes in heart rate were observed; percent changes induced by 100 ng of leukotrienes were 0.8___2.4 (variations: - 4 . 3 to 8.3) for LTC 4 and 0.8___2.3 ( - 6 . 8 to 9.3) for LTD 4. 3.2. Effects of LTC 4 and LTD 4 on isolated guinea pig atria Both LTC 4 and LTD 4, at concentrations of 10-8, 10-7 and 10-6 M, increased the contractile force of electrically paced left atria in a dosedependent manner by 4.2___2.1, 11.8___5.4 and 21.0±7.2% for LTC 4, and 6.1___1.9, 17.2___2.3 and 25.4___2.7% for LTD4, respectively. On the other hand, they showed no significant effect on the spontaneously beating rate of the right atria. 3.3. Release of prostanoids by isolated guinea pig hearts
LTC 4
and
LTD 4
in
Fig. 2 shows the time course of the release of 6-keto-PGFl, into the perfusate by LTC4 and LTD 4. Both leukotrienes released 6-keto-PGF~ dose-dependently at higher doses (10 and 100 ng), but not at lower doses (0.1 and 1 ng); the release by LTC4 was slightly higher than that by LTD 4. Neither leukotriene released a detectable amount of TXB2. Administration of saline containing K + at a dose corresponding to the highest dose of leukotrienes did not affect the coronary flow rate and cardiac parameters in isolated heart and atrial preparations, or the release of prostanoids in the isolated heart preparation.
4. Discussion
Anhut et al. (1977) noted two phases of decrease in the coronary flow rate in sensitized guinea pig hearts challenged with an antigen. They also demonstrated that the early phase of a short lasting decrease in coronary flow rate was mediated by TXA 2. However, mediator(s) of the later phase of a long lasting decrease in coronary flow rate have not been found. In the present study, both LTC4 and LTD4, major components of SRS-A (Lewis et al., 1980), were found to be potent coronary vasospastics in isolated perfused guinea pig hearts. Both leukotrienes also depressed left ventricular systolic pressure and its dp/dt; the time course and magnitude of the depression paralleled those of the decrease in coronary flow rate. In isolated guinea pig atria, however, the leukotrienes showed a slight positive inotropic effect. Therefore, the depression of left ventricular systolic pressure and its d p / d t might result from the decrease in coronary flow rate. With respect to coronary and cardiac actions, LTC4 was equal to or more potent than LTD 4 (fig. 1). In contrast, Drazen et al. (1980) reported that LTD 4 was much more potent than LTC 4 in contracting a parenchymal strip of guinea pig lung. This difference might be attributed to organ specificity. Although the arrhythmia was observed in cardiac anaphylaxis in isolated perfused guinea pig heart (Liebig et al., 1975), the leukotrienes used in the present experiments did not disturb the regular sinus rhythm in isolated heart and atria. Thus, arrhythmia during cardiac anaphylaxis might be mediated through substance(s) other than the leukotrienes. SRS-A is reported to release TXA 2 in isolated perfused guinea pig lung (Berti et al., 1980). The present study demonstrated that, at higher doses, LTC4 and LTD 4 dose-dependently increased the release of 6-keto-PGFl~, but not TXB2, in the perfusate (fig. 2), indicating that these leukotrienes release prostacyclin (PGI2). These findings are interesting, because the leukotrienes are among the arachidonic acid cascade metabolites (Lewis et al., 1980), although the significance and mechanisms of PGI 2 release are unclear. As to the mechanisms
361
of PGI 2 release, the following possibilities might be put forward; first, the release might be due to hypoxia caused by a decrease in coronary flow, as hypoxia causes PG release from perfused heart (Wennmalm et al., 1974). Second, vasoconstriction by the leukotrines per se might be a stimulus of PG release, although there is no supporting evidence for it. Third, the leukotrinenes might stimulate the arachidonic acid cascade directly. In conclusion, LTC 4 and LTD 4 decrease coronary flow rate and depress left ventricular systolic pressure and its dp/dt in isolated perfused guinea pig hearts. The leukotrienes also release prostacyclin (PGI2) , but not TXA 2. These findings suggest that leukotrienes might play a role in coronary vasospasm during cardiac anaphylaxis induced by an antigen.
Acknowledgements We thank Dr. K. Kikuchi (Biology Laboratories) for critical discussion in preparing the manuscript, and Mr. K. Kato for kindly supplying 6-keto-PGFt~ and TXB2.
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